58 results on '"M. Dennis Leo"'
Search Results
2. Diabetic Endothelial Cell Glycogen Synthase Kinase 3β Activation Induces VCAM1 Ectodomain Shedding
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Masuma Akter Brishti, Somasundaram Raghavan, Kennedy Lamar, Udai P. Singh, Daniel M. Collier, and M. Dennis Leo
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diabetic vascular disease ,endothelial cells ,soluble vascular cell adhesion molecule 1 ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Soluble cell adhesion molecules (sCAMs) are secreted ectodomain fragments of surface adhesion molecules, ICAM1 and VCAM1. sCAMs have diverse immune functions beyond their primary function, impacting immune cell recruitment and activation. Elevated sVCAM1 levels have been found to be associated with poor cardiovascular disease (CVD) outcomes, supporting VCAM1’s role as a potential diagnostic marker and therapeutic target. Inhibiting sVCAM1’s release or its interaction with immune cells could offer cardioprotection in conditions such as diabetes. Membrane-bound surface adhesion molecules are widely expressed in a wide variety of cell types with higher expression in endothelial cells (ECs). Still, the source of sCAMs in the circulation is not clear. Hypothesizing that endothelial cells (ECs) could be a potential source of sCAMs, this study investigated whether dysfunctional EC signaling mechanisms during diabetes cause VCAM1 ectodomain shedding. Our results from samples from an inducible diabetic mouse model revealed increased sVCAM1 plasma levels in diabetes. Protein analysis indicated upregulated VCAM1 expression and metalloproteases ADAM10 and ADAM17 in diabetic ECs. ADAMs are known for proteolytic cleavage of adhesion molecules, contributing to inflammation. GSK3β, implicated in EC VCAM1 expression, was found to be activated in diabetic ECs. GSK3β activation in control ECs increased ADAM10/17 and VCAM1. A GSK3β inhibitor reduced active GSK3β and VCAM1 ectodomain shedding. These findings suggest diabetic ECs with elevated GSK3β activity led to VCAM1 upregulation and ADAM10/17-mediated sVCAM1 shedding. This mechanism underscores the potential therapeutic role of GSK3β inhibition in reducing the levels of circulating sVCAM1. The complex roles of sCAMs extend well beyond CVD. Thus, unraveling the intricate involvement of sCAMs in the initiation and progression of vascular disease, particularly in diabetes, holds significant therapeutic potential.
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- 2023
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3. Hypoxia induces purinergic receptor signaling to disrupt endothelial barrier function
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Somasundaram Raghavan, Masuma Akter Brishti, Daniel Mohr Collier, and M. Dennis Leo
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hypoxia ,endothelial cells ,purinergic receptor ,blood-brain barrier ,diabetes ,Physiology ,QP1-981 - Abstract
Blood-brain-barrier permeability is regulated by endothelial junctional proteins and is vital in limiting access to and from the blood to the CNS. When stressed, several cells, including endothelial cells, can release nucleotides like ATP and ADP that signal through purinergic receptors on these cells to disrupt BBB permeability. While this process is primarily protective, unrestricted, uncontrolled barrier disruption during injury or inflammation can lead to serious neurological consequences. Purinergic receptors are broadly classified into two families: the P1 adenosine and P2 nucleotide receptors. The P2 receptors are further sub-classified into the P2XR ion channels and the P2YR GPCRs. While ATP mainly activates P2XRs, P2YRs have a broader range of ligand selectivity. The P2Y1R, essential for platelet function, is reportedly ubiquitous in its expression. Prior studies using gene knockout and specific antagonists have shown that these approaches have neuroprotective effects following occlusive stroke. Here we investigated the expression of P2Y1R in primary cultured brain endothelial cells and its relation to the maintenance of BBB function. Results show that following in vitro hypoxia and reoxygenation, P2Y1R expression is upregulated in both control and diabetic cells. At the same time, endothelial junctional markers, ZO-1 and VE-cadherin, were downregulated, and endothelial permeability increased. siRNA knockdown of P2Y1R and MRS 2500 effectively blocked this response. Thus, we show that P2Y1R signaling in endothelial cells leads to the downregulation of endothelial barrier function.
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- 2022
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4. Histamine Potentiates SARS-CoV-2 Spike Protein Entry Into Endothelial Cells
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Somasundaram Raghavan and M. Dennis Leo
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SARS-CoV-2 ,histamine ,famotidine ,spike ,angiotensin-converting enzyme-2 ,Therapeutics. Pharmacology ,RM1-950 - Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which causes coronavirus disease (COVID-19) is one of the most serious global health crises in recent history. COVID-19 patient symptoms range from life-threatening to mild and asymptomatic, which presents unique problems in identifying, quarantining, and treating the affected individuals. The emergence of unusual symptoms among survivors, now referred to as “Long COVID”, is concerning, especially since much about the condition and the treatment of it is still relatively unknown. Evidence so far also suggests that some of these symptoms can be attributed to vascular inflammation. Although famotidine, the commonly used histamine H2 receptor (H2R) blocker, was shown to have no antiviral activity, recent reports indicate that it could prevent adverse outcomes in COVID-19 patients. Histamine is a classic proinflammatory mediator, the levels of which increase along with other cytokines during COVID-19 infection. Histamine activates H2R signaling, while famotidine specifically blocks H2R activation. Investigating the effects of recombinant SARS-CoV-2 spike protein S1 Receptor-Binding Domain (Spike) on ACE2 expression in cultured human coronary artery endothelial cells, we found that the presence of histamine potentiated spike-mediated ACE2 internalization into endothelial cells. This effect was blocked by famotidine, protein kinase A inhibition, or by H2 receptor protein knockdown. Together, these results indicate that histamine and histamine receptor signaling is likely essential for spike protein to induce ACE2 internalization in endothelial cells and cause endothelial dysfunction and that this effect can be blocked by the H2R blocker, famotidine.
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- 2022
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5. Rab GTPases as Modulators of Vascular Function
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Somasundaram Raghavan, Masuma Akter Brishti, and M. Dennis Leo
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Rab GTPases ,endothelial cells ,vascular smooth muscle ,Cytology ,QH573-671 - Abstract
Rab GTPases, the largest family of small GTPases, are ubiquitously expressed proteins that control various aspects of cellular function, from cell survival to exocytosis. Rabs cycle between the GDP-bound inactive form and the GTP-bound active form. When activated, specific Rab GTPase-positive vesicles mediate cellular networks involved in intracellular trafficking, recycling, and/or exocytosis of cargo proteins. Dysfunctional Rab signaling pathways have been implicated in various disease processes. The precise cellular functions of several members of the Rab GTPase family are still unknown. A lack of pharmacological tools and the lethality of gene knockouts have made more detailed characterizations of their protein interaction networks difficult. Nevertheless, available evidence suggests that these proteins are vital for normal cell function. Endothelial and smooth muscle cells control vascular lumen diameter and modulate blood flow. Endothelial cells also secrete several pro- and antithrombotic factors and vasoactive substances to coordinate local inflammatory responses and angiogenesis. Rab GTPase function in endothelial cells has been relatively well-explored, while only a handful of reports are available on these proteins in vascular smooth muscle. This review summarizes the present knowledge on Rab GTPases in the vasculature.
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- 2022
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6. SARS-CoV-2 Spike Protein Induces Degradation of Junctional Proteins That Maintain Endothelial Barrier Integrity
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Somasundaram Raghavan, Divya Borsandra Kenchappa, and M. Dennis Leo
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endothelial barrier function ,SARS-CoV-2 ,angiotensin converting enzyme-2 ,spike ,junctional proteins ,Diseases of the circulatory (Cardiovascular) system ,RC666-701 - Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses the Angiotensin converting enzyme 2 (ACE2) receptor present on the cell surface to enter cells. Angiotensin converting enzyme 2 is present in many cell types including endothelial cells, where it functions to protect against oxidative damage. There is growing evidence to suggest that coronavirus disease (COVID-19) patients exhibit a wide range of post-recovery symptoms and shows signs related to cardiovascular and specifically, endothelial damage. We hypothesized that these vascular symptoms might be associated with disrupted endothelial barrier integrity. This was investigated in vitro using endothelial cell culture and recombinant SARS-CoV-2 spike protein S1 Receptor-Binding Domain (Spike). Mouse brain microvascular endothelial cells from normal (C57BL/6 mice) and diabetic (db/db) mice were used. An endothelial transwell permeability assay revealed increased permeability in diabetic cells as well as after Spike treatment. The expression of VE-Cadherin, an endothelial adherens junction protein, JAM-A, a tight junctional protein, Connexin-43, a gap junctional protein, and PECAM-1, were all decreased significantly after Spike treatment in control and to a greater extent, in diabetic cells. In control cells, Spike treatment increased association of endothelial junctional proteins with Rab5a, a mediator of the endocytic trafficking compartment. In cerebral arteries isolated from control and diabetic animals, Spike protein had a greater effect in downregulating expression of endothelial junctional proteins in arteries from diabetic animals than from control animals. In conclusion, these experiments reveal that Spike-induced degradation of endothelial junctional proteins affects endothelial barrier function and is the likely cause of vascular damage observed in COVID-19 affected individuals.
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- 2021
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7. Age‐dependent decrease in TRPM4 channel expression but not trafficking alters urinary bladder smooth muscle contractility
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Sarah E. Maxwell, M. Dennis Leo, John Malysz, and Georgi V. Petkov
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channel trafficking ,detrusor ,ion channel ,maturation ,Western blot ,Physiology ,QP1-981 - Abstract
Abstract During development, maturation, or aging, the expression and function of urinary bladder smooth muscle (UBSM) ion channels can change, thus affecting micturition. Increasing evidence supports a novel role of transient receptor potential melastatin‐4 (TRPM4) channels in UBSM physiology. However, it remains unknown whether the functional expression of these key regulatory channels fluctuates in UBSM over different life stages. Here, we examined TRPM4 channel protein expression (Western blot) and the effects of TRPM4 channel inhibitors, 9‐phenanthrol and glibenclamide, on phasic contractions of UBSM isolated strips obtained from juvenile (UBSM‐J, 5–9 weeks old) and adult (UBSM‐A, 6–18 months old) male guinea pigs. Compared to UBSM‐J, UBSM‐A displayed a 50–70% reduction in total TRPM4 protein expression, while the surface‐to‐intracellular expression ratio (channel trafficking) remained the same in both age groups. Consistent with the reduced total TRPM4 protein expression in UBSM‐A, 9‐phenanthrol showed lower potencies and/or maximum efficacies in UBSM‐A than UBSM‐J for inhibiting amplitude and muscle force of spontaneous and 20 mM KCl‐induced phasic contractions. Compared to 9‐phenanthrol, glibenclamide also attenuated both spontaneous and KCl‐induced contractions, but with less pronounced differential effects in UBSM‐A and UBSM‐J. In both age groups, regardless of the overall reduced total TRPM4 protein expression in UBSM‐A, cell surface TRPM4 protein expression (~80%) predominated over its intracellular fraction (~20%), revealing preserved channel trafficking mechanisms toward the cell membrane. Collectively, this study reports novel findings illuminating a fundamental physiological role for TRPM4 channels in UBSM function that fluctuates with age.
