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Mechanisms underlying arteriovenous malformations (AVMs) are poorly understood. Using mice with endothelial cell (EC) expression of constitutively active Notch4 (Notch4* EC ), we show decreased arteriolar tone in vivo during brain AVM initiation. Reduced vascular tone is a primary effect of Notch4* EC , as isolated pial arteries from asymptomatic mice exhibited reduced pressure-induced arterial tone ex vivo. The nitric oxide (NO) synthase (NOS) inhibitor NG-nitro- l -arginine (L-NNA) corrected vascular tone defects in both assays. L-NNA treatment or endothelial NOS ( eNOS ) gene deletion, either globally or specifically in ECs, attenuated AVM initiation, assessed by decreased AVM diameter and delayed time to moribund. Administering nitroxide antioxidant 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl also attenuated AVM initiation. Increased NOS-dependent production of hydrogen peroxide, but not NO, superoxide, or peroxynitrite was detected in isolated Notch4* EC brain vessels during AVM initiation. Our data suggest that eNOS is involved in Notch4* EC -mediated AVM formation by up-regulating hydrogen peroxide and reducing vascular tone, thereby permitting AVM initiation and progression.

The L-type Ca2+ channel CaV1.2 is essential for arterial myocyte excitability, gene expression and contraction. Elevations in extracellular glucose (hyperglycemia) potentiate vascular L-type Ca2+ channel via PKA, but the underlying mechanisms are unclear. Here, we find that cAMP synthesis in response to elevated glucose and the selective P2Y11 agonist NF546 is blocked by disruption of A-kinase anchoring protein 5 (AKAP5) function in arterial myocytes. Glucose and NF546-induced potentiation of L-type Ca2+ channels, vasoconstriction and decreased blood flow are prevented in AKAP5 null arterial myocytes/arteries. These responses are nucleated via the AKAP5-dependent clustering of P2Y11/ P2Y11-like receptors, AC5, PKA and CaV1.2 into nanocomplexes at the plasma membrane of human and mouse arterial myocytes. Hence, data reveal an AKAP5 signaling module that regulates L-type Ca2+ channel activity and vascular reactivity upon elevated glucose. This AKAP5-anchored nanocomplex may contribute to vascular complications during diabetic hyperglycemia., Molecular mechanisms by which glucose modulates L-type Ca2+ channel activity and vascular reactivity are unclear. Here the authors report a nanocomplex orchestrated by AKAP5 that facilitates local purinergic stimulation of L-type Ca2+ channels and vasoconstriction during diabetic hyperglycemia.

Cigarette smoke, including secondhand smoke (SHS), has significant detrimental vascular effects, but its effects on myogenic tone of small resistance arteries and the underlying mechanisms are understudied. Although it is apparent that SHS contributes to endothelial dysfunction, much less is known about how this toxicant alters arterial myocyte contraction, leading to alterations in myogenic tone. The study's goal is to determine the effects of SHS on mesenteric arterial myocyte contractility and excitability. C57BL/6J male mice were randomly assigned to either filtered air (FA) or SHS (6 h/d, 5 d/wk) exposed groups for a 4, 8, or 12-weeks period. Third and fourth-order mesenteric arteries and arterial myocytes were acutely isolated and evaluated with pressure myography and patch clamp electrophysiology, respectively. Myogenic tone was found to be elevated in mesenteric arteries from mice exposed to SHS for 12 wk but not for 4 or 8 wk. These results were correlated with an increase in L-type Ca2+ channel activity in mesenteric arterial myocytes after 12 wk of SHS exposure. Moreover, 12 wk SHS exposed arterial myocytes have reduced total potassium channel current density, which correlates with a depolarized membrane potential (Vm). These results suggest that SHS exposure induces alterations in key ionic conductances that modulate arterial myocyte contractility and myogenic tone. Thus, chronic exposure to an environmentally relevant concentration of SHS impairs mesenteric arterial myocyte electrophysiology and myogenic tone, which may contribute to increased blood pressure and risks of developing vascular complications due to passive exposure to cigarette smoke., Secondhand smoke alters vascular smooth muslce ion channel function and vascular tone. Figure created with BioRender.com

