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BackgroundDespite recanalization after ischemic stroke, neurovascular coupling, i.e., the local hyperaemic response to neuronal activity, is impaired in peri-ischemic brain regions. Reduced neurovascular coupling may contribute to neurological deterioration over time. The mechanism underlying dysfunctional neurovascular coupling following stroke is largely unknown.MethodsMice implanted with chronic cranial windows were trained for awake head-fixation prior to 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 labelling lectin and platelet-derived growth factor receptor β.ResultsArterial occlusion induced on average 11 spreading depressions over one 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 3 and 24 hours after reperfusion, which was associated with constriction of an equivalent proportion of peri-ischemic capillary pericytes. The capillaries in the peri-ischemic cortex that remained perfused showed increased prevalence of dynamic flow stalling. Whisker stimulation led to reduced neurovascular coupling responses in the sensory cortex corresponding to the peri-ischemic region 3 and 24 hours after reperfusion.ConclusionArterial occlusion led to constriction of pericytes in the peri-ischemic cortex associated with long-lasting microcirculatory failure. This reduced capillary capacity may, at least in part, underlie impaired neurovascular coupling in peri-ischemic brain regions after stroke and reperfusion.

BACKGROUND AND PURPOSE The physiological role of vascular β3 -adrenoceptors is not fully understood. Recent evidence suggests cardiac β3 -adrenoceptors are functionally effective after down-regulation of β1 /β2 -adrenoceptors. The functional interaction between the β3 -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 β3 -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 β3 -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 β3 -adrenoceptor agonist SR 58611A and the nonselective β-adrenoceptor agonist isoprenaline caused concentration-dependent dilation. In the presence of β1/2 -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 β3 -adrenoceptor antagonist L 748,337 (1 μM) inhibited vasodilation caused by β3 -adrenoceptor agonists (in the presence of β1/2 -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 β3 -adrenoceptor mediated vasodilation was only evident after blockade of β1/2 -adrenoceptors. This suggests constitutive β1/2 -adrenoceptor activity inhibits β3 -adrenoceptor function in the endothelium of skeletal muscle resistance arteries.

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 signalling in this phenomenon. 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 was 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 recognisedin vivoafter whisker stimulation, were significantly attenuated in mice with altered endothelial gap junctional signalling 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. Our findings establish the integral role of intercellular electrical signalling in transcribing focal stimuli into coordinated changes in cerebrovascular contractile activity and expose, a hitherto unknown mechanism for flow regulation after stroke.SignificanceNeurovascular responses are viewed as a one step process whereby stimuli derived from neural cells focally diffuse to a neighboring vessel, altering its contractile state. While focal changes in tone can subtly tune flow distribution, they can’t substantively change “perfusion magnitude” as vascular resistance is broadly distributed along the cerebral arterial tree. We report that nature overcomes this biophysical constraint by conducting electrical signals among coupled vascular cells, along vessels, and across branch points. Our quantitative exploration of intercellular conduction illustrates how network coordination optimizes blood flow delivery in support of brain function. Diminishing the ability of vascular cells to electrically communicate, mitigates the brain’s ability to regulate perfusion during functional hyperemia and after stroke, the latter advancing tissue injury.

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.

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 Ca2+ signals have been identified in discrete microdomains of vascular endothelial cells. At myoendothelial contacts (between endothelial and smooth muscle cells), both endothelial Ca2+ pulsars (IP3-mediated release of intracellular Ca2+) and Ca2+ sparklets (extracellular Ca2+ entry via TRP channels) contribute to endothelium-dependent hyperpolarization of smooth muscle, vasodilation, and feedback control of vasoconstriction. Ca2+ 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 Ca2+ 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 Ca2+ signals.

The physiological role of vascular βStudies 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 βAll three β-adrenoceptor subtypes were present in the endothelium of the cremaster muscle artery. The βAll three β-adrenoceptor subtypes were present in the endothelium of the rat cremaster muscle artery, but β

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 kV7 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 kV7 channels in ECs would add an extra level of vascular control. This study aims to characterize the expression and function of KV7 channels within rat mesenteric artery ECs.Experimental Approach: In rat mesenteric artery, KCNQ transcript and KV7 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. KV7.1, KV7.4 and KV7.5 protein expression was determined in both isolated EC and VSMC and in whole vessels. Removal of ECs attenuated vasorelaxation to two structurally different KV7.2-5 activators S-1 and ML213. KIR2 blockers ML133, and BaCl2 also attenuated S-1 or ML213-mediated vasorelaxation in an endothelium-dependent process. KV7 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, KV7.4 and KV7.5 channels are expressed, functionally interact with endothelial KIR2.x channels and contribute to endogenous eNOS-mediated relaxation. This study identifies KV7 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.