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- 2021
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8. A plasma membrane-localized polycystin-1/polycystin-2 complex in endothelial cells elicits vasodilation
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Charles E MacKay, Miranda Floen, M Dennis Leo, Raquibul Hasan, Tessa AC Garrud, Carlos Fernández-Peña, Purnima Singh, Kafait U Malik, and Jonathan H Jaggar
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polycystin-1 ,polycystin-2 ,endothelial cell ,vasodilation ,blood pressure ,flow ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
Polycystin-1 (PC-1, PKD1), a receptor-like protein expressed by the Pkd1 gene, is present in a wide variety of cell types, but its cellular location, signaling mechanisms, and physiological functions are poorly understood. Here, by studying tamoxifen-inducible, endothelial cell (EC)-specific Pkd1 knockout (Pkd1 ecKO) mice, we show that flow activates PC-1-mediated, Ca2+-dependent cation currents in ECs. EC-specific PC-1 knockout attenuates flow-mediated arterial hyperpolarization and vasodilation. PC-1-dependent vasodilation occurs over the entire functional shear stress range and via the activation of endothelial nitric oxide synthase (eNOS) and intermediate (IK)- and small (SK)-conductance Ca2+-activated K+ channels. EC-specific PC-1 knockout increases systemic blood pressure without altering kidney anatomy. PC-1 coimmunoprecipitates with polycystin-2 (PC-2, PKD2), a TRP polycystin channel, and clusters of both proteins locate in nanoscale proximity in the EC plasma membrane. Knockout of either PC-1 or PC-2 (Pkd2 ecKO mice) abolishes surface clusters of both PC-1 and PC-2 in ECs. Single knockout of PC-1 or PC-2 or double knockout of PC-1 and PC-2 (Pkd1/Pkd2 ecKO mice) similarly attenuates flow-mediated vasodilation. Flow stimulates nonselective cation currents in ECs that are similarly inhibited by either PC-1 or PC-2 knockout or by interference peptides corresponding to the C-terminus coiled-coil domains present in PC-1 or PC-2. In summary, we show that PC-1 regulates arterial contractility through the formation of an interdependent signaling complex with PC-2 in ECs. Flow stimulates PC-1/PC-2 clusters in the EC plasma membrane, leading to eNOS, IK channel, and SK channel activation, vasodilation, and a reduction in blood pressure.
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- 2022
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9. Correction: Intravascular flow stimulates PKD2 (polycystin-2) channels in endothelial cells to reduce blood pressure
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Charles E MacKay, M Dennis Leo, Carlos Fernández-Peña, Raquibul Hasan, Wen Yin, Alejandro Mata-Daboin, Simon Bulley, Jesse Gammons, Salvatore Mancarella, and Jonathan H Jaggar
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Medicine ,Science ,Biology (General) ,QH301-705.5 - Published
- 2020
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10. Correction: Arterial smooth muscle cell PKD2 (TRPP1) channels regulate systemic blood pressure
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Simon Bulley, Carlos Fernández-Peña, Raquibul Hasan, M Dennis Leo, Padmapriya Muralidharan, Charles E MacKay, Kirk W Evanson, Luiz Moreira-Junior, Alejandro Mata-Daboin, Sarah K Burris, Qian Wang, Korah P Kuruvilla, and Jonathan H Jaggar
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Medicine ,Science ,Biology (General) ,QH301-705.5 - Published
- 2020
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11. Intravascular flow stimulates PKD2 (polycystin-2) channels in endothelial cells to reduce blood pressure
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Charles E MacKay, M Dennis Leo, Carlos Fernández-Peña, Raquibul Hasan, Wen Yin, Alejandro Mata-Daboin, Simon Bulley, Jesse Gammons, Salvatore Mancarella, and Jonathan H Jaggar
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flow mediated-vasodilation ,polycystin-2 ,blood pressure ,endothelial cell ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
PKD2 (polycystin-2, TRPP1), a TRP polycystin channel, is expressed in endothelial cells (ECs), but its physiological functions in this cell type are unclear. Here, we generated inducible, EC-specific Pkd2 knockout mice to examine vascular functions of PKD2. Data show that a broad range of intravascular flow rates stimulate EC PKD2 channels, producing vasodilation. Flow-mediated PKD2 channel activation leads to calcium influx that activates SK/IK channels and eNOS serine 1176 phosphorylation in ECs. These signaling mechanisms produce arterial hyperpolarization and vasodilation. In contrast, EC PKD2 channels do not contribute to acetylcholine-induced vasodilation, suggesting stimulus-specific function. EC-specific PKD2 knockout elevated blood pressure in mice without altering cardiac function or kidney anatomy. These data demonstrate that flow stimulates PKD2 channels in ECs, leading to SK/IK channel and eNOS activation, hyperpolarization, vasodilation and a reduction in systemic blood pressure. Thus, PKD2 channels are a major component of functional flow sensing in the vasculature.
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- 2020
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12. Endothelial cell TMEM16A channels regulate arterial contractility and blood pressure
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Alejandro Mata-Daboin, Tessa Garrud, Carlos Fernandez-Pena, Dieniffer Peixoto-Neves, Angelica Bernardelli, M Dennis Leo, and Jonathan Jaggar
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Physiology - Abstract
Endothelial cells (ECs) are electrically coupled to arterial smooth muscle cells in the vascular wall and can modulate their contractility through direct control of membrane potential and via the production of several vasoactive substances. ECs express several cation channels which are known to regulate arterial contractility. In contrast, physiological functions of anion channels in ECs are unclear. ECs express TMEM16A, a Ca2+-activated Cl-channel. To examine physiological functions of TMEM16A channels in ECs, we generated an inducible, EC-specific TMEM16A knockout (TMEM16A ecKO) mouse. An increase in intracellular Ca2+ concentration ([Ca2+]i) or the application of acetylcholine (ACh) activated Cl‑ currents in fresh-isolated mesenteric artery ECs of control ( TMEM16Afl/fl) mice that were inhibited by tannic acid or benzbromarone, which are TMEM16A channel blockers. In contrast, an increase in [Ca2+]i or ACh did not activate Cl‑ currents in TMEM16A ecKO ECs. HC067047, a TRPV4 channel blocker, inhibited TMEM16A current activation by ACh in TMEM16Afl/fl ECs. GSK101, a TRPV4 channel activator, stimulated TMEM16A currents in TMEM16Afl/fl ECs, but not in TMEM16A ecKO ECs. Super resolution single-molecule localization microscopy demonstrated that surface clusters of TMEM16A and TRPV4 channels locate in nanoscale spatial proximity in the plasma membrane of ECs. ACh produced membrane hyperpolarization in pressurized TMEM16Afl/fl arteries that was attenuated in TMEM16A ecKO arteries. Vasodilation to ACh was smaller in pressurized TMEM16A ecKO arteries than in TMEM16Afl/fl arteries. TMEM16A knockout in ECs increased blood pressure in mice. These data indicate that ACh activates TRPV4-coupled TMEM16A channels in ECs to induce membrane hyperpolarization, vasodilation and a reduction in blood pressure. National Institutes of Health This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
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- 2023
13. Arterial smooth muscle cell PKD2 (TRPP1) channels regulate systemic blood pressure
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Simon Bulley, Carlos Fernández-Peña, Raquibul Hasan, M Dennis Leo, Padmapriya Muralidharan, Charles E Mackay, Kirk W Evanson, Luiz Moreira-Junior, Alejandro Mata-Daboin, Sarah K Burris, Qian Wang, Korah P Kuruvilla, and Jonathan H Jaggar
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PKD2 ,blood pressure ,hypertension ,smooth muscle ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
Systemic blood pressure is determined, in part, by arterial smooth muscle cells (myocytes). Several Transient Receptor Potential (TRP) channels are proposed to be expressed in arterial myocytes, but it is unclear if these proteins control physiological blood pressure and contribute to hypertension in vivo. We generated the first inducible, smooth muscle-specific knockout mice for a TRP channel, namely for PKD2 (TRPP1), to investigate arterial myocyte and blood pressure regulation by this protein. Using this model, we show that intravascular pressure and α1-adrenoceptors activate PKD2 channels in arterial myocytes of different systemic organs. PKD2 channel activation in arterial myocytes leads to an inward Na+ current, membrane depolarization and vasoconstriction. Inducible, smooth muscle cell-specific PKD2 knockout lowers both physiological blood pressure and hypertension and prevents pathological arterial remodeling during hypertension. Thus, arterial myocyte PKD2 controls systemic blood pressure and targeting this TRP channel reduces high blood pressure.
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- 2018
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14. Vasodilators stimulate SK3 channel anterograde trafficking in endothelial cells to elicit vasodilation
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Dieniffer Peixoto‐Neves, Shambhu Yadav, Charles Mackay, M. Dennis Leo, Alejandro Mata‐Daboin, Ulrich C. Mbiakop, and Jonathan H. Jaggar
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Genetics ,Molecular Biology ,Biochemistry ,Biotechnology - Published
- 2022
15. Author response: A plasma membrane-localized polycystin-1/polycystin-2 complex in endothelial cells elicits vasodilation
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Charles E MacKay, Miranda Floen, M Dennis Leo, Raquibul Hasan, Tessa AC Garrud, Carlos Fernández-Peña, Purnima Singh, Kafait U Malik, and Jonathan H Jaggar
- Published
- 2022
16. SUMO1 modification of PKD2 channels regulates arterial contractility
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M. Dennis Leo, Simon Bulley, Wen Yin, Carlos Fernández-Peña, Padmapriya Muralidharan, Alejandro Mata-Daboin, Jonathan H. Jaggar, Raquibul Hasan, and Charles E. Mackay
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education.field_of_study ,Multidisciplinary ,Contraction (grammar) ,Cell division ,urogenital system ,Chemistry ,Vasodilation ,Membrane hyperpolarization ,Biological Sciences ,urologic and male genital diseases ,female genital diseases and pregnancy complications ,Cell biology ,Contractility ,Polycystin 2 ,medicine ,Myocyte ,medicine.symptom ,education ,Vasoconstriction - Abstract
PKD2 (polycystin-2, TRPP1) channels are expressed in a wide variety of cell types and can regulate functions, including cell division and contraction. Whether posttranslational modification of PKD2 modifies channel properties is unclear. Similarly uncertain are signaling mechanisms that regulate PKD2 channels in arterial smooth muscle cells (myocytes). Here, by studying inducible, cell-specific Pkd2 knockout mice, we discovered that PKD2 channels are modified by SUMO1 (small ubiquitin-like modifier 1) protein in myocytes of resistance-size arteries. At physiological intravascular pressures, PKD2 exists in approximately equal proportions as either nonsumoylated (PKD2) or triple SUMO1-modifed (SUMO-PKD2) proteins. SUMO-PKD2 recycles, whereas unmodified PKD2 is surface-resident. Intravascular pressure activates voltage-dependent Ca 2+ influx that stimulates the return of internalized SUMO-PKD2 channels to the plasma membrane. In contrast, a reduction in intravascular pressure, membrane hyperpolarization, or inhibition of Ca 2+ influx leads to lysosomal degradation of internalized SUMO-PKD2 protein, which reduces surface channel abundance. Through this sumoylation-dependent mechanism, intravascular pressure regulates the surface density of SUMO-PKD2−mediated Na + currents (I Na ) in myocytes to control arterial contractility. We also demonstrate that intravascular pressure activates SUMO-PKD2, not PKD2, channels, as desumoylation leads to loss of I Na activation in myocytes and vasodilation. In summary, this study reveals that PKD2 channels undergo posttranslational modification by SUMO1, which enables physiological regulation of their surface abundance and pressure-mediated activation in myocytes and thus control of arterial contractility.
