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Summary The retrograde axonal transport of neurotrophins occurs after receptor-mediated endocytosis into vesicles at the nerve terminal. We have been investigating the process of targeting these vesicles for retrograde transport, by examining the transport of [125I]-labelled neurotrophins from the eye to sympathetic and sensory ganglia. With the aid of confocal microscopy, we examined the phenomena further in cultures of dissociated sympathetic ganglia to which rhodamine-labelled nerve growth factor (NGF) was added. We found the label in large vesicles in the growth cone and axons. Light microscopic examination of the sympathetic nerve trunk in vivo also showed the retrogradely transported material to be sporadically located in large structures in the axons. Ultrastructural examination of the sympathetic nerve trunk after the transport of NGF bound to gold particles showed the label to be concentrated in relatively few large organelles that consisted of accumulations of multivesicular bodies. These results suggest that in vivo NGF is transported in specialized organelles that require assembly in the nerve terminal. [ABSTRACT FROM AUTHOR]

Coupling red blood cell (RBC) supply to O 2 demand is an intricate process requiring O 2 sensing, generation of a stimulus, and signal transduction that alters upstream arteriolar tone. Although actively debated, this process has been theorized to be induced by hypoxia and to involve activation of endothelial inwardly rectifying K + channels (K IR ) 2.1 by elevated extracellular K + to trigger conducted hyperpolarization via connexin40 (Cx40) gap junctions to upstream resistors. This concept was tested in resting healthy skeletal muscle of Cx40 -/- and endothelial K IR 2.1 -/- mice using state-of-the-art live animal imaging where the local tissue O 2 environment was manipulated using a custom gas chamber. Second-by-second capillary RBC flow responses were recorded as O 2 was altered. A stepwise drop in PO 2 at the muscle surface increased RBC supply in capillaries of control animals while elevated O 2 elicited the opposite response; capillaries were confirmed to express Cx40. The RBC flow responses were rapid and tightly coupled to O 2 ; computer simulations did not support hypoxia as a driving factor. In contrast, RBC flow responses were significantly diminished in Cx40 -/- mice. Endothelial K IR 2.1 -/- mice, on the other hand, reacted normally to O 2 changes, even when the O 2 challenge was targeted to a smaller area of tissue with fewer capillaries. Conclusively, microvascular O 2 responses depend on coordinated electrical signaling via Cx40 gap junctions, and endothelial K IR 2.1 channels do not initiate the event. These findings reconceptualize the paradigm of blood flow regulation in skeletal muscle and how O 2 triggers this process in capillaries independent of extracellular K + ., Competing Interests: Competing interests statement:The authors declare no competing interest.

Objective: This study investigated the actions of advanced glycated end-products (AGE), their receptors (RAGE), and NAD(P)H oxidase (Nox) subtypes 1, 2, and 4 on mechanisms of endothelium-dependent dilation of the rat cremaster muscle artery (CMA)., Methods: Immunofluorescence studies were used to examine expression of RAGE in rat arteries. ROS accumulation was measured using luminescence and fluorescence assays. Functional studies were performed using pressure myography., Results: High levels of RAGE expression were shown in the endothelial cells of the CMA, compared with low endothelial expression in middle cerebral and mesenteric arteries and the aorta. Exogenous AGE (in vitro glycated bovine serum albumin) stimulated H2O2 accumulation in CMA, which was prevented by the RAGE antagonist FPS-ZM1, the NAD(P)H oxidase (Nox) inhibitor apocynin and inhibited by the Nox1/4 inhibitor setanaxib, but not the Nox2 inhibitor GSK2795039. In functional studies, AGE inhibited vasodilation of CMA stimulated by acetylcholine, sodium nitroprusside, and the BKCa activator NS1619, but not adenosine-induced dilation. FPS-ZM1, apocynin, and setanaxib prevented the inhibitory effects of AGE on responses to acetylcholine and NS-1619., Conclusion: These observations suggest RAGE are constitutively expressed in the endothelium of the rat CMA and may be activated by AGE to stimulate Nox1/4 and ROS formation with resulting inhibition of NO and BKCa-mediated endothelium-dependent dilation., (© 2023 The Authors. Microcirculation published by John Wiley & Sons Ltd.)

Background Normal brain function depends on the ability of the vasculature to increase blood flow to regions with high metabolic demands. Impaired neurovascular coupling, such as the local hyperemic response to neuronal activity, may contribute to poor neurological outcome after stroke despite successful recanalization, that is, futile recanalization. Methods and Results Mice implanted with chronic cranial windows were trained for awake head-fixation before experiments. One-hour occlusion of the anterior middle cerebral artery branch was induced using single-vessel photothrombosis. Cerebral perfusion and neurovascular coupling were assessed by optical coherence tomography and laser speckle contrast imaging. Capillaries and pericytes were studied in perfusion-fixed tissue by labeling lectin and platelet-derived growth factor receptor β. Arterial occlusion induced multiple spreading depolarizations over 1 hour associated with substantially reduced blood flow in the peri-ischemic cortex. Approximately half of the capillaries in the peri-ischemic area were no longer perfused at the 3- and 24-hour follow-up (45% [95% CI, 33%-58%] and 53% [95% CI, 39%-66%] reduction, respectively; P <0.0001), which was associated with contraction of an equivalent proportion of peri-ischemic capillary pericytes. The capillaries in the peri-ischemic cortex that remained perfused showed increased point prevalence of dynamic flow stalling (0.5% [95% CI, 0.2%-0.7%] at baseline, 5.1% [95% CI, 3.2%-6.5%] and 3.2% [95% CI, 1.1%-5.3%] at 3- and 24-hour follow-up, respectively; P =0.001). Whisker stimulation at the 3- and 24-hour follow-up led to reduced neurovascular coupling responses in the sensory cortex corresponding to the peri-ischemic region compared with that observed at baseline. Conclusions Arterial occlusion led to contraction of capillary pericytes and capillary flow stalling in the peri-ischemic cortex. Capillary dysfunction was associated with neurovascular uncoupling. Neurovascular coupling impairment associated with capillary dysfunction may be a mechanism that contributes to futile recanalization. Hence, the results from this study suggest a novel treatment target to improve neurological outcome after stroke.