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 kV7 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 kV7 channels in ECs would add an extra level of vascular control. This study aims to characterize the expression and function of KV7 channels within rat mesenteric artery ECs. Experimental Approach: In rat mesenteric artery, KCNQ transcript and KV7 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. KV7.1, KV7.4 and KV7.5 protein expression was determined in both isolated EC and VSMC and in whole vessels. Removal of ECs attenuated vasorelaxation to two structurally different KV7.2-5 activators S-1 and ML213. KIR2 blockers ML133, and BaCl2 also attenuated S-1 or ML213-mediated vasorelaxation in an endothelium-dependent process. KV7 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, KV7.4 and KV7.5 channels are expressed, functionally interact with endothelial KIR2.x channels and contribute to endogenous eNOS-mediated relaxation. This study identifies KV7 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.

Background and purpose KCNQ-encoded KV7 channels are expressed within vascular smooth muscle cells (VSMCs) and are key regulators of vascular reactivity, regulating resting tone and as functional targets of endogenous responses. Endothelial cells (ECs) form a paracrine signaling platform that line all blood vessels and regulate tone, but little is known of KV7 channels in vascular ECs. This study aims to characterize the expression and function of KV7 channels within rat mesenteric artery ECs. Experimental approach In rat mesenteric artery, KCNQ transcript and KV7 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 EC marker expressing cells using a reductive approach. KV7.4 and KV7.5 protein expression was determined in both isolated EC and VSMC and in whole vessels. Removal of ECs attenuated vasorelaxation to two structurally different KV7.2-5 activators S-1 and ML213. KIR2 blockers ML133 and BaCl also attenuated S-1 or ML213-mediated vasorelaxation in an endothelium-dependent process. KV7 inhibition attenuated receptor-dependent nitric oxide (NO)-mediated vasorelaxation to carbachol, but had no impact on relaxation to the NO donor, SNP. Conclusions and implications In rat mesenteric artery ECs, KV7.4 and KV7.5 channels are expressed, functionally interact with endothelial KIR2.x channels and contribute to endogenous eNOS-mediated relaxation. This study identifies KV7 channels as novel functional channels within rat mesenteric ECs and suggests that these channels are involved in NO release from the endothelium.

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.

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 Ca2+and membrane potential were measured simultaneously. Protein expression was semi-quantified by immunohistochemistry. Protein phosphorylation was analysed by Western blot. MSA from Atp1a2+/−G301Rand WT showed similar contractile responses. The Atp1a2+/−G301RMCA constricted stronger to U46619, endothelin and potassium compared to WT. This was associated with an increased depolarization, although the Ca2+change was smaller than in WT. The enhanced constriction of Atp1a2+/−G301RMCA was associated with increased cSrc activation, stronger sensitization to [Ca2+]iand increased MYPT1 phosphorylation. These differences were abolished by cSrc inhibition. Atp1a2+/−G301Rmice 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 Ca2+, which is mediated via Na,K-ATPase/Src-kinase/MYPT1 signalling.

Objective- Inward rectifying K

Localized, oscillating Ca

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.

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.

Myocardial perfusion and coronary vascular resistance are regulated by signalling metabolites released from the local myocardium that act either directly on the vascular smooth muscle cells (VSMC) or indirectly via stimulation of the endothelium. A prominent mechanism of vasodilation is endothelium-derived hyperpolarization (EDH) of the arteriolar smooth muscle, with epoxyeicosatrienoic acids (EETs) and hydrogen peroxide (H2O2) playing important roles in EDH in the coronary microcirculation. In some cases, EETs and H2O2 are released as transferable hyperpolarizing factors (EDHFs) that act directly on the VSMCs. By contrast, EETs and H2O2 can also promote endothelial Ca2+-activated K+ channel activity secondary to the amplification of extracellular Ca2+ influx and Ca2+ 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 signalling involving EETs and H2O2 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 H2O2 regulate vessel tone, and also examines the hypothesis that myoendothelial microdomain signalling facilitates EDH activity in the human heart.

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.