- Published
- 2019
17. K V 7 Channel Subtype Expression and Physiological Roles in Rat Urinary Bladder Smooth Muscle Excitability and Contractility
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M. Dennis Leo, John Malysz, Wenkuan Xin, Georgi V. Petkov, Sarah Cawan Maxwell, and Frank Park
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Contractility ,Smooth muscle ,Chemistry ,Genetics ,Channel (broadcasting) ,Rat Urinary Bladder ,Molecular Biology ,Biochemistry ,Biotechnology ,Cell biology - Published
- 2021
18. Large conductance Ca2+-activated K+ channel (BKCa) α-subunit splice variants in resistance arteries from rat cerebral and skeletal muscle vasculature.
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Zahra Nourian, Min Li, M Dennis Leo, Jonathan H Jaggar, Andrew P Braun, and Michael A Hill
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Medicine ,Science - Abstract
Previous studies report functional differences in large conductance Ca2+ activated-K+ channels (BKCa) of smooth muscle cells (VSMC) from rat cerebral and cremaster muscle resistance arteries. The present studies aimed to determine if this complexity in BKCa activity may, in part, be due to splice variants in the pore-forming α-subunit. BKCa variants in the intracellular C terminus of the α-subunit, and their relative expression to total α-subunit, were examined by qPCR. Sequencing of RT-PCR products showed two α-subunit variants, ZERO and STREX, to be identical in cremaster and cerebral arteries. Levels of STREX mRNA expression were, however, significantly higher in cremaster VSMCs (28.9±4.2% of total α-BKCa) compared with cerebral vessels (16.5±0.9%). Further, a low level of BKCa SS4 α-subunit variant was seen in cerebral arteries, while undetectable in cremaster arteries. Protein biotinylation assays, in expression systems and arterial preparations, were used to determine whether differences in splice variant mRNA expression affect surface membrane/cytosolic location of the channel. In AD-293 and CHO-K1 cells, rat STREX was more likely to be located at the plasma membrane compared to ZERO, although the great majority of channel protein was in the membrane in both cases. Co-expression of β1-BKCa subunit with STREX or ZERO did not influence the dominant membrane expression of α-BKCa subunits, whereas in the absence of α-BKCa, a significant proportion of β1-subunit remained cytosolic. Biotinylation assays of cremaster and cerebral arteries showed that differences in STREX/ZERO expression do not alter membrane/cytosolic distribution of the channel under basal conditions. These data, however, revealed that the amount of α-BKCa in cerebral arteries is approximately 20X higher than in cremaster vessels. Thus, the data support the major functional differences in BKCa activity in cremaster, as compared to cerebral VSMCs, being related to total α-BKCa expression, regardless of differences in splice variant expression.
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- 2014
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19. Cholesterol activates BK channels by increasing KCNMB1 protein levels in the plasmalemma
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M. Dennis Leo, Alex M. Dopico, Anna N. Bukiya, and Jonathan H. Jaggar
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0301 basic medicine ,Male ,BK channel ,Vascular smooth muscle ,Potassium Channels ,BK, large conductance ,slo1 ,Large-Conductance Calcium-Activated Potassium Channel beta Subunits ,BFA, brefeldin A ,Biochemistry ,Calcium in biology ,Muscle, Smooth, Vascular ,Rats, Sprague-Dawley ,chemistry.chemical_compound ,Mice ,MβCD, methyl-β-cyclodextrin ,Myocyte ,biology ,Smooth muscle contraction ,Brefeldin A ,Coronary Vessels ,Potassium channel ,Cell biology ,Cholesterol ,CA, coronary artery, Ca2+-and voltage-gated potassium (channels) ,DM, dissociation medium ,wt, wild type ,Research Article ,BK beta1 subunit ,MaxiK ,BA, basilar artery ,PBS, phosphate-buffered saline ,03 medical and health sciences ,CRAC, cholesterol recognition amino acid consensus (motif) ,Homomeric ,Animals ,Large-Conductance Calcium-Activated Potassium Channels ,Molecular Biology ,Muscle Cells ,KO, knockout ,030102 biochemistry & molecular biology ,Cell Membrane ,Membrane Proteins ,Cell Biology ,Cerebral Arteries ,Rats ,Mice, Inbred C57BL ,030104 developmental biology ,chemistry ,Vasoconstriction ,vascular smooth muscle ,NO•, nitric oxide ,biology.protein ,CLR, cholesterol ,Calcium Channels ,MCA, middle cerebral artery - Abstract
Calcium-/voltage-gated, large-conductance potassium channels (BKs) control critical physiological processes, including smooth muscle contraction. Numerous observations concur that elevated membrane cholesterol (CLR) inhibits the activity of homomeric BKs consisting of channel-forming alpha subunits. In mammalian smooth muscle, however, native BKs include accessory KCNMB1 (β1) subunits, which enable BK activation at physiological intracellular calcium. Here, we studied the effect of CLR enrichment on BK currents from rat cerebral artery myocytes. Using inside-out patches from middle cerebral artery (MCA) myocytes at [Ca2+]free=30 μM, we detected BK activation in response to in vivo and in vitro CLR enrichment of myocytes. While a significant increase in myocyte CLR was achieved within 5 min of CLR in vitro loading, this brief CLR enrichment of membrane patches decreased BK currents, indicating that BK activation by CLR requires a protracted cellular process. Indeed, blocking intracellular protein trafficking with brefeldin A (BFA) not only prevented BK activation but led to channel inhibition upon CLR enrichment. Surface protein biotinylation followed by Western blotting showed that BFA blocked the increase in plasmalemmal KCNMB1 levels achieved via CLR enrichment. Moreover, CLR enrichment of arteries with naturally high KCNMB1 levels, such as basilar and coronary arteries, failed to activate BK currents. Finally, CLR enrichment failed to activate BK channels in MCA myocytes from KCNMB1-/- mouse while activation was detected in their wild-type (C57BL/6) counterparts. In conclusion, the switch in CLR regulation of BK from inhibition to activation is determined by a trafficking-dependent increase in membrane levels of KCNMB1 subunits.
- Published
- 2020
20. Correction: Arterial smooth muscle cell PKD2 (TRPP1) channels regulate systemic blood pressure
- Author
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Luiz Moreira-Junior, Simon Bulley, Carlos Fernández-Peña, Sarah K. Burris, Korah P. Kuruvilla, Alejandro Mata-Daboin, M. Dennis Leo, Jonathan H. Jaggar, Kirk W. Evanson, Qian Wang, Padmapriya Muralidharan, Raquibul Hasan, and Charles E. Mackay
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medicine.medical_specialty ,hypertension ,Mouse ,QH301-705.5 ,PKD2 ,Science ,Cell ,Physics of Living Systems ,General Biochemistry, Genetics and Molecular Biology ,smooth muscle ,Smooth muscle ,Internal medicine ,medicine ,Biology (General) ,General Immunology and Microbiology ,business.industry ,General Neuroscience ,blood pressure ,Systemic blood pressure ,General Medicine ,medicine.anatomical_structure ,Cardiology ,Medicine ,business ,Research Article ,Neuroscience - Abstract
Systemic blood pressure is determined, in part, by arterial smooth muscle cells (myocytes). Several Transient Receptor Potential (TRP) channels are proposed to be expressed in arterial myocytes, but it is unclear if these proteins control physiological blood pressure and contribute to hypertension in vivo. We generated the first inducible, smooth muscle-specific knockout mice for a TRP channel, namely for PKD2 (TRPP1), to investigate arterial myocyte and blood pressure regulation by this protein. Using this model, we show that intravascular pressure and α1-adrenoceptors activate PKD2 channels in arterial myocytes of different systemic organs. PKD2 channel activation in arterial myocytes leads to an inward Na+ current, membrane depolarization and vasoconstriction. Inducible, smooth muscle cell-specific PKD2 knockout lowers both physiological blood pressure and hypertension and prevents pathological arterial remodeling during hypertension. Thus, arterial myocyte PKD2 controls systemic blood pressure and targeting this TRP channel reduces high blood pressure.
- Published
- 2020
21. Author response: Intravascular flow stimulates PKD2 (polycystin-2) channels in endothelial cells to reduce blood pressure
- Author
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Charles E MacKay, M Dennis Leo, Carlos Fernández-Peña, Raquibul Hasan, Wen Yin, Alejandro Mata-Daboin, Simon Bulley, Jesse Gammons, Salvatore Mancarella, and Jonathan H Jaggar
- Published
- 2020
22. Intravascular flow stimulates PKD2 (polycystin-2) channels in endothelial cells to reduce blood pressure
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Jonathan H. Jaggar, Charles E. Mackay, Simon Bulley, Carlos Fernández-Peña, Alejandro Mata-Daboin, Jesse Gammons, M. Dennis Leo, Raquibul Hasan, Salvatore Mancarella, and Wen Yin
- Subjects
Male ,0301 basic medicine ,Mouse ,Small-Conductance Calcium-Activated Potassium Channels ,Structural Biology and Molecular Biophysics ,Vasodilation ,urologic and male genital diseases ,Mechanotransduction, Cellular ,Membrane Potentials ,0302 clinical medicine ,Enos ,Phosphorylation ,Biology (General) ,Mice, Knockout ,education.field_of_study ,biology ,Chemistry ,General Neuroscience ,blood pressure ,General Medicine ,Hyperpolarization (biology) ,Intermediate-Conductance Calcium-Activated Potassium Channels ,female genital diseases and pregnancy complications ,Mesenteric Arteries ,Cell biology ,Endothelial stem cell ,Polycystin 2 ,Hypertension ,Knockout mouse ,endothelial cell ,Medicine ,Flow-Mediated Vasodilation ,Research Article ,TRPP Cation Channels ,Nitric Oxide Synthase Type III ,QH301-705.5 ,Science ,Nitric Oxide ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Animals ,Arterial Pressure ,Calcium Signaling ,education ,General Immunology and Microbiology ,urogenital system ,flow mediated-vasodilation ,Endothelial Cells ,Correction ,biology.organism_classification ,030104 developmental biology ,Regional Blood Flow ,polycystin-2 ,030217 neurology & neurosurgery - Abstract
PKD2 (polycystin-2, TRPP1), a TRP polycystin channel, is expressed in endothelial cells (ECs), but its physiological functions in this cell type are unclear. Here, we generated inducible, EC-specific Pkd2 knockout mice to examine vascular functions of PKD2. Data show that a broad range of intravascular flow rates stimulate EC PKD2 channels, producing vasodilation. Flow-mediated PKD2 channel activation leads to calcium influx that activates SK/IK channels and eNOS serine 1176 phosphorylation in ECs. These signaling mechanisms produce arterial hyperpolarization and vasodilation. In contrast, EC PKD2 channels do not contribute to acetylcholine-induced vasodilation, suggesting stimulus-specific function. EC-specific PKD2 knockout elevated blood pressure in mice without altering cardiac function or kidney anatomy. These data demonstrate that flow stimulates PKD2 channels in ECs, leading to SK/IK channel and eNOS activation, hyperpolarization, vasodilation and a reduction in systemic blood pressure. Thus, PKD2 channels are a major component of functional flow sensing in the vasculature.