Background: Within the vasculature platelets and endothelial cells play crucial roles in hemostasis and thrombosis. Platelets, like endothelial cells, possess intermediate conductance Ca 2+ -activated K + (IK Ca ) channels and generate nitric oxide (NO). Although NO limits platelet aggregation, the role of IK Ca channels in platelet function and NO generation has not yet been explored., Objectives: We investigated whether IK Ca channel activation inhibits platelet aggregation, and per endothelial cells, enhances platelet NO production., Methods: Platelets were isolated from human volunteers. Aggregometry, confocal microscopy, and a novel flow chamber model, the Quartz Crystal Microbalance (QCM) were used to assess platelet function. Flow cytometry was used to measure platelet NO production, calcium signaling, membrane potential, integrin α IIb /β 3 activation, granule release, and procoagulant platelet formation., Results: Platelet IK Ca channel activation with SKA-31 inhibited aggregation in a concentration-dependent manner, an effect reversed by the selective IK Ca channel blocker TRAM-34. The QCM model along with confocal microscopy demonstrated that SKA-31 inhibited platelet aggregation under flow conditions. Surprisingly, IK Ca activation by SKA-31 inhibited platelet NO generation, but this could be explained by a concomitant reduction in platelet calcium signaling. IK Ca activation by SKA-31 also inhibited dense and alpha-granule secretion and integrin α IIb /β 3 activation, but maintained platelet phosphatidylserine surface exposure as a measure of procoagulant response., Conclusions: Platelet IK Ca channel activation inhibits aggregation by reducing calcium-signaling and granule secretion, but not by enhancing platelet NO generation. IK Ca channels may be novel targets for the development of antiplatelet drugs that limit atherothrombosis, but not coagulation., (© 2022 International Society on Thrombosis and Haemostasis.)

Background and Purpose: The physiological role of vascular β 3 -adrenoceptors is not fully understood. Recent evidence suggests cardiac β 3 -adrenoceptors are functionally effective after down-regulation of β 1 /β 2 -adrenoceptors. The functional interaction between the β 3 -adrenoceptor and other β-adrenoceptor subtypes in rat striated muscle arteries was investigated., Experimental Approach: Studies were performed in cremaster muscle arteries isolated from male Sprague-Dawley rats. β-adrenoceptor expression was assessed through RT-PCR and immunofluorescence. Functional effects of β 3 -adrenoceptor agonists and antagonists and other β-adrenoceptor ligands were measured using pressure myography., Key Results: All three β-adrenoceptor subtypes were present in the endothelium of the cremaster muscle artery. The β 3 -adrenoceptor agonists mirabegron and CL 316,243 had no effect on the diameter of pressurized (70 mmHg) cremaster muscle arterioles with myogenic tone, while the β 3 -adrenoceptor agonist SR 58611A and the nonselective β-adrenoceptor agonist isoprenaline caused concentration-dependent dilation. In the presence of β 1/2 -adrenoceptor antagonists nadolol (10 μM), atenolol (1 μM) and ICI 118,551 (0.1 μM) both mirabegron and CL 316,243 were effective in causing vasodilation and the potency of SR 58611A was enhanced, while responses to isoprenaline were inhibited. The β 3 -adrenoceptor antagonist L 748,337 (1 μM) inhibited vasodilation caused by β 3 -adrenoceptor agonists (in the presence of β 1/2 -adrenoceptor blockade), but L 748,337 had no effect on isoprenaline-induced vasodilation., Conclusion and Implications: All three β-adrenoceptor subtypes were present in the endothelium of the rat cremaster muscle artery, but β 3 -adrenoceptor mediated vasodilation was only evident after blockade of β 1/2 -adrenoceptors. This suggests constitutive β 1/2 -adrenoceptor activity inhibits β 3 -adrenoceptor function in the endothelium of skeletal muscle resistance arteries., (© 2021 The British Pharmacological Society.)

Background and Purpose: Arterial diameter is dictated by the contractile state of the vascular smooth muscle cells (VSMCs), which is modulated by direct and indirect inputs from endothelial cells (ECs). Modulators of KCNQ-encoded k V 7 channels have considerable impact on arterial diameter and these channels are known to be expressed in VSMCs but not yet defined in ECs. However, expression of k V 7 channels in ECs would add an extra level of vascular control. This study aims to characterize the expression and function of K V 7 channels within rat mesenteric artery ECs. Experimental Approach: In rat mesenteric artery, KCNQ transcript and K V 7 channel protein expression were determined via RT-qPCR, immunocytochemistry, immunohistochemistry and immunoelectron microscopy. Wire myography was used to determine vascular reactivity. Key Results: KCNQ transcript was identified in isolated ECs and VSMCs. K V 7.1, K V 7.4 and K V 7.5 protein expression was determined in both isolated EC and VSMC and in whole vessels. Removal of ECs attenuated vasorelaxation to two structurally different K V 7.2-5 activators S-1 and ML213. K IR 2 blockers ML133, and BaCl 2 also attenuated S-1 or ML213-mediated vasorelaxation in an endothelium-dependent process. K V 7 inhibition attenuated receptor-dependent nitric oxide (NO)-mediated vasorelaxation to carbachol, but had no impact on relaxation to the NO donor, SNP. Conclusion and Implications: In rat mesenteric artery ECs, K V 7.4 and K V 7.5 channels are expressed, functionally interact with endothelial K IR 2.x channels and contribute to endogenous eNOS-mediated relaxation. This study identifies K V 7 channels as novel functional channels within rat mesenteric ECs and suggests that these channels are involved in NO release from the endothelium of these vessels., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Baldwin, Sandow, Mondéjar-Parreño, Stott and Greenwood.)

Aims: Acute migraine attack in familial hemiplegic migraine type 2 (FHM2) patients is characterized by sequential hypo- and hyperperfusion. FHM2 is associated with mutations in the Na, K-ATPase α2 isoform. Heterozygous mice bearing one of these mutations (α2+/G301R mice) were shown to have elevated cerebrovascular tone and, thus, hypoperfusion that might lead to elevated concentrations of local metabolites. We hypothesize that these α2+/G301R mice also have increased cerebrovascular hyperaemic responses to these local metabolites leading to hyperperfusion in the affected part of the brain., Methods and Results: Neurovascular coupling was compared in α2+/G301R and matching wild-type (WT) mice using Laser Speckle Contrast Imaging. In brain slices, parenchymal arteriole diameter and intracellular calcium changes in neuronal tissue, astrocytic endfeet, and smooth muscle cells in response to neuronal excitation were assessed. Wall tension and smooth muscle membrane potential were measured in isolated middle cerebral arteries. Quantitative polymerase chain reaction, western blot, and immunohistochemistry were used to assess the molecular background underlying the functional changes. Whisker stimulation induced larger increase in blood perfusion, i.e. hyperaemic response, of the somatosensory cortex of α2+/G301R than WT mice. Neuronal excitation was associated with larger parenchymal arteriole dilation in brain slices from α2+/G301R than WT mice. These hyperaemic responses in vivo and ex vivo were inhibited by BaCl2, suggesting involvement of inward-rectifying K+ channels (Kir). Relaxation to elevated bath K+ was larger in arteries from α2+/G301R compared to WT mice. This difference was endothelium-dependent. Endothelial Kir2.1 channel expression was higher in arteries from α2+/G301R mice. No sex difference in functional responses and Kir2.1 expression was found., Conclusion: This study suggests that an abnormally high cerebrovascular hyperaemic response in α2+/G301R mice is a result of increased endothelial Kir2.1 channel expression. This may be initiated by vasospasm-induced accumulation of local metabolites and underlie the hyperperfusion seen in FHM2 patients during migraine attack., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