Inward rectifying (KIR) K+channels are present in cerebral arterial smooth muscle and endothelial cells, a tandem arrangement suggestive of a dynamic yet undiscovered role for this channel. We explored whether vascular KIRchannels were uniquely modulated by membrane lipids and hemodynamic stimuli. A KIRcurrent was isolated in smooth muscle and endothelial cells of rat cerebral arteries and molecular analyses confirmed KIR2.1/KIR2.2 mRNA and protein expression. Electrophysiology next revealed that endothelial KIRwas sensitive to phosphatidylinositol 4,5- bisphosphate (PIP2), with depletion impairing flow-induced activation of the channel. In contrast, smooth muscle KIRwas sensitive to membrane cholesterol, with sequestration blocking pressure’s ability to inhibit this channel. Membrane lipids helped confer KIRmechanosensitivity to intact arteries; virtual models then reconceptualised KIRas a dynamic regulator of basal tone development. We conclude that specific membrane lipid-KIRinteractions enable unique channel populations to sense hemodynamic stimuli and set brain perfusion.

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 Ca2+. 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 [Ca2+]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 Ca2+, 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 Ca2+ 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 Ca2+ sensitization of vascular smooth muscle cells by MYPT1 phosphorylation. This mechanism may be critical for acute control of vascular tone. © 2018 Scandinavian Physiological Society.

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 10Panx1 (100 μM) and P2X7R antagonist A438079 (100 μM). The effects of 10Panx1 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 (10Panx1, A438079, or 10Panx1 + 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 10Panx1 (P = 0.04). Likewise, 10Panx1 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.

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.

Key points Electrical signalling in blood vessels is important in acute and long-term flow regulation. As myoendothelial gap junctions (MEGJs) provide electrical contacts between endothelial cells and smooth muscle cells (SMCs), a high MEGJ expression is thought to be important for intercellular communication despite observations of heterogeneous MEGJ expression. Using a computational approach, we find that a minimal but sufficient number of heterogeneously distributed MEGJs optimize electrical cell–cell communication. Tailoring SMC–SMC coupling to the MEGJ distribution and permeability ensures a smooth electrical response while retaining the ability to display local responses. As heterogeneous MEGJ distributions allow for different conduction profiles along the SMC and endothelial cell layers, it is possible to observe an apparently non-decaying conduction profile in the SMC layer of a vessel with passive electrical properties. These findings expand our understanding of how heterogeneity in MEGJ distribution affects local and global flow. Abstract 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.

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.

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 mmH2O; decanoic acid: 27 ± 2–14 ± 2 mmH2O; and l-phenylalanine: 30 ± 3–14 ± 3mmH2O. 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.

In pregnancy, α-adrenoceptor-mediated vasoconstriction is augmented in uterine radial arteries and is accompanied by underlying changes in smooth muscle (SM) Ca2+ activity. This study aims to determine the Ca2+ 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 Ca2+ 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)-1 H-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 Ca2+ 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.

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 hyperpolari- zation (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 vaso- dilator mechanisms. Indeed, the transfer of hyperpolarization initiated in the endothelium to the adjacent smooth muscle via gap junction connexins occurs separately or synergisti- cally 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 compo- nents of the microdomain signalling complex are the inositol 1,4,5-trisphosphate receptor-mediated endoplasmic reticu- lum 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, endog- enous EETs enhance extracellular Ca 2+ influx (thereby amplifying and prolonging KCa-mediated endothelial hyper- polarization) and also facilitate the conduction of this hyper- polarization to spatially remote vessel regions. The contri- bution of EETs and the receptor and channel subtypes in- volved in EDH-related microdomain signalling, as a candi- date for a universal EDH-mediated vasodilator mechanism, vary with vascular bed, species, development and disease and thus represent potentially selective targets for modulat- ing specific artery function.

Regulation of blood flow in microcirculatory networks depends on spread of local vasodilatation to encompass upstream arteries; a process mediated by endothelial conduction of hyperpolarization. Given that endothelial coupling is reduced in hypertension, we used hypertensive Cx40ko mice, in which endothelial coupling is attenuated, to investigate the contribution of the renin-angiotensin system and reduced endothelial cell coupling to conducted vasodilatation of cremaster arterioles in vivo. When the endothelium was disrupted by light dye treatment, conducted vasodilatation, following ionophoresis of acetylcholine, was abolished beyond the site of endothelial damage. In the absence of Cx40, sparse immunohistochemical staining was found for Cx37 in the endothelium, and endothelial, myoendothelial and smooth muscle gap junctions were identified by electron microscopy. Hyperpolarization decayed more rapidly in arterioles from Cx40ko than wild-type mice. This was accompanied by a shift in the threshold potential defining the linear relationship between voltage and diameter, increased T-type calcium channel expression and increased contribution of T-type (3 μmol l(-1) NNC 55-0396), relative to L-type (1 μmol l(-1) nifedipine), channels to vascular tone. The change in electromechanical coupling was reversed by inhibition of the renin-angiotensin system (candesartan, 1.0 mg kg(-1) day(-1) for 2 weeks) or by acute treatment with the superoxide scavenger tempol (1 mmol l(-1)). Candesartan and tempol treatments also significantly improved conducted vasodilatation. We conclude that conducted vasodilatation in Cx40ko mice requires the endothelium, and attenuation results from both a reduction in endothelial coupling and an angiotensin II-induced increase in oxidative stress. We suggest that during cardiovascular disease, the ability of microvascular networks to maintain tissue integrity may be compromised due to oxidative stress-induced changes in electromechanical coupling.