- Published
- 2020
23. Impaired Trafficking of β1 Subunits Inhibits BK Channels in Cerebral Arteries of Hypertensive Rats
- Author
-
Wen Yin, Jonathan H. Jaggar, M. Dennis Leo, and Xue Zhai
- Subjects
Male ,Nitroprusside ,0301 basic medicine ,medicine.medical_specialty ,BK channel ,Bisindolylmaleimide ,Indoles ,Myocytes, Smooth Muscle ,Cerebral arteries ,Vasodilation ,Rats, Inbred WKY ,Muscle, Smooth, Vascular ,Article ,Nitric oxide ,Maleimides ,Contractility ,03 medical and health sciences ,chemistry.chemical_compound ,Rats, Inbred SHR ,Internal medicine ,Internal Medicine ,medicine ,Animals ,Myocyte ,Nitric Oxide Donors ,Large-Conductance Calcium-Activated Potassium Channels ,cardiovascular diseases ,Protein Kinase C ,Protein kinase C ,biology ,Cerebral Arteries ,Protein Subunits ,Protein Transport ,030104 developmental biology ,Endocrinology ,chemistry ,Hypertension ,biology.protein - Abstract
Hypertension is a risk factor for cerebrovascular diseases, including stroke and dementia. During hypertension, arteries become constricted and are less responsive to vasodilators, including nitric oxide (NO). The regulation of arterial contractility by smooth muscle cell (myocyte) large-conductance calcium (Ca 2+ )-activated potassium (BK) channels is altered during hypertension, although mechanisms involved are unclear. We tested the hypothesis that dysfunctional trafficking of pore-forming BK channel (BKα) and auxiliary β1 subunits contributes to changes in cerebral artery contractility of stroke-prone spontaneously hypertensive rats (SP-SHRs). Our data indicate that the amounts of total and surface BKα and β1 proteins are similar in unstimulated arteries of age-matched SP-SHRs and normotensive Wistar-Kyoto rats. In contrast, stimulated surface-trafficking of β1 subunits by NO or membrane depolarization is inhibited in SP-SHR myocytes. PKCα (protein kinase C α) and PKCβII total protein and activity were both higher in SP-SHR than in Wistar-Kyoto rat arteries. NO or depolarization robustly activated Rab11, a small trafficking GTPase, in Wistar-Kyoto rat arteries but weakly activated Rab11 in SP-SHRs. Bisindolylmaleimide, a PKC inhibitor, and overexpression of a PKC phosphorylation-deficient Rab11A mutant (Rab11A S177A) restored stimulated β1 subunit surface-trafficking in SP-SHR myocytes. BK channel activation by NO was inhibited in SP-SHR myocytes and restored by Rab11A S177A expression. Vasodilation to NO and lithocholate, a BKα/β1 channel activator, was inhibited in pressurized SP-SHR arteries and reestablished by bisindolylmaleimide. In summary, data indicate that spontaneously active PKC inhibits Rab11A-mediated β1 subunit trafficking in arterial myocytes of SP-SHRs, leading to dysfunctional NO-induced BK channel activation and vasodilation.
- Published
- 2018
24. Endothelin-1 Stimulates Vasoconstriction Through Rab11A Serine 177 Phosphorylation
- Author
-
Xue Zhai, Jonathan H. Jaggar, and M. Dennis Leo
- Subjects
0301 basic medicine ,medicine.hormone ,BK channel ,biology ,Physiology ,PKC Phosphorylation Site ,Vasodilation ,Potassium channel ,Cell biology ,Endothelins ,03 medical and health sciences ,030104 developmental biology ,Biochemistry ,medicine ,biology.protein ,Phosphorylation ,medicine.symptom ,Cardiology and Cardiovascular Medicine ,Vasoconstriction ,Protein kinase C - Abstract
Rationale: Large-conductance calcium-activated potassium channels (BK) are composed of pore-forming BKα and auxiliary β1 subunits in arterial smooth muscle cells (myocytes). Vasoconstrictors, including endothelin-1 (ET-1), inhibit myocyte BK channels, leading to contraction, but mechanisms involved are unclear. Recent evidence indicates that BKα is primarily plasma membrane localized, whereas the cellular location of β1 can be rapidly altered by Rab11A-positive recycling endosomes. Whether vasoconstrictors regulate the multisubunit composition of surface BK channels to stimulate contraction is unclear. Objective: Test the hypothesis that ET-1 inhibits BK channels by altering BKα and β1 surface trafficking in myocytes, identify mechanisms involved, and determine functional significance in myocytes of small cerebral arteries. Methods and Results: ET-1, through activation of PKC (protein kinase C), reduced surface β1 abundance and the proximity of β1 to surface BKα in myocytes. In contrast, ET-1 did not alter surface BKα, total β1, or total BKα proteins. ET-1 stimulated Rab11A phosphorylation, which reduced Rab11A activity. Rab11A serine 177 was identified as a high-probability PKC phosphorylation site. Expression of a phosphorylation-incapable Rab11A construct (Rab11A S177A) blocked the ET-1–induced Rab11A phosphorylation, reduction in Rab11A activity, and decrease in surface β1 protein. ET-1 inhibited single BK channels and transient BK currents in myocytes and stimulated vasoconstriction via a PKC-dependent mechanism that required Rab11A S177. In contrast, NO-induced Rab11A activation, surface trafficking of β1 subunits, BK channel and transient BK current activation, and vasodilation did not involve Rab11A S177. Conclusions: ET-1 stimulates PKC-mediated phosphorylation of Rab11A at serine 177, which inhibits Rab11A and Rab11A-dependent surface trafficking of β1 subunits. The decrease in surface β1 subunits leads to a reduction in BK channel calcium-sensitivity, inhibition of transient BK currents, and vasoconstriction. We describe a unique mechanism by which a vasoconstrictor inhibits BK channels and identify Rab11A serine 177 as a modulator of arterial contractility.
- Published
- 2017
25. Cholesterol-induced Trafficking of beta1 Subunits Switches Modulation of BK Function by this Steroid from Inhibition to Activation
- Author
-
M. Dennis Leo, Anna N. Bukiya, Jonathan H. Jaggar, and Alex M. Dopico
- Subjects
chemistry.chemical_compound ,Chemistry ,Modulation ,Cholesterol ,medicine.medical_treatment ,Biophysics ,medicine ,Function (biology) ,Steroid ,Cell biology - Published
- 2020
26. Author response: Arterial smooth muscle cell PKD2 (TRPP1) channels regulate systemic blood pressure
- Author
-
Charles E. Mackay, M. Dennis Leo, Luiz Moreira-Junior, Qian Wang, Sarah K. Burris, Raquibul Hasan, Alejandro Mata-Daboin, Jonathan H. Jaggar, Padmapriya Muralidharan, Kirk W. Evanson, Korah P. Kuruvilla, Simon Bulley, and Carlos Fernández-Peña
- Subjects
medicine.medical_specialty ,medicine.anatomical_structure ,Smooth muscle ,business.industry ,Internal medicine ,Cell ,Cardiology ,medicine ,Systemic blood pressure ,business - Published
- 2018
27. Local coupling of TRPC6 to ANO1/TMEM16A channels in smooth muscle cells amplifies vasoconstriction in cerebral arteries
- Author
-
Qian Wang, Damodaran Narayanan, Korah P. Kuruvilla, Jonathan H. Jaggar, and M. Dennis Leo
- Subjects
Male ,0301 basic medicine ,medicine.medical_specialty ,Physiology ,Myocytes, Smooth Muscle ,G protein-gated ion channel ,Biology ,Transfection ,Muscle, Smooth, Vascular ,Membrane Potentials ,Rats, Sprague-Dawley ,Tissue Culture Techniques ,03 medical and health sciences ,Transient receptor potential channel ,TRPC3 ,Chloride Channels ,Internal medicine ,medicine ,Animals ,Vasoconstrictor Agents ,Calcium Signaling ,Anoctamin-1 ,Calcium Chelating Agents ,TRPC Cation Channels ,Cardiac action potential ,Cell Biology ,Cerebral Arteries ,Inositol trisphosphate receptor ,Stretch-activated ion channel ,030104 developmental biology ,Endocrinology ,Vasoconstriction ,Call for Papers ,Chloride channel ,Biophysics ,RNA Interference - Abstract
Anoctamin-1 [ANO1, also known as transmembrane protein 16A (TMEM16A)] is a Ca2+-activated Cl− channel expressed in arterial myocytes that regulates membrane potential and contractility. Signaling mechanisms that control ANO1 activity in arterial myocytes are poorly understood. In cerebral artery myocytes, ANO1 channels are activated by local Ca2+ signals generated by plasma membrane nonselective cation channels, but the molecular identity of these proteins is unclear. Arterial myocytes express several different nonselective cation channels, including multiple members of the transient receptor potential receptor (TRP) family. The goal of this study was to identify localized ion channels that control ANO1 currents in cerebral artery myocytes. Coimmunoprecipitation and immunofluorescence resonance energy transfer microscopy experiments indicate that ANO1 and canonical TRP 6 (TRPC6) channels are present in the same macromolecular complex and localize in close spatial proximity in the myocyte plasma membrane. In contrast, ANO1 is not near TRPC3, TRP melastatin 4, or inositol trisphosphate receptor 1 channels. Hyp9, a selective TRPC6 channel activator, stimulated Cl− currents in myocytes that were blocked by T16Ainh-A01, an ANO1 inhibitor, ANO1 knockdown using siRNA, and equimolar replacement of intracellular EGTA with BAPTA, a fast Ca2+ chelator that abolishes local Ca2+ signaling. Hyp9 constricted pressurized cerebral arteries, and this response was attenuated by T16Ainh-A01. In contrast, T16Ainh-A01 did not alter depolarization-induced (60 mM K+) vasoconstriction. These data indicate that TRPC6 channels generate a local intracellular Ca2+ signal that activates nearby ANO1 channels in myocytes to stimulate vasoconstriction.
- Published
- 2016
28. Arterial smooth muscle cell PKD2 (TRPP1) channels regulate systemic blood pressure
- Author
-
Charles E. Mackay, Qian Wang, M. Dennis Leo, Alejandro Mata-Daboin, Kirk W. Evanson, Korah P. Kuruvilla, Simon Bulley, Jonathan H. Jaggar, Carlos Fernández-Peña, Raquibul Hasan, Luiz Moreira-Junior, Padmapriya Muralidharan, and Sarah K. Burris
- Subjects
0301 basic medicine ,PKD2 ,Cell ,Blood Pressure ,Physics of Living Systems ,Membrane Potentials ,smooth muscle ,Transient receptor potential channel ,Mice ,Myocyte ,Biology (General) ,Mice, Knockout ,Chemistry ,General Neuroscience ,Depolarization ,General Medicine ,Arteries ,Cations, Monovalent ,3. Good health ,Hindlimb ,medicine.anatomical_structure ,Knockout mouse ,Hypertension ,Medicine ,medicine.symptom ,Signal Transduction ,medicine.medical_specialty ,TRPP Cation Channels ,QH301-705.5 ,Science ,Myocytes, Smooth Muscle ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,In vivo ,Internal medicine ,Receptors, Adrenergic, alpha-1 ,medicine ,Animals ,Ion Transport ,General Immunology and Microbiology ,Sodium ,Correction ,030104 developmental biology ,Blood pressure ,Endocrinology ,Gene Expression Regulation ,Vasoconstriction ,Neuroscience - Abstract
Systemic blood pressure is determined, in part, by arterial smooth muscle cells (myocytes). Several Transient Receptor Potential (TRP) channels are proposed to be expressed in arterial myocytes, but it is unclear if these proteins control physiological blood pressure and contribute to hypertension in vivo. We generated the first inducible, smooth muscle-specific knockout mice for a TRP channel, namely for PKD2 (TRPP1), to investigate arterial myocyte and blood pressure regulation by this protein. Using this model, we show that intravascular pressure and α1-adrenoceptors activate PKD2 channels in arterial myocytes of different systemic organs. PKD2 channel activation in arterial myocytes leads to an inward Na+ current, membrane depolarization and vasoconstriction. Inducible, smooth muscle cell-specific PKD2 knockout lowers both physiological blood pressure and hypertension and prevents pathological arterial remodeling during hypertension. Thus, arterial myocyte PKD2 controls systemic blood pressure and targeting this TRP channel reduces high blood pressure.