Objective: Cerebral arterial networks match blood flow delivery with neural activity. Neurovascular response begins with a stimulus and a focal change in vessel diameter, which by themselves is inconsequential to blood flow magnitude, until they spread and alter the contractile status of neighboring arterial segments. We sought to define the mechanisms underlying integrated vascular behavior and considered the role of intercellular electrical signaling in this phenomenon. Approach and Results: Electron microscopic and histochemical analysis revealed the structural coupling of cerebrovascular cells and the expression of gap junctional subunits at the cell interfaces, enabling intercellular signaling among vascular cells. Indeed, robust vasomotor conduction was detected in human and mice cerebral arteries after focal vessel stimulation: a response attributed to endothelial gap junctional communication, as its genetic alteration attenuated this behavior. Conducted responses were observed to ascend from the penetrating arterioles, influencing the contractile status of cortical surface vessels, in a simulated model of cerebral arterial network. Ascending responses recognized in vivo after whisker stimulation were significantly attenuated in mice with altered endothelial gap junctional signaling confirming that gap junctional communication drives integrated vessel responses. The diminishment in vascular communication also impaired the critical ability of the cerebral vasculature to maintain blood flow homeostasis and hence tissue viability after stroke., Conclusions: Our findings highlight the integral role of intercellular electrical signaling in transcribing focal stimuli into coordinated changes in cerebrovascular contractile activity and expose, a hitherto unknown mechanism for flow regulation after stroke.

Familial hemiplegic migraine type 2 (FHM2) is associated with inherited point-mutations in the Na,K-ATPase α2 isoform, including G301R mutation. We hypothesized that this mutation affects specific aspects of vascular function, and thus compared cerebral and systemic arteries from heterozygote mice bearing the G301R mutation (Atp1a2 +/-G301R ) with wild type (WT). Middle cerebral (MCA) and mesenteric small artery (MSA) function was compared in an isometric myograph. Cerebral blood flow was assessed with Laser speckle analysis. Intracellular Ca 2+ and membrane potential were measured simultaneously. Protein expression was semi-quantified by immunohistochemistry. Protein phosphorylation was analysed by Western blot. MSA from Atp1a2 +/-G301R and WT showed similar contractile responses. The Atp1a2 +/-G301R MCA constricted stronger to U46619, endothelin and potassium compared to WT. This was associated with an increased depolarization, although the Ca 2+ change was smaller than in WT. The enhanced constriction of Atp1a2 +/-G301R MCA was associated with increased cSrc activation, stronger sensitization to [Ca 2+ ] i and increased MYPT1 phosphorylation. These differences were abolished by cSrc inhibition. Atp1a2 +/-G301R mice had reduced resting blood flow through MCA in comparison with WT mice . FHM2-associated mutation leads to elevated contractility of MCA due to sensitization of the contractile machinery to Ca 2+ , which is mediated via Na,K-ATPase/Src-kinase/MYPT1 signalling.

Objective- Inward rectifying K + (K IR ) channels are present in cerebral arterial smooth muscle and endothelial cells, a tandem arrangement suggestive of a dynamic yet undiscovered role for this channel. This study defined whether distinct pools of cerebral arterial K IR channels were uniquely modulated by membrane lipids and hemodynamic stimuli. Approach and Results- A Ba 2+ -sensitive K IR current was isolated in smooth muscle and endothelial cells of rat cerebral arteries; molecular analyses subsequently confirmed K IR 2.1/K IR 2.2 mRNA and protein expression in both cells. Patch-clamp electrophysiology next demonstrated that each population of K IR channels was sensitive to key membrane lipids and hemodynamic stimuli. In this regard, endothelial K IR was sensitive to phosphatidylinositol 4,5-bisphosphate content, with depletion impairing the ability of laminar shear stress to activate this channel pool. In contrast, smooth muscle K IR was sensitive to membrane cholesterol content, with sequestration blocking the ability of pressure to inhibit channel activity. The idea that membrane lipids help confer shear stress and pressure sensitivity of K IR channels was confirmed in intact arteries using myography. Virtual models integrating structural/electrical observations reconceptualized K IR as a dynamic regulator of membrane potential working in concert with other currents to set basal tone across a range of shear stresses and intravascular pressures. Conclusions- The data show for the first time that specific membrane lipid-K IR interactions enable unique channel populations to sense hemodynamic stimuli and drive vasomotor responses to set basal perfusion in the cerebral circulation.