Elevated blood glucose (hyperglycemia) is a hallmark metabolic abnormality in diabetes. Hyperglycemia is associated with protein kinase A–dependent (PKA-dependent) stimulation of L-type Ca(2+) channels in arterial myocytes resulting in increased vasoconstriction. However, the mechanisms by which glucose activates PKA remain unclear. Here, we showed that elevating extracellular glucose stimulates cAMP production in arterial myocytes, and that this was specifically dependent on adenylyl cyclase 5 (AC5) activity. Super-resolution imaging suggested nanometer proximity between subpopulations of AC5 and the L-type Ca(2+) channel pore-forming subunit Ca(V)1.2. In vitro, in silico, ex vivo, and in vivo experiments revealed that this close association is critical for stimulation of L-type Ca(2+) channels in arterial myocytes and increased myogenic tone upon acute hyperglycemia. This pathway supported the increase in L-type Ca(2+) channel activity and myogenic tone in 2 animal models of diabetes. Our collective findings demonstrate a unique role for AC5 in PKA-dependent modulation of L-type Ca(2+) channel activity and vascular reactivity during acute hyperglycemia and diabetes.

Elevated glucose increases vascular reactivity by promoting L-type CaV1.2 channel (LTCC) activity by protein kinase A (PKA). Yet, how glucose activates PKA is unknown. We hypothesized that a Gs-coupled P2Y receptor is an upstream activator of PKA mediating LTCC potentiation during diabetic hyperglycemia. Experiments in apyrase-treated cells suggested involvement of a P2Y receptor underlying the glucose effects on LTTCs. Using human tissue, expression for P2Y11, the only Gs-coupled P2Y receptor, was detected in nanometer proximity to CaV1.2 and PKA. FRET-based experiments revealed that the selective P2Y11 agonist NF546 and elevated glucose stimulate cAMP production resulting in enhanced PKA-dependent LTCC activity. These changes were blocked by the selective P2Y11 inhibitor NF340. Comparable results were observed in mouse tissue, suggesting that a P2Y11-like receptor is mediating the glucose response in these cells. These findings established a key role for P2Y11 in regulating PKA-dependent LTCC function and vascular reactivity during diabetic hyperglycemia.

Large-conductance Ca2+-activated potassium (BKCa) channels are key determinants of vascular smooth muscle excitability. Impaired BKCa channel function through remodeling of BKCa β1 expression and function contributes to vascular complications in animal models of diabetes. Yet, whether similar alterations occur in native vascular smooth muscle from humans with type 2 diabetes is unclear. In this study, we evaluated BKCa function in vascular smooth muscle from small resistance adipose arteries of non-diabetic and clinically diagnosed type 2 diabetic patients. We found that BKCa channel activity opposes pressure-induced constriction in human small resistance adipose arteries, and this is compromised in arteries from diabetic patients. Consistent with impairment of BKCa channel function, the amplitude and frequency of spontaneous BKCa currents, but not Ca2+ sparks were lower in cells from diabetic patients. BKCa channels in diabetic cells exhibited reduced Ca2+ sensitivity, single-channel open probability and tamoxifen sensitivity. These effects were associated with decreased functional coupling between BKCa α and β1 subunits, but no change in total protein abundance. Overall, results suggest impairment in BKCa channel function in vascular smooth muscle from diabetic patients through unique mechanisms, which may contribute to vascular complications in humans with type 2 diabetes.