- Published
- 2018
29. Ion Channel Trafficking in Arterial Smooth Muscle Cells and Functional Significance
- Author
-
Jonathan H. Jaggar and M. Dennis Leo
- Subjects
Chemistry ,Biophysics ,Functional significance ,Ion channel ,Arterial smooth muscle cells - Published
- 2018
30. Arterial smooth muscle cell PKD2 (TRPP1) channels control systemic blood pressure
- Author
-
Jonathan H. Jaggar, Sarah K. Burris, Qian Wang, Kirk W. Evanson, Charles E. Mackay, Padmapriya Muralidharan, Raquibul Hasan, Simon Bulley, M. Dennis Leo, Carlos Fernández-Peña, and Korah P. Kuruvilla
- Subjects
0303 health sciences ,medicine.medical_specialty ,Chemistry ,Cell ,Depolarization ,030204 cardiovascular system & hematology ,03 medical and health sciences ,Transient receptor potential channel ,0302 clinical medicine ,medicine.anatomical_structure ,Blood pressure ,Smooth muscle ,In vivo ,Internal medicine ,medicine ,Cardiology ,Myocyte ,medicine.symptom ,Vasoconstriction ,030304 developmental biology - Abstract
Systemic blood pressure is determined, in part, by arterial smooth muscle cells (myocytes). Several Transient Receptor Potential (TRP) channels are proposed to be expressed in arterial myocytes, but it is unclear if these proteins control physiological blood pressure and contribute to hypertensionin vivo. We generated the first inducible, smooth muscle-specific knockout for a TRP channel, namely for PKD2 (TRPP1), to investigate arterial myocyte and blood pressure regulation by this protein. Using this model, we show that intravascular pressure and α1-receptors activate PKD2 channels in arterial myocytes of different systemic organs. PKD2 channel activation in arterial myocytes leads to an inward Na+ current, membrane depolarization and vasoconstriction. Inducible, smooth muscle cell-specific PKD2 knockout lowers both physiological blood pressure and hypertension and prevents pathological arterial remodeling during hypertension. In summary, we show for the first time that arterial myocyte PKD2 channels control systemic blood pressure and targeting reduces high blood pressure.
- Published
- 2018
31. Arterial smooth muscle cell PKD2 (TRPP1) channels control systemic blood pressure
- Author
-
Charles R. Mackay, Simon Bulley, Qian Wang, Carlos Fernández-Peña, Kirk W. Evanson, Raquibul Hasan, Jonathan H. Jaggar, M. Dennis Leo, Padmapriya Muralidharan, Sarah K. Burris, and Korah P. Kuruvilla
- Subjects
medicine.medical_specialty ,business.industry ,Cell ,Systemic blood pressure ,Biochemistry ,medicine.anatomical_structure ,Smooth muscle ,Internal medicine ,Genetics ,medicine ,Cardiology ,business ,Molecular Biology ,Biotechnology - Abstract
Systemic blood pressure is determined, in part, by arterial smooth muscle cells (myocytes). Several Transient Receptor Potential (TRP) channels are proposed to be expressed in arterial myocytes, but it is unclear if these proteins control physiological blood pressure and contribute to hypertension in vivo . We generated the first inducible, smooth muscle-specific knockout for a TRP channel, namely for PKD2 (TRPP1), to investigate arterial myocyte and blood pressure regulation by this protein. Using this model, we show that intravascular pressure and α 1 -receptors activate PKD2 channels in arterial myocytes of different systemic organs. PKD2 channel activation in arterial myocytes leads to an inward Na+ current, membrane depolarization and vasoconstriction. Inducible, smooth muscle cell-specific PKD2 knockout lowers both physiological blood pressure and hypertension and prevents pathological arterial remodeling during hypertension. In summary, we show for the first time that arterial myocyte PKD2 channels control systemic blood pressure and targeting reduces high blood pressure.
- Published
- 2018
32. Membrane depolarization activates BK channels through ROCK-mediated β1 subunit surface trafficking to limit vasoconstriction
- Author
-
M. Dennis Leo, Padmapriya Muralidharan, Xue Zhai, Frederick A. Boop, Korah P. Kuruvilla, Simon Bulley, and Jonathan H. Jaggar
- Subjects
0301 basic medicine ,Male ,BK channel ,Large-Conductance Calcium-Activated Potassium Channel beta Subunits ,Vasodilation ,030204 cardiovascular system & hematology ,Biochemistry ,Article ,Muscle, Smooth, Vascular ,Membrane Potentials ,Rats, Sprague-Dawley ,03 medical and health sciences ,0302 clinical medicine ,Cell Movement ,medicine ,Animals ,Molecular Biology ,Membrane potential ,rho-Associated Kinases ,biology ,Voltage-gated ion channel ,Chemistry ,Cell Membrane ,Cardiac action potential ,Depolarization ,Cell Biology ,Hyperpolarization (biology) ,Rats ,Protein Subunits ,Protein Transport ,030104 developmental biology ,Vasoconstriction ,rab GTP-Binding Proteins ,Biophysics ,biology.protein ,Calcium ,medicine.symptom ,Signal Transduction - Abstract
Membrane depolarization of smooth muscle cells (myocytes) in the small arteries that regulate regional organ blood flow leads to vasoconstriction. Membrane depolarization also activates large-conductance calcium (Ca2+)-activated potassium (BK) channels, which limits Ca2+ channel activity that promotes vasoconstriction, thus leading to vasodilation. We showed that in human and rat arterial myocytes, membrane depolarization rapidly increased the cell surface abundance of auxiliary BK β1 subunits but not that of the pore-forming BKα channels. Membrane depolarization stimulated voltage-dependent Ca2+ channels, leading to Ca2+ influx and the activation of Rho kinase (ROCK) 1 and 2. ROCK1/2-mediated activation of Rab11A promoted the delivery of β1 subunits to the plasma membrane by Rab11A-positive recycling endosomes. These additional β1 subunits associated with BKα channels already at the plasma membrane, leading to an increase in apparent Ca2+ sensitivity and activation of the channels in pressurized arterial myocytes and vasodilation. Thus, membrane depolarization activates BK channels through stimulation of ROCK- and Rab11A-dependent trafficking of β1 subunits to the surface of arterial myocytes.
- Published
- 2017
33. Trafficking of BK channel subunits controls arterial contractility
- Author
-
Jonathan H. Jaggar and M. Dennis Leo
- Subjects
vasoconstrictors ,0301 basic medicine ,BK channel ,biology ,030204 cardiovascular system & hematology ,Pharmacology ,Nitric oxide ,Contractility ,03 medical and health sciences ,chemistry.chemical_compound ,Editorial ,030104 developmental biology ,0302 clinical medicine ,Oncology ,chemistry ,nitric oxide ,biology.protein ,intravascular pressure - Published
- 2017
34. Smooth muscle cell transient receptor potential polycystin-2 (TRPP2) channels contribute to the myogenic response in cerebral arteries
- Author
-
Kyle S. Gabrick, Jonathan H. Jaggar, Damodaran Narayanan, M. Dennis Leo, Sarah K. Burris, Frederick A. Boop, and Simon Bulley
- Subjects
medicine.medical_specialty ,education.field_of_study ,biology ,Voltage-dependent calcium channel ,Physiology ,Myogenic contraction ,Cerebral arteries ,ANO1 ,Endocrinology ,Polycystin 2 ,TRPC3 ,medicine.anatomical_structure ,Internal medicine ,medicine ,biology.protein ,medicine.symptom ,education ,Mesenteric arteries ,Vasoconstriction - Abstract
Intravascular pressure-induced vasoconstriction is a smooth muscle cell-specific mechanism that controls systemic blood pressure and organ regional blood flow. Smooth muscle cell polycystin-1 and -2 (TRPP1 and -2) proteins modulate the myogenic response in mesenteric arteries, but involvement in other vascular beds is unclear. Here, we examined TRPP2 expression, cellular distribution, cation currents (ICat), and physiological functions in smooth muscle cells of rat and human cerebral arteries. We demonstrate that TRPP2 is the major TRPP isoform expressed in cerebral artery smooth muscle cells, with message levels higher than those of TRPP1. Arterial biotinylation and immunofluorescence indicated that TRPP2 is located primarily (∼88%) in the smooth muscle cell plasma membrane. RNA interference reduced TRPP2 expression by ∼55% compared to control, but did not alter levels of TRPP1, TRPC1, TRPC3, TRPC6, TRPM4, ANO1/TMEM16A, or voltage-dependent Ca(2+) (CaV1.2) channels, other ion channel proteins that modulate myogenic tone. Cell swelling induced by hyposmotic (250 osmol (l solution)(-1)) bath solution stimulated Gd(3+)-sensitive ICat in smooth muscle cells that were reduced by selective TRPP2 knockdown. TRPP2 knockdown did not alter myogenic tone at 20 mmHg but reduced tone between ∼28 and 39% over an intravascular pressure range between 40 and 100 mmHg. In contrast, TRPP2 knockdown did not alter depolarization-induced (60 mmol l K(+)) vasoconstriction. In summary, we show that TRPP2 is expressed in smooth muscle cells of resistance-size cerebral arteries, resides primarily in the plasma membrane, and contributes to the myogenic response. Data also suggest that TRPP2 differentially regulates the myogenic response in cerebral and mesenteric arteries.