Localized, oscillating Ca 2+ signals have been identified in discrete microdomains of vascular endothelial cells. At myoendothelial contacts (between endothelial and smooth muscle cells), both endothelial Ca 2+ pulsars (IP 3 -mediated release of intracellular Ca 2+ ) and Ca 2+ sparklets (extracellular Ca 2+ entry via TRP channels) contribute to endothelium-dependent hyperpolarization of smooth muscle, vasodilation, and feedback control of vasoconstriction. Ca 2+ sparklets occurring at close-contact domains between endothelial cells are possibly involved in conducted hyperpolarization and spreading vasodilation in arterial networks. This review summarizes these Ca 2+ signalling phenomena, examines the proposed mechanisms leading to their generation by G-protein-coupled receptor agonists, and explores the proposed physiological roles of these localized and specialized Ca 2+ signals., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Aims: The Na,K-ATPase is involved in a large number of regulatory activities including cSrc-dependent signalling. Upon inhibition of the Na,K-ATPase with ouabain, cSrc activation is shown to occur in many cell types. This study tests the hypothesis that acute potentiation of agonist-induced contraction by ouabain is mediated through Na,K-ATPase-cSrc signalling-dependent sensitization of vascular smooth muscle cells to Ca 2+ ., Methods: Agonist-induced rat mesenteric small artery contraction was examined in vitro under isometric conditions and in vivo in anaesthetized rats. Arterial wall tension and [Ca 2+ ] i in vascular smooth muscle cells were measured simultaneously. Changes in cSrc and myosin phosphatase targeting protein 1 (MYPT1) phosphorylation were analysed by Western blot. Protein expression was examined with immunohistochemistry. The α1 and α2 isoforms of the Na,K-ATPase were transiently downregulated by siRNA transfection in vivo., Results: Ten micromolar ouabain, but not digoxin, potentiated contraction to noradrenaline. This effect was not endothelium-dependent. Ouabain sensitized smooth muscle cells to Ca 2+ , and this was associated with increased phosphorylation of cSrc and MYPT1. Inhibition of tyrosine kinase by genistein, PP2 or pNaKtide abolished the potentiating effect of ouabain on arterial contraction and Ca 2+ sensitization. Downregulation of the Na,K-ATPase α2 isoform made arterial contraction insensitive to ouabain and tyrosine kinase inhibition., Conclusion: Data suggest that micromolar ouabain potentiates agonist-induced contraction of rat mesenteric small artery via Na,K-ATPase-dependent cSrc activation, which increases Ca 2+ sensitization of vascular smooth muscle cells by MYPT1 phosphorylation. This mechanism may be critical for acute control of vascular tone., (© 2018 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Introduction: The pannexin-1 (Panx1) channels are found in many cell types, and ATP released from these channels can act on nearby cells activating purinergic P2X7 receptors (P2X7R) which lead to inflammation. Although Panx1 and P2X7R are implicated in the process of inflammation and cell death, few studies have looked at the role they play in inflammatory bowel disease in human. Hence, the aim of the present study was to investigate the function of Panx1 and P2X7R in an ex vivo colitis model developed from human colonic mucosal explants. Materials and Methods: Healthy human colonic mucosal strips (4 × 10 mm) were incubated in carbogenated culture medium at 37°C for 16 h. Proinflammatory cytokines TNFα and IL-1β (each 10 ng/mL) were used to induce colitis in mucosal strips, and the effects of Panx1 and P2X7R on cytokines-induced tissue damage were determined in the presence of the Panx1 channel blocker 10 Panx1 (100 μM) and P2X7R antagonist A438079 (100 μM). The effects of 10 Panx1 and A438079 on cytokines-enhanced epithelial permeability were also studied using Caco-2 cells. Results: Histological staining showed that the mucosal strips had severe structural damage in the cytokines-only group but not in the incubation-control group ( P < 0.01). Compared to the cytokines-only group, crypt damage was significantly decreased in groups receiving cytokines with inhibitors ( 10 Panx1, A438079, or 10 Panx1 + A438079, P < 0.05). The immunoreactive signals of tight junction protein zonula occludens-1 (ZO-1) were abundant in all control tissues but were significantly disrupted and lost in the cytokines-only group ( P < 0.01). The diminished ZO-1 immunoreactivity induced by cytokines was prevented in the presence of 10 Panx1 ( P = 0.04). Likewise, 10 Panx1 significantly attenuated the cytokines-evoked increase in paracellular permeability of Caco-2 cells. Although the inhibition of P2X7R activity by A438079 diminished cytokines-induced crypt damage, its effect on the maintenance of ZO-1 immunoreactivity and Caco-2 epithelial cell integrity was less evident. Conclusion: The blockade of Panx1 and P2X7R reduced the inflammatory cytokines-induced crypt damage, loss of tight junctions and increase in cell permeability. Thus, Panx1 and P2X7R may have roles in causing mucosal damage, a common clinical feature of inflammatory bowel disease.

Transient receptor potential canonical type 3 channels (TRPC3) are expressed in neural, cardiac, respiratory and vascular tissues, with both similarities and differences between human and animal models for the same cell types. In common with all members of the six subfamilies of TRP channels, TRPC3 are non-voltage gated, non-selective cation channels that are mainly permeated by Ca 2+ , and have distinct molecular, biophysical, anatomical and functional properties. TRP channels are present in excitable and non-excitable cells where they sense and respond to a wide variety of physical and chemical stimuli. TRPC3 are expressed in the endothelium and/or smooth muscle of specific intact arteries, such as mesenteric, cerebral and myometrial, where they are critical for the control of vascular tone, and show altered activity in development and disease. In artery endothelium, TRPC3 contributes to endothelium-derived hyperpolarization and nitric oxide-mediated vasodilation. In artery smooth muscle, TRPC3 contributes to constrictor mechanisms. In both endothelium and smooth muscle, TRPC3 contributes to function via caveolae-caveolin dependent and independent mechanisms. In different cell types and states, like other TRP channels, TRPC3 can form complexes with other TRP proteins and associated channels and accessory proteins, including those associated with endo(sarco)plasmic reticulum (ER/SR), thereby facilitating Ca 2+ channel activation and/or refilling ER/SR Ca 2+ stores. The diversity of TRPC3 interactions with other vascular signaling components is a potential target for artery specific control mechanisms. This brief perspective highlights recent advances in understanding the functional diversity of TRPC3, with an emphasis on vascular health and disease., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Essential hypertension is a complex multifactorial disease process that involves the interaction of multiple genes at various loci throughout the genome, and the influence of environmental factors such as diet and lifestyle, to ultimately determine long-term arterial pressure. These factors converge with physiological signaling pathways to regulate the set-point of long-term blood pressure. In hypertension, structural changes in arteries occur and show differences within and between vascular beds, between species, models and sexes. Such changes can also reflect the development of hypertension, and the levels of circulating humoral and vasoactive compounds. The role of perivascular adipose tissue in the modulation of vascular structure under various disease states such as hypertension, obesity and metabolic syndrome is an emerging area of research, and is likely to contribute to the heterogeneity described in this review. Diversity in structure and related function is the norm, with morphological changes being causative in some beds and states, and in others, a consequence of hypertension. Specific animal models of hypertension have advantages and limitations, each with factors influencing the relevance of the model to the human hypertensive state/s. However, understanding the fundamental properties of artery function and how these relate to signalling mechanisms in real (intact) tissues is key for translating isolated cell and model data to have an impact and relevance in human disease etiology. Indeed, the ultimate aim of developing new treatments to correct vascular dysfunction requires understanding and recognition of the limitations of the methodologies used.