Calcium (Ca2+) plays a central role in excitation, contraction, transcription, and proliferation of vascular smooth muscle cells (VSMs). Precise regulation of intracellular Ca2+ concentration ([Ca2+]i) is crucial for proper physiological VSM function. Studies over the last several decades have revealed that VSMs express a variety of Ca2+-permeable channels that orchestrate a dynamic, yet finely tuned regulation of [Ca2+]i. In this review, we discuss the major Ca2+-permeable channels expressed in VSM and their contribution to vascular physiology and pathology.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a potent vasodilator of numerous vascular beds, including cerebral arteries. Although PACAP-induced cerebral artery dilation is suggested to be cyclic AMP (cAMP)-dependent, the downstream intracellular signaling pathways are still not fully understood. In this study, we examined the role of smooth muscle K(+) channels and hypothesized that PACAP-mediated increases in cAMP levels and protein kinase A (PKA) activity result in the coordinate activation of ATP-sensitive K(+) (KATP) and large-conductance Ca(2+)-activated K(+) (BK) channels for cerebral artery dilation. Using patch-clamp electrophysiology, we observed that PACAP enhanced whole-cell KATP channel activity and transient BK channel currents in freshly isolated rat cerebellar artery myocytes. The increased frequency of transient BK currents following PACAP treatment is indicative of increased intracellular Ca(2+) release events termed Ca(2+) sparks. Consistent with the electrophysiology data, the PACAP-induced vasodilations of cannulated cerebellar artery preparations were attenuated by approximately 50 % in the presence of glibenclamide (a KATP channel blocker) or paxilline (a BK channel blocker). Further, in the presence of both blockers, PACAP failed to cause vasodilation. In conclusion, our results indicate that PACAP causes cerebellar artery dilation through two mechanisms: (1) KATP channel activation and (2) enhanced BK channel activity, likely through increased Ca(2+) spark frequency.

The homeostatic control of cranial and cerebral blood flow is essential for normal brain function. Brain surface (cerebral) and meningeal (cranial) arteries/arterioles are densely innervated by sensory and autonomic fibers containing a variety of vasoactive peptides including pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) which has been shown to exert potent vasodilatory effects in a variety of vascular beds. Recent studies have demonstrated that PACAP potency, activation of PACAP receptor subtypes, and signaling to potassium channels to mediate vasodilatory responses are different between cerebral and cranial arteries, such as the cerebellar artery and the middle meningeal artery (MMA). PACAP demonstrates exquisite picomolar potency at the PACAP-selective PAC1 receptor (Adcyap1r1) to activate ATP-sensitive potassium (KATP) channels of the MMA causing vasodilation. Although PACAP frequently colocalizes with the vasodilatory calcitonin gene related peptide (CGRP) in the fiber networks innervating the MMA, PACAP potency is nearly three orders of magnitude greater than that of CGRP. CGRP is equipotent in the MMA and cerebral arteries. In contrast, PACAP is a less potent vasodilator of cerebral arteries compared to the MMA, with nanomolar concentrations required to activate VPAC receptors leading to KATP and large-conductance calcium-dependent potassium (BK) channel activation and cerebellar artery dilation. Further, PACAP’s effects on the brain are multidimensional. PACAP signaling also exerts direct neurotrophic and neuroprotective effects in the central nervous system against degenerative processes or physiological insults. Thus, PACAP regulation of cerebral blood flow helps sustains brain metabolic demands, maintains neural physiological functions—including learning and memory, and protects neurons from neurodegeneration in disease and trauma. Coherent with CGRP vascular responses and functions, the vasodilatory effects of PACAP in the MMA also implicate a role in genesis of migraine disorders.

Voltage-gated potassium (KV ) channels are key regulators of vascular smooth muscle contractility and vascular tone, and thus have major influence on the microcirculation. KV channels are important determinants of vascular smooth muscle membrane potential (Em ). A number of KV subunits are expressed in the plasma membrane of smooth muscle cells. Each subunit confers distinct kinetics and regulatory properties that allow for fine control of Em to orchestrate vascular tone. Modifications in KV subunit expression and/or channel activity can contribute to changes in vascular smooth muscle contractility in response to different stimuli and in diverse pathological conditions. Consistent with this, a number of studies suggest alterations in KV subunit expression and/or function as underlying contributing mechanisms for small resistance artery dysfunction in pathologies such as hypertension and metabolic disorders, including diabetes. Here, we review our current knowledge on the effects of these pathologies on KV channel expression and function in vascular smooth muscle cells, and the repercussions on (micro)vascular function.