- Published
- 2013
35. An Elevation in Physical Coupling of Type 1 Inositol 1,4,5-Trisphosphate (IP 3 ) Receptors to Transient Receptor Potential 3 (TRPC3) Channels Constricts Mesenteric Arteries in Genetic Hypertension
- Author
-
Jonathan H. Jaggar, Candice M. Thomas-Gatewood, Zachary P. Neeb, Michael W. Kidd, Adebowale Adebiyi, and M. Dennis Leo
- Subjects
medicine.medical_specialty ,Chemistry ,Cerebral arteries ,Vasodilation ,TRPC6 ,TRPC1 ,Endocrinology ,TRPC3 ,medicine.anatomical_structure ,Internal medicine ,Internal Medicine ,medicine ,medicine.symptom ,Receptor ,Mesenteric arteries ,Vasoconstriction - Abstract
Hypertension is associated with an elevation in agonist-induced vasoconstriction, but mechanisms involved require further investigation. Many vasoconstrictors bind to phospholipase C–coupled receptors, leading to an elevation in inositol 1,4,5-trisphosphate (IP 3 ) that activates sarcoplasmic reticulum IP 3 receptors. In cerebral artery myocytes, IP 3 receptors release sarcoplasmic reticulum Ca 2+ and can physically couple to canonical transient receptor potential 3 (TRPC3) channels in a caveolin-1-containing macromolecular complex, leading to cation current activation that stimulates vasoconstriction. Here, we investigated mechanisms by which IP 3 receptors control vascular contractility in systemic arteries and IP 3 R involvement in elevated agonist-induced vasoconstriction during hypertension. Total and plasma membrane-localized TRPC3 protein was ≈2.7- and 2-fold higher in mesenteric arteries of spontaneously hypertensive rats (SHRs) than in Wistar-Kyoto (WKY) rat controls, respectively. In contrast, IP 3 R1, TRPC1, TRPC6, and caveolin-1 expression was similar. TRPC3 expression was also similar in arteries of pre-SHRs and WKY rats. Control, IP 3 -induced and endothelin-1 (ET-1)-induced fluorescence resonance energy transfer between IP3R1 and TRPC3 was higher in SHR than WKY myocytes. IP3-induced cation current was ≈3-fold larger in SHR myocytes. Pyr3, a selective TRPC3 channel blocker, and calmodulin and IP 3 receptor binding domain peptide, an IP 3 R-TRP physical coupling inhibitor, reduced IP 3 -induced cation current and ET-1–induced vasoconstriction more in SHR than WKY myocytes and arteries. Thapsigargin, a sarcoplasmic reticulum Ca 2+ -ATPase blocker, did not alter ET-1–stimulated vasoconstriction in SHR or WKY arteries. These data indicate that ET-1 stimulates physical coupling of IP 3 R1 to TRPC3 channels in mesenteric artery myocytes, leading to vasoconstriction. Furthermore, an elevation in IP 3 R1 to TRPC3 channel molecular coupling augments ET-1–induced vasoconstriction during hypertension.
- Published
- 2012
36. Ion Channel Trafficking and Control of Arterial Contractility
- Author
-
Jonathan H. Jaggar and M. Dennis Leo
- Subjects
Membrane potential ,Contractility ,Transient receptor potential channel ,Vascular smooth muscle ,chemistry ,Extracellular ,Biophysics ,chemistry.chemical_element ,Myocyte ,Calcium ,Ion channel - Abstract
Ion channels control many cellular processes, including neuronal excitability and arterial contractility. Vascular smooth muscle cell (myocyte) plasma membrane ion channels regulate membrane potential and extracellular calcium (Ca2+) influx, which alters regional blood flow and systemic blood pressure. Ion channels can be homomers or heteromers of several pore-forming subunits and may associate with auxiliary subunits that can modify channel properties, including activity and surface expression. Recent studies have investigated pathways that control the surface expression of some plasma membrane ion channels in myocytes. These findings have also found that vasoregulatory stimuli can control the number of plasma membrane ion channels to regulate contractility. Here, we review current literature describing mechanisms of trafficking of voltage-gated K+ (Kv), voltage-gated Ca2+ (Cav1.2), large-conductance Ca2+-activated potassium (BKCa) and transient receptor potential (TRP) channels in arterial myocytes. These studies indicate that regulated ion channel trafficking is a functional mechanism to control vascular contractility.
- Published
- 2016
37. Rab25 influences functional Cav1.2 channel surface expression in arterial smooth muscle cells
- Author
-
Michael W. Kidd, John P. Bannister, Simon Bulley, M. Dennis Leo, and Jonathan H. Jaggar
- Subjects
0301 basic medicine ,Male ,Proteasome Endopeptidase Complex ,Calcium Channels, L-Type ,Physiology ,Cerebral arteries ,Myocytes, Smooth Muscle ,Transfection ,Cav1.2 ,Muscle, Smooth, Vascular ,Membrane Potentials ,Rats, Sprague-Dawley ,03 medical and health sciences ,medicine ,Myocyte ,Animals ,Cells, Cultured ,Gene knockdown ,biology ,Voltage-dependent calcium channel ,Depolarization ,Cell Biology ,Cerebral Arteries ,Molecular biology ,Cell biology ,Protein Transport ,030104 developmental biology ,Vasoconstriction ,rab GTP-Binding Proteins ,Proteolysis ,biology.protein ,Call for Papers ,RNA Interference ,Rab ,medicine.symptom ,Lysosomes ,Signal Transduction - Abstract
Plasma membrane-localized CaV1.2 channels are the primary calcium (Ca2+) influx pathway in arterial smooth muscle cells (myocytes). CaV1.2 channels regulate several cellular functions, including contractility and gene expression, but the trafficking pathways that control the surface expression of these proteins are unclear. Similarly, expression and physiological functions of small Rab GTPases, proteins that control vesicular trafficking in arterial myocytes, are poorly understood. Here, we investigated Rab proteins that control functional surface abundance of CaV1.2 channels in cerebral artery myocytes. Western blotting indicated that Rab25, a GTPase previously associated with apical recycling endosomes, is expressed in cerebral artery myocytes. Immunofluorescence Förster resonance energy transfer (immunoFRET) microscopy demonstrated that Rab25 locates in close spatial proximity to CaV1.2 channels in myocytes. Rab25 knockdown using siRNA reduced CaV1.2 surface and intracellular abundance in arteries, as determined using arterial biotinylation. In contrast, CaV1.2 was not located nearby Rab11A or Rab4 and CaV1.2 protein was unaltered by Rab11A or Rab4A knockdown. Rab25 knockdown resulted in CaV1.2 degradation by a mechanism involving both lysosomal and proteasomal pathways and reduced whole cell CaV1.2 current density but did not alter voltage dependence of current activation or inactivation in isolated myocytes. Rab25 knockdown also inhibited depolarization (20–60 mM K+) and pressure-induced vasoconstriction (myogenic tone) in cerebral arteries. These data indicate that Rab25 is expressed in arterial myocytes where it promotes surface expression of CaV1.2 channels to control pressure- and depolarization-induced vasoconstriction.
- Published
- 2015
38. Vasoconstriction resulting from dynamic membrane trafficking of TRPM4 in vascular smooth muscle cells
- Author
-
M. Dennis Leo, Jonathan H. Jaggar, Michael M. Tamkun, Scott Earley, Albert L. Gonzales, Rachael Crnich, and Gregory C. Amberg
- Subjects
Male ,Vascular smooth muscle ,Physiology ,Myocytes, Smooth Muscle ,Cell ,Cerebral arteries ,Enzyme Activators ,TRPM Cation Channels ,Biology ,Muscle, Smooth, Vascular ,Membrane Potentials ,Rats, Sprague-Dawley ,Mice ,Transient receptor potential channel ,Vascular Biology ,medicine ,Animals ,Cells, Cultured ,Protein kinase C ,Cell Membrane ,Depolarization ,Cell Biology ,Cerebral Arteries ,Membrane transport ,Rats ,Cell biology ,Protein Kinase C-delta ,Protein Transport ,medicine.anatomical_structure ,Biochemistry ,Tetradecanoylphorbol Acetate ,medicine.symptom ,Vasoconstriction ,Muscle Contraction - Abstract
The melastatin (M) transient receptor potential (TRP) channel TRPM4 mediates pressure and protein kinase C (PKC)-induced smooth muscle cell depolarization and vasoconstriction of cerebral arteries. We hypothesized that PKC causes vasoconstriction by stimulating translocation of TRPM4 to the plasma membrane. Live-cell confocal imaging and fluorescence recovery after photobleaching (FRAP) analysis was performed using a green fluorescent protein (GFP)-tagged TRPM4 (TRPM4-GFP) construct expressed in A7r5 cells. The surface channel was mobile, demonstrating a FRAP time constant of 168 +/- 19 s. In addition, mobile intracellular trafficking vesicles were readily detected. Using a cell surface biotinylation assay, we showed that PKC activation with phorbol 12-myristate 13-acetate (PMA) increased (approximately 3-fold) cell surface levels of TRPM4-GFP protein in10 min. Similarly, total internal reflection fluorescence microscopy demonstrated that stimulation of PKC activity increased (approximately 3-fold) the surface fluorescence of TRPM4-GFP in A7r5 cells and primary cerebral artery smooth muscle cells. PMA also caused an elevation of cell surface TRPM4 protein levels in intact arteries. PMA-induced translocation of TRPM4 to the plasma membrane was independent of PKCalpha and PKCbeta activity but was inhibited by blockade of PKCdelta with rottlerin. Pressure-myograph studies of intact, small interfering RNA (siRNA)-treated cerebral arteries demonstrate that PKC-induced constriction of cerebral arteries requires expression of both TRPM4 and PKCdelta. In addition, pressure-induced arterial myocyte depolarization and vasoconstriction was attenuated in arteries treated with siRNA against PKCdelta. We conclude that PKCdelta activity causes smooth muscle depolarization and vasoconstriction by increasing the number of TRPM4 channels in the sarcolemma.
- Published
- 2010
39. Type 1 IP3 receptors activate BKCa channels via local molecular coupling in arterial smooth muscle cells
- Author
-
Guiling Zhao, Zachary P. Neeb, Kunfu Ouyang, Ju Chen, Judith Pachuau, M. Dennis Leo, Adebowale Adebiyi, and Jonathan H. Jaggar
- Subjects
Male ,BK channel ,Adenosine ,Patch-Clamp Techniques ,Potassium Channels ,Time Factors ,Journal Club ,Physiology ,Fluorescent Antibody Technique ,Receptors, Cytoplasmic and Nuclear ,Muscle, Smooth, Vascular ,Membrane Potentials ,Rats, Sprague-Dawley ,Mice ,chemistry.chemical_compound ,0302 clinical medicine ,Fluorescence Resonance Energy Transfer ,Inositol 1,4,5-Trisphosphate Receptors ,Myocyte ,Large-Conductance Calcium-Activated Potassium Channel alpha Subunits ,Egtazic Acid ,Aorta ,Chelating Agents ,Mice, Knockout ,Membrane potential ,0303 health sciences ,biology ,Voltage-dependent calcium channel ,Antibodies, Monoclonal ,Potassium channel ,Sarcoplasmic Reticulum ,Biochemistry ,Ion Channel Gating ,Protein Binding ,Myocytes, Smooth Muscle ,03 medical and health sciences ,BAPTA ,Animals ,Immunoprecipitation ,Calcium Signaling ,Patch clamp ,030304 developmental biology ,Heparin ,Endoplasmic reticulum ,Cell Membrane ,Cerebral Arteries ,Rats ,chemistry ,biology.protein ,Biophysics ,Calcium Channels ,030217 neurology & neurosurgery - Abstract
Plasma membrane large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels and sarcoplasmic reticulum inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) are expressed in a wide variety of cell types, including arterial smooth muscle cells. Here, we studied BK(Ca) channel regulation by IP(3) and IP(3)Rs in rat and mouse cerebral artery smooth muscle cells. IP(3) activated BK(Ca) channels both in intact cells and in excised inside-out membrane patches. IP(3) caused concentration-dependent BK(Ca) channel activation with an apparent dissociation constant (K(d)) of approximately 4 microM at physiological voltage (-40 mV) and intracellular Ca(2+) concentration ([Ca(2+)](i); 10 microM). IP(3) also caused a leftward-shift in BK(Ca) channel apparent Ca(2+) sensitivity and reduced the K(d) for free [Ca(2+)](i) from approximately 20 to 12 microM, but did not alter the slope or maximal P(o). BAPTA, a fast Ca(2+) buffer, or an elevation in extracellular Ca(2+) concentration did not alter IP(3)-induced BK(Ca) channel activation. Heparin, an IP(3)R inhibitor, and a monoclonal type 1 IP(3)R (IP(3)R1) antibody blocked IP(3)-induced BK(Ca) channel activation. Adenophostin A, an IP(3)R agonist, also activated BK(Ca) channels. IP(3) activated BK(Ca) channels in inside-out patches from wild-type (IP(3)R1(+/+)) mouse arterial smooth muscle cells, but had no effect on BK(Ca) channels of IP(3)R1-deficient (IP(3)R1(-/-)) mice. Immunofluorescence resonance energy transfer microscopy indicated that IP(3)R1 is located in close spatial proximity to BK(Ca) alpha subunits. The IP(3)R1 monoclonal antibody coimmunoprecipitated IP(3)R1 and BK(Ca) channel alpha and beta1 subunits from cerebral arteries. In summary, data indicate that IP(3)R1 activation elevates BK(Ca) channel apparent Ca(2+) sensitivity through local molecular coupling in arterial smooth muscle cells.