Abdominal aortic aneurysm (AAA) is an inflammatory disease associated with macrophage accumulation in the adventitia, oxidative stress, medial elastin degradation and aortic dilation. Progression of AAA is linked to increased risk of rupture, which carries a high mortality rate. Drug therapies trialled to date lack efficacy and although aneurysm repair is available for patients with large aneurysm, peri-surgical morbidity and mortality have been widely reported. Recent studies using rodent models of AAA suggest that long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs) and their metabolites can moderate inflammation and oxidative stress perpetuated by infiltrating macrophages and intervene in the destruction of medial elastin. This review examines evidence from these animal studies and related reports of inhibition of inflammation and arrest of aneurysm development following prophylactic supplementation with LC n-3 PUFAs. The efficacy of LC n-3 PUFAs for management of existing aneurysm is unclear and further investigations involving human clinical trials are warranted., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

This study investigated the expression and function of transient receptor potential vanilloid type-3 ion channels (TRPV3) in uterine radial arteries isolated from non-pregnant and twenty-day pregnant rats. Immunohistochemistry (IHC) suggested TRPV3 is primarily localized to the smooth muscle in arteries from both non-pregnant and pregnant rats. IHC using C' targeted antibody, and qPCR of TRPV3 mRNA, suggested pregnancy increased arterial TRPV3 expression. The TRPV3 activator carvacrol caused endothelium-independent dilation of phenylephrine-constricted radial arteries, with no difference between vessels from non-pregnant and pregnant animals. Carvacrol-induced dilation was reduced by the TRPV3-blockers isopentenyl pyrophosphate and ruthenium red, but not by the TRPA1 or TRPV4 inhibitors HC-030031 or HC-067047, respectively. In radial arteries from non-pregnant rats only, inhibition of NOS and sGC, or PKG, enhanced carvacrol-mediated vasodilation. Carvacrol-induced dilation of arteries from both non-pregnant and pregnant rats was prevented by the IKCa blocker TRAM-34. TRPV3 caused an endothelium-independent, IKCa-mediated dilation of the uterine radial artery. NO-PKG-mediated modulation of TRPV3 activity is lost in pregnancy, but this did not alter the response to carvacrol., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Myocardial perfusion and coronary vascular resistance are regulated by signaling metabolites released from the local myocardium that act either directly on the VSMC or indirectly via stimulation of the endothelium. A prominent mechanism of vasodilation is EDH of the arteriolar smooth muscle, with EETs and H(2)O(2) playing important roles in EDH in the coronary microcirculation. In some cases, EETs and H(2)O(2) are released as transferable hyperpolarizing factors (EDHFs) that act directly on the VSMCs. By contrast, EETs and H(2)O(2) can also promote endothelial KCa activity secondary to the amplification of extracellular Ca(2+) influx and Ca(2+) mobilization from intracellular stores, respectively. The resulting endothelial hyperpolarization may subsequently conduct to the media via myoendothelial gap junctions or potentially lead to the release of a chemically distinct factor(s). Furthermore, in human isolated coronary arterioles dilator signaling involving EETs and H(2)O(2) may be integrated, being either complimentary or inhibitory depending on the stimulus. With an emphasis on the human coronary microcirculation, this review addresses the diverse and integrated mechanisms by which EETs and H(2)O(2) regulate vessel tone and also examines the hypothesis that myoendothelial microdomain signaling facilitates EDH activity in the human heart., (© 2015 John Wiley & Sons Ltd.)

Changes in diet are a challenge to the gastrointestinal tract which needs to alter its processing mechanisms to continue to process nutrients and maintain health. In particular, the enteric nervous system (ENS) needs to adapt its motor and secretory programs to deal with changes in nutrient type and load in order to optimise nutrient absorption.The nerve circuits in the gut are complex, and the numbers and types of neurons make recordings of specific cell types difficult, time-consuming, and prone to sampling errors. Nonetheless, traditional research methods like intracellular electrophysiological approaches have provided the basis for our understanding of the ENS circuitry. In particular, animal models of intestinal inflammation have shown us that we can document changes to neuronal excitability and synaptic transmission.Recent studies examining diet-induced changes to ENS programming have opted to use fast imaging techniques to reveal changes in neuron function. Advances in imaging techniques using voltage- or calcium-sensitive dyes to record neuronal activity promise to overcome many limitations inherent to electrophysiological approaches. Imaging techniques allow access to a wide range of ENS phenotypes and to the changes they undergo during dietary challenges. These sorts of studies have shown that dietary variation or obesity can change how the ENS processes information-in effect reprogramming the ENS. In this review, the data gathered from intracellular recordings will be compared with measurements made using imaging techniques in an effort to determine if the lessons learnt from inflammatory changes are relevant to the understanding of diet-induced reprogramming.

Agonist-induced vasoconstriction triggers a negative feedback response whereby movement of charged ions through gap junctions and/or release of endothelium-derived (NO) limit further reductions in diameter, a mechanism termed myoendothelial feedback. Recent studies indicate that electrical myoendothelial feedback can be accounted for by flux of inositol trisphosphate (IP3) through myoendothelial gap junctions resulting in localized increases in endothelial Ca(2+) to activate intermediate conductance calcium-activated potassium (IKCa) channels, the resultant hyperpolarization then conducting back to the smooth muscle to attenuate agonist-induced depolarization and tone. In the present study we tested the hypothesis that activation of IKCa channels underlies NO-mediated myoendothelial feedback. Functional experiments showed that block of IP3 receptors, IKCa channels, gap junctions and transient receptor potential canonical type-3 (TRPC3) channels caused endothelium-dependent potentiation of agonist-induced increase in tone which was not additive with that caused by inhibition of NO synthase supporting a role for these proteins in NO-mediated myoendothelial feedback. Localized densities of IKCa and TRPC3 channels occurred at the internal elastic lamina/endothelial-smooth muscle interface in rat basilar arteries, potential communication sites between the two cell layers. Smooth muscle depolarization to contractile agonists was accompanied by IKCa channel-mediated endothelial hyperpolarization providing the first demonstration of IKCa channel-mediated hyperpolarization of the endothelium in response to contractile agonists. Inhibition of IKCa channels, gap junctions, TRPC3 channels or NO synthase potentiated smooth muscle depolarization to agonists in a non-additive manner. Together these data indicate that rather being distinct pathways for the modulation of smooth muscle tone, NO and endothelial IKCa channels are involved in an integrated mechanism for the regulation of agonist-induced vasoconstriction., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Preeclampsia is a systemic vascular disorder of pregnancy and is associated with increased sensitivity to angiotensin II (AngII) and hypertension. The cause of preeclampsia remains unknown. We identified the role of regulator of G protein (heterotrimeric guanine nucleotide-binding protein) signaling 5 (RGS5) in blood pressure regulation during pregnancy and preeclampsia. RGS5 expression in human myometrial vessels is markedly suppressed in gestational hypertension and/or preeclampsia. In pregnant RGS5-deficient mice, reduced vascular RGS5 expression causes gestational hypertension by enhancing vascular sensitivity to AngII. Further challenge by increasing AngII results in preeclampsia-like symptoms, namely, more severe hypertension, proteinuria, placental pathology, and reduced birth weight. In pregnant heterozygote null mice, treatment with peroxisome proliferator-activated receptor (PPAR) agonists normalizes vascular function and blood pressure through effects on RGS5. These findings highlight a key role of RGS5 at the interface between AngII and PPAR signaling. Because preeclampsia is refractory to current standard therapies, our study opens an unrecognized and urgently needed opportunity for treatment of gestational hypertension and preeclampsia., (Copyright © 2015, American Association for the Advancement of Science.)