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.

OBJECTIVE: The authors probed endothelium-dependent dilation and endothelial cell Ca2+ handling in myogenically active resistance arteries. METHODS: First-order arteries were removed from rat cremaster muscles, cannulated, and pressurized (75 mmHg). Vessel diameter and endothelial cell Ca2+ were monitored using confocal microscopy, and arterial ultrastructure was determined using electron microscopy. RESULTS: Acetylcholine (ACh) stimulated elevations and oscillations in endothelial cell Ca2+, and concentration-dependently dilated arteries with myogenic tone. NO-independent dilation was blocked by 35 mM K+. Combined IK(Ca) (1 microM TRAM-34) and SK(Ca) (100 nM apamin) blockade partially inhibited NO-independent relaxations, with residual relaxations sensitive to BK(Ca) or cytochrome P-450 inhibition (100 nM iberiotoxin, and 20 microM 17-ODYA or 10 microM MS-PPOH). 11,12-EET stimulated iberiotoxin-sensitive dilation, but did not affect endothelial cell Ca2+. 15 mM K+ evoked dilation sensitive to inhibition of K(IR) (30 microM Ba2+) and Na+/K+-ATPase (10 microM ouabain), whereas these blockers did not affect ACh-mediated dilations. Homo- and heterocellular gap junctions were identified in radial sections through arteries. CONCLUSION: These data suggest that rises in endothelial cell Ca2+ stimulate SK(Ca) and IK(Ca) channels, leading to hyperpolarization and dilation, likely due to electrical coupling. In addition, a component was unmasked following SK(Ca) and IK(Ca) blockade, attributable to activation of BK(Ca) channels by cytochrome P-450 metabolites.

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.

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.

Diet-induced obesity induces changes in mechanisms that are essential for the regulation of normal artery function, and in particular the function of the vascular endothelium. Using a rodent model that reflects the characteristics of human dietary obesity, in the rat saphenous artery we have previously demonstrated that endothelium-dependent vasodilation shifts from an entirely nitric oxide (NO)-mediated mechanism to one involving upregulation of myoendothelial gap junctions and intermediate conductance calcium-activated potassium channel activity and expression. This study investigates the changes in NO-mediated mechanisms that accompany this shift. In saphenous arteries from controls fed a normal chow diet, acetylcholine-mediated endothelium-dependent vasodilation was blocked by NO synthase and soluble guanylyl cyclase inhibitors, but in equivalent arteries from obese animals sensitivity to these agents was reduced. The expression of endothelial NO synthase (eNOS) and caveolin-3 in rat saphenous arteries was unaffected by obesity, whilst that of caveolin-1 monomer and large oligomeric complexes of caveolins-1 and -2 were increased in membrane-enriched samples. The density of caveolae was increased at the membrane and cytoplasm of endothelial and smooth muscle cells of saphenous arteries from obese rats. Dissociation of eNOS from caveolin-1, as a prerequisite for activation of the enzyme, may be compromised and thereby impair NO-mediated vasodilation in the saphenous artery from diet-induced obese rats. Such altered signaling mechanisms in obesity-related vascular disease represent significant potential targets for therapeutic intervention.