- Published
- 2010
40. Intravascular pressure enhances the abundance of functional Kv1.5 channels at the surface of arterial smooth muscle cells
- Author
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M. Dennis Leo, Jonathan H. Jaggar, Michael W. Kidd, and John P. Bannister
- Subjects
Male ,Patch-Clamp Techniques ,Blotting, Western ,Myocytes, Smooth Muscle ,In Vitro Techniques ,Biochemistry ,Article ,Membrane Potentials ,Cell membrane ,Rats, Sprague-Dawley ,Kv1.5 Potassium Channel ,medicine ,Myocyte ,Animals ,Humans ,Patch clamp ,Molecular Biology ,Mesenteric arteries ,Cells, Cultured ,Membrane potential ,Chemistry ,Reverse Transcriptase Polymerase Chain Reaction ,Cell Membrane ,Depolarization ,Cell Biology ,Anatomy ,Mesenteric Arteries ,medicine.anatomical_structure ,HEK293 Cells ,Vasoconstriction ,Biophysics ,medicine.symptom ,Intracellular ,Signal Transduction - Abstract
Voltage-dependent potassium (K(v)) channels are present in various cell types, including smooth muscle cells (myocytes) of resistance-sized arteries that control systemic blood pressure and regional organ blood flow. Intravascular pressure depolarizes arterial myocytes, stimulating calcium (Ca(2+)) influx through voltage-dependent Ca(2+) (Ca(v)) channels that results in vasoconstriction and also K(+) efflux through K(v) channels that oppose vasoconstriction. We hypothesized that pressure-induced depolarization may not only increase the open probability of plasma membrane-resident K(v) channels but also increase the abundance of these channels at the surface of arterial myocytes to limit vasoconstriction. We found that K(v)1.5 and K(v)2.1 proteins were abundant in the myocytes of resistance-sized mesenteric arteries. K(v)1.5, but not K(v)2.1, continuously recycled between the intracellular compartment and the plasma membrane in contractile arterial myocytes. Using ex vivo preparations of intact arteries, we showed that physiological intravascular pressure through membrane depolarization or membrane depolarization in the absence of pressure inhibited the degradation of internalized K(v)1.5 and increased recycling of K(v)1.5 to the plasma membrane. Accordingly, by stimulating the activity of Ca(v)1.2, membrane depolarization increased whole-cell K(v)1.5 current density in myocytes and K(v)1.5 channel activity in pressurized arteries. In contrast, the total amount and cell surface abundance of K(v)2.1 were independent of intravascular pressure or membrane potential. Thus, our data indicate that intravascular pressure-induced membrane depolarization selectively increased K(v)1.5 surface abundance to increase K(v) currents in arterial myocytes, which would limit vasoconstriction.
- Published
- 2015
41. Angiotensin II stimulates internalization and degradation of arterial myocyte plasma membrane BK channels to induce vasoconstriction
- Author
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Korah P. Kuruvilla, Simon Bulley, Damodaran Narayanan, John P. Bannister, Jonathan H. Jaggar, and M. Dennis Leo
- Subjects
Male ,medicine.medical_specialty ,BK channel ,Physiology ,media_common.quotation_subject ,Myocytes, Smooth Muscle ,Endosomes ,Muscle, Smooth, Vascular ,Membrane Potentials ,Contractility ,Rats, Sprague-Dawley ,Internal medicine ,medicine ,Myocyte ,Animals ,Vasoconstrictor Agents ,Large-Conductance Calcium-Activated Potassium Channels ,Internalization ,Arterial smooth muscle cells ,media_common ,Muscle Cells ,biology ,Chemistry ,Angiotensin II ,Cell Membrane ,Cell Biology ,Cerebral Arteries ,Cell biology ,Rats ,Endocrinology ,Membrane ,Vasoconstriction ,Proteolysis ,biology.protein ,Call for Papers ,medicine.symptom - Abstract
Arterial smooth muscle cells (myocytes) express large-conductance Ca2+-activated K+ (BK) channel α and auxiliary β1 subunits that modulate arterial contractility. In arterial myocytes, β1 subunits are stored within highly mobile rab11A-positive recycling endosomes. In contrast, BKα subunits are primarily plasma membrane-localized. Trafficking pathways for BKα and whether physiological stimuli that regulate arterial contractility alter BKα localization in arterial myocytes are unclear. Here, using biotinylation, immunofluorescence resonance energy transfer (immunoFRET) microscopy, and RNAi-mediated knockdown, we demonstrate that rab4A-positive early endosomes traffic BKα to the plasma membrane in myocytes of resistance-size cerebral arteries. Angiotensin II (ANG II), a vasoconstrictor, reduced both surface and total BKα, an effect blocked by bisindolylmaleimide-II, concanavalin A, and dynasore, protein kinase C (PKC), internalization, and endocytosis inhibitors, respectively. In contrast, ANG II did not reduce BKα mRNA, and sodium nitroprusside, a nitric oxide donor, did not alter surface BKα protein over the same time course. MG132 and bafilomycin A, proteasomal and lysosomal inhibitors, respectively, also inhibited the ANG II-induced reduction in surface and total BKα, resulting in intracellular BKα accumulation. ANG II-mediated BK channel degradation reduced BK currents in isolated myocytes and functional responses to iberiotoxin, a BK channel blocker, and NS1619, a BK activator, in pressurized (60 mmHg) cerebral arteries. These data indicate that rab4A-positive early endosomes traffic BKα to the plasma membrane in arterial myocytes. We also show that ANG II stimulates PKC-dependent BKα internalization and degradation. These data describe a unique mechanism by which ANG II inhibits arterial myocyte BK currents, by reducing surface channel number, to induce vasoconstriction.
- Published
- 2015
42. Angiotensin II Stimulates Internalization and Degradation of BK Channels in Arterial Myocytes to Induce Vasoconstriction
- Author
-
M. Dennis Leo, John P. Bannister, Simon Bulley, and Jonathan H. Jaggar
- Subjects
BK channel ,biology ,Chemistry ,media_common.quotation_subject ,Biochemistry ,Angiotensin II ,Cell biology ,Genetics ,medicine ,biology.protein ,Myocyte ,medicine.symptom ,Internalization ,Molecular Biology ,Vasoconstriction ,Biotechnology ,media_common - Published
- 2015
43. Localized TRPA1 channel Ca 2+ signals stimulated by reactive oxygen species promote cerebral artery dilation
- Author
-
M. Dennis Leo, Allison Bruhl, Frederick A. Boop, Donald G. Welsh, Paulo W. Pires, Yumei Feng, Wencheng Li, Agathe Oulidi, Albert L. Gonzales, Jonathan H. Jaggar, Michelle N. Sullivan, and Scott Earley
- Subjects
Cerebral arteries ,Vasodilation ,Biochemistry ,Membrane Potentials ,Rats, Sprague-Dawley ,Mice ,Cerebral circulation ,chemistry.chemical_compound ,Transient receptor potential channel ,Transient Receptor Potential Channels ,0302 clinical medicine ,TRPA1 Cation Channel ,Mice, Knockout ,0303 health sciences ,Membrane Glycoproteins ,NADPH oxidase ,biology ,Voltage-dependent calcium channel ,Reverse Transcriptase Polymerase Chain Reaction ,food and beverages ,Anatomy ,Hyperpolarization (biology) ,Immunohistochemistry ,NADPH Oxidase 2 ,psychological phenomena and processes ,Nicotinamide adenine dinucleotide phosphate ,Blotting, Western ,Nerve Tissue Proteins ,Article ,03 medical and health sciences ,Animals ,Humans ,Immunoprecipitation ,Calcium Signaling ,Molecular Biology ,030304 developmental biology ,Aldehydes ,NADPH Oxidases ,Cell Biology ,Cerebral Arteries ,Rats ,HEK293 Cells ,chemistry ,Biophysics ,biology.protein ,Calcium Channels ,Lipid Peroxidation ,Reactive Oxygen Species ,030217 neurology & neurosurgery - Abstract
Reactive oxygen species (ROS) can have divergent effects in cerebral and peripheral circulations. We found that Ca(2+)-permeable transient receptor potential ankyrin 1 (TRPA1) channels were present and colocalized with NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase 2 (NOX2), a major source of ROS, in the endothelium of cerebral arteries but not in other vascular beds. We recorded and characterized ROS-triggered Ca(2+) signals representing Ca(2+) influx through single TRPA1 channels, which we called "TRPA1 sparklets." TRPA1 sparklet activity was low under basal conditions but was stimulated by NOX-generated ROS. Ca(2+) entry during a single TRPA1 sparklet was twice that of a TRPV4 sparklet and ~200 times that of an L-type Ca(2+) channel sparklet. TRPA1 sparklets representing the simultaneous opening of two TRPA1 channels were more common in endothelial cells than in human embryonic kidney (HEK) 293 cells expressing TRPA1. The NOX-induced TRPA1 sparklets activated intermediate-conductance, Ca(2+)-sensitive K(+) channels, resulting in smooth muscle hyperpolarization and vasodilation. NOX-induced activation of TRPA1 sparklets and vasodilation required generation of hydrogen peroxide and lipid-peroxidizing hydroxyl radicals as intermediates. 4-Hydroxy-nonenal, a metabolite of lipid peroxidation, also increased TRPA1 sparklet frequency and dilated cerebral arteries. These data suggest that in the cerebral circulation, lipid peroxidation metabolites generated by ROS activate Ca(2+) influx through TRPA1 channels in the endothelium of cerebral arteries to cause dilation.