The dynamic adjustment of hearing sensitivity and frequency selectivity is mediated by the medial olivocochlear efferent reflex, which suppresses the gain of the 'cochlear amplifier' in each ear. Such efferent feedback is important for promoting discrimination of sounds in background noise, sound localization and protecting the cochleae from acoustic overstimulation. However, the sensory driver for the olivocochlear reflex is unknown. Here, we resolve this longstanding question using a mouse model null for the gene encoding the type III intermediate filament peripherin (Prph). Prph((-/-)) mice lacked type II spiral ganglion neuron innervation of the outer hair cells, whereas innervation of the inner hair cells by type I spiral ganglion neurons was normal. Compared with Prph((+/+)) controls, both contralateral and ipsilateral olivocochlear efferent-mediated suppression of the cochlear amplifier were absent in Prph((-/-)) mice, demonstrating that outer hair cells and their type II afferents constitute the sensory drive for the olivocochlear efferent reflex.

Background: Pannexin-2 (Panx2) is a member of the novel group of membrane spanning protein channels present in the central nervous system. Limited studies have examined Panx2 in the intestine, where it may have important physiological roles. The present study characterized Panx2 expression and localization in the human colon in health and disease states., Methods: Immunofluorescence determined Panx2 localization and co-localization, and quantitative real-time PCR and Western blot determined gene and protein expression in ulcerative colitis (UC), Crohn's disease (CD), and control human colon., Key Results: Panx2 was widely expressed in myenteric and submucosal ganglia, particularly in the cytoplasm of neurons. Panx2 was also expressed on smooth muscle of the muscularis and blood vessels, some non-lymphoid leukocytes, mast cells, and mucosal epithelial cells. Co-localization of Panx2 occurred with β-tubulin, neuronal nitric oxide synthase, substance P, vesicular acetylcholine transporter, and calcitonin gene-related peptide, indicating widespread Panx2 expression in extrinsic and intrinsic neurons. Molecular studies revealed a 3.4-fold higher level of Panx2 mRNA in ascending compared to sigmoid muscularis (p < 0.05), despite similar protein levels. Similarly, UC muscularis showed a 35-fold up-regulation in Panx2 mRNA, but not in protein (p < 0.05)., Conclusions & Inferences: Here, we demonstrated the dense expression of Panx2 in the enteric nervous system and the co-localization of Panx2 with a spectrum of neuronal markers, indicating that Panx2 may be involved in mediating neurotransmission in the colon. The substantial increase in Panx2 mRNA in UC muscle but not protein suggests that the Panx2 translation process may be disrupted in UC., (© 2015 John Wiley & Sons Ltd.)

Dysfunctional electrical signalling within the arteriolar wall is a major cause of cardiovascular disease. The endothelial cell layer constitutes the primary electrical pathway, co-ordinating contraction of the overlying smooth muscle cell (SMC) layer. As myoendothelial gap junctions (MEGJs) provide direct contact between the cell layers, proper vasomotor responses are thought to depend on a high, uniform MEGJ density. However, MEGJs are observed to be expressed heterogeneously within and among vascular beds. This discrepancy is addressed in the present study. As no direct measures of MEGJ conductance exist, we employed a computational modelling approach to vary the number, conductance and distribution of MEGJs. Our simulations demonstrate that a minimal number of randomly distributed MEGJs augment arteriolar cell-cell communication by increasing conduction efficiency and ensuring appropriate membrane potential responses in SMCs. We show that electrical coupling between SMCs must be tailored to the particular MEGJ distribution. Finally, observation of non-decaying mechanical conduction in arterioles without regeneration has been a long-standing controversy in the microvascular field. As heterogeneous MEGJ distributions provide for different conduction profiles along the cell layers, we demonstrate that a non-decaying conduction profile is possible in the SMC layer of a vessel with passive electrical properties. These intriguing findings redefine the concept of efficient electrical communication in the microcirculation, illustrating how heterogeneous properties, ubiquitous in biological systems, may have a profound impact on system behaviour and how acute local and global flow control is explained from the biophysical foundations., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