The cellular mechanisms contributing to migraine headache are poorly understood, but may involve prolonged dilation of cranial arteries, specifically the MMA. Agonist-induced activation of ATP-sensitive potassium (KATP) channels is known to play an important role in the modulation of arterial diameter in a number of vascular beds. The objective of the current study was to determine if a tonic vasodilator influence resulting from basal KATP channel activity is present in isolated, pressurized rat MMA. At an intravascular pressure of 40 mmHg, arteries developed pressure-induced constriction representing an approximate 40 % decrease in diameter. Treatment of arteries with either of the KATP channel inhibitors glibenclamide (10 µM) or PNU37883 (10 µM) induced a further decrease in diameter of ∼ 20 %. Also consistent with basal KATP activity, glibenclamide induced a membrane potential depolarization of ∼ 14 mV in MMA segments at an intravascular pressure of 40 mmHg. Further, in MMA loaded with the ratiometric ...

The causes of migraine and cluster headaches are not clear, but may involve dilation of the meningeal vasculature. Recent evidence indicates that transient receptor potential channel V1 (TRPV1) ago...

Migraine is a debilitating neurological disorder characterized by mild to severe headache that is often accompanied by aura and other neurological symptoms. Among proposed mechanisms, dilation of the dural vasculature especially the middle meningeal artery (MMA) has been implicated as one component underlying this disorder. Several regulatory peptides from trigeminal sensory and sphenopalatine postganglionic parasympathetic fibers innervating these vessels have been implicated in the process including pituitary adenylate cyclase-activating polypeptide (PACAP). Although PACAP has been well described as a potent dilator in many vascular beds, the effects of PACAP on the dural vasculature are unclear. In the current study, we examined the ability of PACAP to dilate MMAs that were isolated from rats and pressurized ex vivo. PACAP38 potently dilated pressurized MMAs with an EC(50) of 1 pM. The PAC1 receptor antagonist, PACAP(6-38), abolished MMA dilation caused by picomolar concentrations of PACAP. In contrast, cerebellar arteries isolated from the brain surface were ~1,000-fold less sensitive to PACAP than MMAs. Although cerebellar arteries expressed transcripts for all three PACAP receptor subtypes (PAC1, VPAC1, and VPAC2 receptors) by RT-PCR analyses, MMA demonstrated only PAC1 and VPAC2 receptor expression. Further, multiple variants of the PAC1 receptor were identified in the MMA. The expression of PAC1 receptors and the high potency of PACAP to induce MMA vasodilation are consistent with their potential roles in the etiology of migraine.

In the present study we report on the use of speed congenics to generate a C57BL/6J congenic line of HD-model R6/2 mice carrying 110 CAG repeats, which uniquely exhibits minimal intergenerational instability. We also report the first identification of the R6/2 transgene insertion site. The relatively stable line of 110 CAG R6/2 mice was characterized for the onset of behavioral impairments in motor, cognitive and psychiatric-related phenotypes as well as the progression of disease-related impairments from 4 to 10 weeks of age. 110Q mice exhibited many of the phenotypes commonly associated with the R6/2 model including reduced activity and impairments in rotarod performance. The onset of many of the phenotypes occurred around 6 weeks and was progressive across age. In addition, some phenotypes were observed in mice as early as 4 weeks of age. The present study also reports the onset and progression of changes in several molecular phenotypes in the novel R6/2 mice and the association of these changes with behavioral symptom onset and progression. Data from TR-FRET suggest an association of mutant protein state changes (soluble versus aggregated) in disease onset and progression.