- Published
- 2015
44. LRRC26 is a functional BK channel auxiliary γ subunit in arterial smooth muscle cells
- Author
-
Jonathan H. Jaggar, M. Dennis Leo, Kirk W. Evanson, and John P. Bannister
- Subjects
Male ,BK channel ,Physiology ,Protein subunit ,Vasodilator Agents ,Blotting, Western ,Myocytes, Smooth Muscle ,Vasodilation ,Biology ,Transfection ,Muscle, Smooth, Vascular ,Article ,Membrane Potentials ,Rats, Sprague-Dawley ,Tissue Culture Techniques ,medicine ,Myocyte ,Animals ,Immunoprecipitation ,Vasoconstrictor Agents ,Calcium Signaling ,Large-Conductance Calcium-Activated Potassium Channels ,RNA, Messenger ,Reverse Transcriptase Polymerase Chain Reaction ,Depolarization ,Iberiotoxin ,Cerebral Arteries ,Molecular biology ,Potassium channel ,Cell biology ,Neoplasm Proteins ,Protein Subunits ,Gene Expression Regulation ,Vasoconstriction ,biology.protein ,RNA Interference ,medicine.symptom ,Cardiology and Cardiovascular Medicine - Abstract
Rationale : Smooth muscle cell (myocyte) large-conductance calcium (Ca) 2+ -activated potassium (BK) channels are functionally significant modulators of arterial contractility. Arterial myocytes express both pore-forming BKα and auxiliary β1 subunits, which increase channel Ca 2+ sensitivity. Recently, several leucine-rich repeat containing (LRRC) proteins have been identified as auxiliary γ subunits that elevate the voltage sensitivity of recombinant and prostate adenocarcinoma BK channels. LRRC expression and physiological functions in native cell types are unclear. Objective : Investigate the expression and physiological functions of leucine-rich repeat containing protein 26 (LRRC26) in arterial myocytes. Methods and Results : Reverse transcription polymerase chain reaction and Western blotting detected LRRC26 mRNA and protein in cerebral artery myocytes. Biotinylation, immunofluorescence resonance energy transfer microscopy, and coimmunoprecipitation indicated that LRRC26 was located in close spatial proximity to, and associated with, plasma membrane BKα subunits. LRRC26 knockdown (RNAi) reduced total and surface LRRC26, but did not alter BKα or β1, proteins in arteries. LRRC26 knockdown did not alter Ca 2+ sparks but reduced BK channel voltage sensitivity, which decreased channel apparent Ca 2+ sensitivity and transient BK current frequency and amplitude in myocytes. LRRC26 knockdown also increased myogenic tone over a range (40–100 mm Hg) of intravascular pressures, and reduced vasoconstriction to iberiotoxin and vasodilation to NS1619, BK channel inhibitors and activators, respectively. In contrast, LRRC26 knockdown did not alter depolarization (60 mmol/L K + )–induced vasoconstriction. Conclusions : LRRC26 is expressed, associates with BKα subunits, and elevates channel voltage- and apparent Ca 2+ sensitivity in arterial myocytes to induce vasodilation. This study indicates that arterial myocytes express a functional BK channel γ subunit.
- Published
- 2014
45. Involvement of inducible nitric oxide synthase and dimethylarginine dimethylaminohydrolase in Nω‐Nitro‐L‐arginine methyl ester (L‐NAME)‐induced hypertension (LB676)
- Author
-
Dinesh Kumar, M. Dennis Leo, Kathirvel Kandasamy, Surendra K. Tandan, and Jaganathan Subramani
- Subjects
Aorta ,medicine.medical_specialty ,Mean arterial pressure ,biology ,Chemistry ,biology.organism_classification ,Biochemistry ,Nitric oxide ,Nitric oxide synthase ,chemistry.chemical_compound ,Basal (phylogenetics) ,Real-time polymerase chain reaction ,Endocrinology ,Enos ,medicine.artery ,Internal medicine ,Genetics ,medicine ,biology.protein ,Soluble guanylyl cyclase ,Molecular Biology ,Biotechnology - Abstract
Chronic administration of L-NAME in rats is an excellent model to study the induction and progression of a NO deficiency-induced endothelial dysfunctional state. This study was performed to determine how different vascular beds compensate for the loss in nitric oxide. Male Wistar rats received L-NAME (50mg/kg/day in drinking water) or no drug for 6 weeks. Aorta, pulmonary and renal arteries were isolated for determination of basal cGMP production, PDE5 activity, dimethylarginine dimethylaminohydrolase (DDAH) and hemeoxygenase (HO) activity. mRNA expression studies were done by real time PCR. Plasma IL-1β level was measured by ELISA. Results: L-NAME-induced hypertension was associated with an increase in mean arterial pressure and plasma IL-1β. The treatment had varied effects on mRNA expression levels of eNOS, soluble guanylyl cyclase, PDE5 and HO1 but showed an increased iNOS and decreased DDAH2 expression in all three tissues studied. Basal cGMP levels were decreased in all tissues, but PDE5 activity wa...
- Published
- 2014
46. Arterial smooth muscle cells express segment a‐deficient TMEM16A channels (1077.7)
- Author
-
Sarah K. Burris, M. Dennis Leo, Wanchana Jangsangthong, Jonathan H. Jaggar, and Damodaran Narayanan
- Subjects
Contractility ,Chemistry ,cardiovascular system ,Genetics ,Molecular Biology ,Biochemistry ,Transmembrane protein ,Biotechnology ,Arterial smooth muscle cells ,Cell biology - Abstract
Transmembrane protein A (TMEM16A) Cl- channels are expressed in arterial smooth muscle cells (SMCs) where they regulate physiological functions, including contractility and proliferation. TMEM16A a...
- Published
- 2014
47. Dynamic regulation of β1 subunit trafficking controls vascular contractility
- Author
-
Frederick A. Boop, Kyle S. Gabrick, Jonathan H. Jaggar, Damodaran Narayanan, Jordan E. Grubbs, John P. Bannister, M. Dennis Leo, and Anitha Nair
- Subjects
Male ,BK channel ,Multidisciplinary ,Ion Transport ,Patch-Clamp Techniques ,biology ,Endosome ,Protein subunit ,Biological Sciences ,Cell biology ,Rats ,Contractility ,Rats, Sprague-Dawley ,Potassium Channels, Calcium-Activated ,Biochemistry ,biology.protein ,Fluorescence Resonance Energy Transfer ,Myocyte ,Animals ,Blood Vessels ,Protein kinase A ,Intracellular ,Ion channel - Abstract
Ion channels composed of pore-forming and auxiliary subunits control physiological functions in virtually all cell types. A conventional view is that channels assemble with their auxiliary subunits before anterograde plasma membrane trafficking of the protein complex. Whether the multisubunit composition of surface channels is fixed following protein synthesis or flexible and open to acute and, potentially, rapid modulation to control activity and cellular excitability is unclear. Arterial smooth muscle cells (myocytes) express large-conductance Ca(2+)-activated potassium (BK) channel α and auxiliary β1 subunits that are functionally significant modulators of arterial contractility. Here, we show that native BKα subunits are primarily (∼95%) plasma membrane-localized in human and rat arterial myocytes. In contrast, only a small fraction (∼10%) of total β1 subunits are located at the cell surface. Immunofluorescence resonance energy transfer microscopy demonstrated that intracellular β1 subunits are stored within Rab11A-postive recycling endosomes. Nitric oxide (NO), acting via cGMP-dependent protein kinase, and cAMP-dependent pathways stimulated rapid (≤1 min) anterograde trafficking of β1 subunit-containing recycling endosomes, which increased surface β1 almost threefold. These β1 subunits associated with surface-resident BKα proteins, elevating channel Ca(2+) sensitivity and activity. Our data also show that rapid β1 subunit anterograde trafficking is the primary mechanism by which NO activates myocyte BK channels and induces vasodilation. In summary, we show that rapid β1 subunit surface trafficking controls functional BK channel activity in arterial myocytes and vascular contractility. Conceivably, regulated auxiliary subunit trafficking may control ion channel activity in a wide variety of cell types.
- Published
- 2014
48. Smooth muscle cell transient receptor potential polycystin-2 (TRPP2) channels contribute to the myogenic response in cerebral arteries
- Author
-
Damodaran, Narayanan, Simon, Bulley, M Dennis, Leo, Sarah K, Burris, Kyle S, Gabrick, Frederick A, Boop, and Jonathan H, Jaggar
- Subjects
Male ,TRPP Cation Channels ,Adolescent ,Calcium Channels, L-Type ,Cell Membrane ,Infant ,Cerebral Arteries ,Cardiovascular ,Muscle, Smooth, Vascular ,Rats ,Rats, Sprague-Dawley ,Protein Transport ,HEK293 Cells ,Vasoconstriction ,Animals ,Humans ,Protein Isoforms ,Female ,RNA, Messenger ,Child - Abstract
Intravascular pressure-induced vasoconstriction is a smooth muscle cell-specific mechanism that controls systemic blood pressure and organ regional blood flow. Smooth muscle cell polycystin-1 and -2 (TRPP1 and -2) proteins modulate the myogenic response in mesenteric arteries, but involvement in other vascular beds is unclear. Here, we examined TRPP2 expression, cellular distribution, cation currents (ICat), and physiological functions in smooth muscle cells of rat and human cerebral arteries. We demonstrate that TRPP2 is the major TRPP isoform expressed in cerebral artery smooth muscle cells, with message levels higher than those of TRPP1. Arterial biotinylation and immunofluorescence indicated that TRPP2 is located primarily (∼88%) in the smooth muscle cell plasma membrane. RNA interference reduced TRPP2 expression by ∼55% compared to control, but did not alter levels of TRPP1, TRPC1, TRPC3, TRPC6, TRPM4, ANO1/TMEM16A, or voltage-dependent Ca(2+) (CaV1.2) channels, other ion channel proteins that modulate myogenic tone. Cell swelling induced by hyposmotic (250 osmol (l solution)(-1)) bath solution stimulated Gd(3+)-sensitive ICat in smooth muscle cells that were reduced by selective TRPP2 knockdown. TRPP2 knockdown did not alter myogenic tone at 20 mmHg but reduced tone between ∼28 and 39% over an intravascular pressure range between 40 and 100 mmHg. In contrast, TRPP2 knockdown did not alter depolarization-induced (60 mmol l K(+)) vasoconstriction. In summary, we show that TRPP2 is expressed in smooth muscle cells of resistance-size cerebral arteries, resides primarily in the plasma membrane, and contributes to the myogenic response. Data also suggest that TRPP2 differentially regulates the myogenic response in cerebral and mesenteric arteries.
- Published
- 2013
49. Smooth muscle cell transient receptor potential polycystin (TRPP)2 channels contribute to the myogenic response in cerebral arteries
- Author
-
Simon Bulley, M. Dennis Leo, Kyle S. Gabrick, Frederick A. Boop, Jonathan H. Jaggar, and Damodaran Narayanan
- Subjects
Chemistry ,Myogenic contraction ,Cell ,Cerebral arteries ,TRPP ,Biochemistry ,Cell biology ,Transient receptor potential channel ,medicine.anatomical_structure ,Smooth muscle ,Genetics ,medicine ,Molecular Biology ,Biotechnology - Published
- 2013
50. Intravascular pressure stimulates functional Kv1.5 surface expression in mesenteric artery smooth muscle cells
- Author
-
Michael William Kidd, M. Dennis Leo, Damodaran Narayanan, John P. Bannister, and Jonathan H. Jaggar
- Subjects
Genetics ,Molecular Biology ,Biochemistry ,Biotechnology - Published
- 2013
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