This study examined the effect of diet-induced obesity on the functional role of large-conductance Ca²⁺-activated K⁺ channels (BK(Ca)) in rat middle cerebral arteries. Male Sprague-Dawley rats were fed a control (chow) or high-fat diet for 16-20 weeks. Diet-induced obesity decreased maximum bradykinin-induced dilation of isolated, pressurized (80 mmHg) arteries, but vasodilation induced by sodium nitroprusside (SNP) was unaltered. Responses to bradykinin and SNP in arteries from both control and obese rats were abolished by combination of the nitric oxide synthase (NOS) and guanylate cyclase (sGC) inhibitors L-NAME (100 μmol/L) and ODQ (10 μmol/L) respectively, or by the BK(Ca) blocker iberiotoxin (IBTX, 0.1 μmol/L). Vasodilation induced by the PAR2 agonist SLIGRL in arteries from control-diet rats was abolished by L-NAME/ODQ, but unaffected by IBTX. Obesity greatly reduced the inhibitory effect of L-NAME/ODQ on SLIGRL-induced dilation, whereas IBTX alone now inhibited responses to SLIGRL. Neither obesity nor IBTX altered the responsiveness of the arteries to vasoconstrictors 5-hydroxytryptamine (5-HT) or angiotensin II (Ang II). Obesity had variable effects on the functional role of BK(Ca) in the middle-cerebral artery depending upon the agent used to stimulate the channel, reflecting the variety of mechanisms by which BK(Ca) may be activated., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Low-voltage-activated T-type calcium channels play an important role in regulating cellular excitability and are implicated in conditions, such as epilepsy and neuropathic pain. T-type channels, especially Cav3.1 and Cav3.2, are also expressed in the vasculature, although patch clamp studies of isolated vascular smooth muscle cells have in general failed to demonstrate these low-voltage-activated calcium currents. By contrast, the channels which are blocked by T-type channel antagonists are high-voltage activated but distinguishable from their L-type counterparts by their T-type biophysical properties and small negative shifts in activation and inactivation voltages. These changes in T-channel properties may result from vascular-specific expression of splice variants of Cav3 genes, particularly in exon 25/26 of the III-IV linker region. Recent physiological studies suggest that T-type channels make a small contribution to vascular tone at low intraluminal pressures, although the relevance of this contribution is unclear. By contrast, these channels play a larger role in vascular tone of small arterioles, which would be expected to function at lower intra-vascular pressures. Upregulation of T-type channel function following decrease in nitric oxide bioavailability and increase in oxidative stress, which occurs during cardiovascular disease, suggests that a more important role could be played by these channels in pathophysiological situations. The ability of T-type channels to be rapidly recruited to the plasma membrane, coupled with their subtype-specific localisation in signalling microdomains where they could modulate the function of calcium-dependent ion channels and pathways, provides a mechanism for rapid up- and downregulation of vasoconstriction. Future investigation into the molecules which govern these changes may illuminate novel targets for the treatment of conditions such as therapy-resistant hypertension and vasospasm.

Background: Dietary iron supplementation is associated with gastrointestinal (GI) side effects including vomiting, nausea, and diarrhea. Although inorganic iron in high concentrations may be damaging to the intestinal mucosa, we hypothesize that there are physiological effects on the GI tract that occur at concentrations achieved by supplementation. Thus, our aim was to investigate the effect of intraluminal ferrous sulfate (FeSO4 ) on jejunal motility., Methods: Segments of guinea pig jejunum were cannulated and the intraluminal pressure recorded with a transducer, while movements were recorded with a video camera. Peristaltic threshold was the oral pressure that evoked four consecutive propulsive contractions. The nutrients decanoic acid (1 mM), l-phenylalanine (50 mM), or the micronutrient FeSO4 (1 mM) were infused intraluminally. We also tested the effect of FeSO4 on electrochemically detected serotonin (5-HT, 5-hydroxytryptamine) released from in vitro tissues, both at rest and following mechanical stimulation., Key Results: The jejuna peristaltic threshold was significantly decreased by all three nutrients: FeSO4 : 31 ± 2-23 ± 3 mmH2 O; decanoic acid: 27 ± 2-14 ± 2 mmH2 O; and l-phenylalanine: 30 ± 3-14 ± 3mmH2 O. Of the three, only decanoic acid induced segmentation, while FeSO4 inhibited decanoic acid-induced segmentation. Resting 5-HT release was increased by FeSO4 (128% of control), but mechanically evoked 5-HT release was reduced (70% of control)., Conclusions & Inferences: These data suggest that some luminal effects of inorganic iron on jejunal motility could be mediated through a pathway involving altered release of 5-HT. A better understanding of the interaction between luminal iron and 5-HT containing enterochromaffin cells could improve iron supplementation strategies, thus reducing side effects., (© 2013 John Wiley & Sons Ltd.)

Endothelium-derived epoxyeicosatrienoic acids (EETs) are fatty acid epoxides that play an important role in the control of vascular tone in selected coronary, renal, carotid, cerebral and skeletal muscle arteries. Vasodilation due to endothelium-dependent smooth muscle hyperpolarization (EDH) has been suggested to involve EETs as a transferable endothelium-derived hyperpolarizing factor. However, this activity may also be due to EETs interacting with the components of other primary EDH-mediated vasodilator mechanisms. Indeed, the transfer of hyperpolarization initiated in the endothelium to the adjacent smooth muscle via gap junction connexins occurs separately or synergistically with the release of K(+) ions at discrete myoendothelial microdomain signalling sites. The net effects of such activity are smooth muscle hyperpolarization, closure of voltage-dependent Ca(2+) channels, phospholipase C deactivation and vasodilation. The spatially localized and key components of the microdomain signalling complex are the inositol 1,4,5-trisphosphate receptor-mediated endoplasmic reticulum Ca(2+) store, Ca(2+)-activated K(+) (KCa), transient receptor potential (TRP) and inward-rectifying K(+) channels, gap junctions and the smooth muscle Na(+)/K(+)-ATPase. Of these, TRP channels and connexins are key endothelial effector targets modulated by EETs. In an integrated manner, endogenous EETs enhance extracellular Ca(2+) influx (thereby amplifying and prolonging KCa-mediated endothelial hyperpolarization) and also facilitate the conduction of this hyperpolarization to spatially remote vessel regions. The contribution of EETs and the receptor and channel subtypes involved in EDH-related microdomain signalling, as a candidate for a universal EDH-mediated vasodilator mechanism, vary with vascular bed, species, development and disease and thus represent potentially selective targets for modulating specific artery function.

Relaxin is a potent vasodilator of small resistance arteries and modifies arterial compliance in some systemic vascular beds, yet receptors for relaxin, such as RXFP1, have only been localized to vascular smooth muscle. This study first aimed to localize RXFP1 in rat arteries and veins from different organ beds and determine whether receptors are present in endothelial cells. We then tested the hypothesis that region-specific vascular effects of relaxin may be influenced by the cellular localization of RXFP1 within different blood vessels. The aorta, vena cava, mesenteric artery, and vein had significantly higher (P<0.05) RXFP1 immunostaining in endothelial cells compared with vascular smooth muscle, whereas the femoral artery and vein and small pulmonary arteries had higher (P<0.01) RXFP1 immunostaining in the vascular smooth muscle. Male rats were treated subcutaneously with recombinant human relaxin-2 (serelaxin; 4 μg/h) for 5 d; vasodilation and compliance in mesenteric and femoral arteries and veins were compared with placebo controls. Serelaxin significantly (P=0.04) reduced wall stiffness and increased volume compliance in mesenteric arteries but not in the other vessels examined. This was associated with changes in geometrical properties, and not compositional changes in the extracellular matrix. Serelaxin treatment had no effect on acetylcholine-mediated relaxation but significantly (P<0.001) enhanced bradykinin (BK)-mediated relaxation in mesenteric arteries, involving enhanced nitric oxide but not endothelium-derived hyperpolarization or vasodilatory prostanoids. In conclusion, there is differential distribution of RXFP1 on endothelial and smooth muscle across the vasculature. In rats, mesenteric arteries exhibit the greatest functional response to chronic serelaxin treatment.

The role of melatonin in the gastrointestinal (GI) tract had previously been limited to its well-described anti-oxidant properties. Recent studies have, however, expanded the role of melatonin in the intestine, showing that it acts as a hormone with local paracrine actions to modulate GI function and the release of other hormones. The GI epithelium produces melatonin from the active precursor serotonin, which is thought to come from the serotonin synthesising enterochromaffin cells (EC). The receptors for melatonin, the membrane bound melatonin receptors 1 and 2, are present on some smooth muscles, neurons, and epithelium. Endogenous release of melatonin has been linked with secretory reflexes and the ileal brake reflex, while exogenous application of melatonin in pharmacological doses has been associated with reduced inflammation in a variety of animal models. Recent studies have begun to look at melatonin release from the GI epithelium using real-time electrochemical detection methods. Using these techniques, the time course of melatonin production shows similarities to that of 5-HT release while the ratio of 5-HT to melatonin is altered during aging. In addition, the effects of melatonin supplementation on the production of endogenous melatonin and its precursor serotonin are suppressed. In summary, the role of melatonin in the GI tract is coming of age. There are many studies showing a clear role for endogenously produced melatonin and clear effects of melatonin supplementation. Newly developed electrochemical techniques for exploring melatonin availability in real-time promise to accelerate our understanding of GI melatonin in the years to come.

In pregnancy, the vasculature of the uterus undergoes rapid remodelling to increase blood flow and maintain perfusion to the fetus. The present study determines the distribution and density of caveolae, transient receptor potential vanilloid type 4 channels (TRPV4) and myoendothelial gap junctions, and the relative contribution of related endothelium-dependent vasodilator components in uterine radial arteries of control virgin non-pregnant and 20-day late-pregnant rats. The hypothesis examined is that specific components of endothelium-dependent vasodilator mechanisms are altered in pregnancy-related uterine radial artery remodelling. Conventional and serial section electron microscopy were used to determine the morphological characteristics of uterine radial arteries from control and pregnant rats. TRPV4 distribution and expression was examined using conventional confocal immunohistochemistry, and the contribution of endothelial TRPV4, nitric oxide (NO) and endothelium-derived hyperpolarization (EDH)-type activity determined using pressure myography with pharmacological intervention. Data show outward hypertrophic remodelling occurs in uterine radial arteries in pregnancy. Further, caveolae density in radial artery endothelium and smooth muscle from pregnant rats was significantly increased by ~94% and ~31%, respectively, compared with control, whereas caveolae density did not differ in endothelium compared with smooth muscle from control. Caveolae density was significantly higher by ~59% on the abluminal compared with the luminal surface of the endothelium in uterine radial artery of pregnant rats but did not differ at those surfaces in control. TRPV4 was present in endothelium and smooth muscle, but not associated with internal elastic lamina hole sites in radial arteries. TRPV4 fluorescence intensity was significantly increased in the endothelium and smooth muscle of radial artery of pregnant compared with control rats by ~2.6- and 5.5-fold, respectively. The TRPV4 signal was significantly higher in the endothelium compared with the smooth muscle in radial artery of both control and pregnant rats, by ~5.7- and 2.7-fold, respectively. Myoendothelial gap junction density was significantly decreased by ~37% in radial artery from pregnant compared with control rats. Pressure myography with pharmacological intervention showed that NO contributes ~80% and ~30%, and the EDH-type component ~20% and ~70% of the total endothelium-dependent vasodilator response in radial arteries of control and pregnant rats, respectively. TRPV4 plays a functional role in radial arteries, with a greater contribution in those from pregnant rats. The correlative association of increased TRPV4 and caveolae density and role of EDH-type activity in uterine radial artery of pregnant rats is suggestive of their causal relationship. The decreased myoendothelial gap junction density and lack of TRPV4 density at such sites is consistent with their having an integral, albeit complex, interactive role in uterine vascular signalling and remodelling in pregnancy., (© 2013 Anatomical Society.)

In pregnancy, α-adrenoceptor-mediated vasoconstriction is augmented in uterine radial arteries and is accompanied by underlying changes in smooth muscle (SM) Ca(2+) activity. This study aims to determine the Ca(2+) entry channels associated with altered vasoconstriction in pregnancy, with the hypothesis that augmented vasoconstriction involves transient receptor potential canonical type-3 (TRPC3) and L- and T-type voltage-dependent Ca(2+) channels. Immunohistochemistry showed TRPC3, L-type Cav1.2 (as the α1C subunit), T-type Cav3.1 (α1G), and Cav3.2 (α1H) localization to the uterine radial artery SM. Fluorescence intensity of TRPC3, Cav1.2, and Cav3.2 was increased, and Cav3.1 decreased in radial artery SM from pregnant rats. Western blot analysis confirmed increased TRPC3 protein expression in the radial artery from pregnant rats. Pressure myography incorporating pharmacological intervention to examine the role of these channels in uterine radial arteries showed an attenuation of phenylephrine (PE)-induced constriction with Pyr3 {1-[4-[(2,3,3-trichloro-1-oxo-2-propen-1-yl)amino]phenyl]-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic acid}-mediated TRPC3 inhibition or with nifedipine-mediated L-type channel block alone in vessels from pregnant rats; both effects of which were diminished in radial arteries from nonpregnant rats. Combined TRPC3 and L-type inhibition attenuated PE-induced constriction in radial arteries, and the residual vasoconstriction was reduced and abolished with T-type channel block with NNC 55-0396 in arteries from nonpregnant and pregnant rats, respectively. With SM Ca(2+) stores depleted and in the presence of PE, nifedipine, and NNC 55-0396, blockade of TRPC3 reversed PE-induced constriction. These data suggest that TRPC3 channels act synergistically with L- and T-type channels to modulate radial artery vasoconstriction, with the mechanism being augmented in pregnancy.