Constipation and slowed transit are associated with diet-induced obesity, although the mechanisms by which this occurs are unclear. Enterochromaffin (EC) cells within the intestinal epithelium respond to mechanical stimulation with the release of serotonin [5-hydroxytryptamine (5-HT)], which promotes transit. Thus our aim was to characterize 5-HT availability in the rat colon of a physiologically relevant model of diet-induced obesity. EC cell numbers were determined immunohistochemically in chow-fed (CF) and Western diet-fed (WD) rats, while electrochemical methods were used to measure mechanically evoked (peak) and steady-state (SS) 5-HT levels. Fluoxetine was used to block the 5-HT reuptake transporter (SERT), and the levels of mRNA for tryptophan hydroxylase 1 and SERT were determined by quantitative PCR, and SERT protein was determined by Western blot. In WD rats, there was a significant decrease in the total number of EC cells per crypt (0.86 ± 0.06 and 0.71 ± 0.05 in CF and WD, respectively), which was supported by a reduction in the levels of 5-HT in WD rats (2.9 ± 1.0 and 10.5 ± 2.6 μM at SS and peak, respectively) compared with CF rats (7.3 ± 0.4 and 18.4 ± 3.4 μM at SS and peak, respectively). SERT-dependent uptake of 5-HT was unchanged, which was supported by a lack of change in SERT protein levels. In WD rats, there was no change in tryptophan hydroxylase 1 mRNA but an increase in SERT mRNA. In conclusion, our data show that foods typical of a WD are associated with decreased 5-HT availability in rat colon. Decreased 5-HT availability is driven primarily by a reduction in the numbers and/or 5-HT content of EC cells, which are likely to be associated with decreased intestinal motility in vivo. © 2012 the American Physiological Society.

In several species and in many vascular beds, ultrastructural studies describe close contact sites between the endothelium and smooth muscle of

Blood flow is adjusted to tissue demand through rapidly ascending vasodilatations resulting from conduction of hyperpolarisation through vascular gap junctions. We investigated how these dilatations can spread without attenuation if mediated by an electrical signal. Cremaster muscle arterioles were studied in vivo by simultaneously measuring membrane potential and vessel diameter. Focal application of acetylcholine elicited hyperpolarisations which decayed passively with distance from the local site,while dilatation spread upstream without attenuation. Analysis of simultaneous recordings at the local site revealed that hyperpolarisation and dilatation were only linearly related over a restricted voltage range to a threshold potential, beyond which dilatation was maximal. Experimental data could be simulated in a computational model with electrotonic decay of hyperpolarisation but imposition of this threshold. The model was tested by reducing the amplitude of the local hyperpolarisation which led to entry into the linear range closer to the local site and decay of dilatation. Serial section electron microscopy and light dye treatment confirmed that the spread of dilatation occurred through the endothelium and that the two cell layers were tightly coupled. Generality of the mechanism was demonstrated by applying the model to the attenuated propagation of dilatation found in larger arteries.We conclude that long distance spread of locally initiated dilatations is not due to a regenerative electrical phenomenon, but rather a restricted linear relationship between voltage and vessel tone, which minimises the impact of electrotonic decay of voltage. Disease-related alterations in endothelial coupling or ion channel expression could therefore decrease the ability to adjust blood flow to meet metabolic demand.

Myoendothelial microdomain signaling via localized calcium-activated potassium channel (K(Ca)) and gap junction connexins (Cx) is critical for endothelium-dependent vasodilation in rat mesenteric artery. The present study determines the relative contribution of NO and gap junction-K(Ca) mediated microdomain signaling to endothelium-dependent vasodilation in human mesenteric artery. The hypothesis tested was that such activity is due to NO and localized K(Ca) and Cx activity. In mesenteric arteries from intestinal surgery patients, endothelium-dependent vasodilation was characterized using pressure myography with pharmacological intervention. Vessel morphology was examined using immunohistochemical and ultrastructural techniques. In vessel segments at 80 mm Hg, the intermediate (I)K(Ca) blocker 1-[(2-chlorophenyl)diphenyl-methyl]-1H-pyrazole (TRAM-34; 1 μM) inhibited bradykinin (0.1 nM-3 μM)-induced vasodilation, whereas the small (S) K(Ca) blocker apamin (50 and 100 nM) had no effect. Direct IK(Ca) activation with 1-ethyl-2-benzimidazolinone (1-EBIO; 10-300 μM) induced vasodilation, whereas cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (1-30 μM), the SK(Ca) activator, failed to dilate arteries, whereas dilation induced by 1-EBIO (10-100 μM) was blocked by TRAM-34. Bradykinin-mediated vasodilation was attenuated by putative gap junction block with carbenoxolone (100 μM), with remaining dilation blocked by N-nitro l-arginine methyl ester (100 μM) and [1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one] (10 μM), NO synthase and soluble guanylate cyclase blockers, respectively. In human mesenteric artery, myoendothelial gap junction and IK(Ca) activity are consistent with Cx37 and IK(Ca) microdomain expression and distribution. Data suggest that endothelium-dependent vasodilation is primarily mediated by NO, IK(Ca), and gap junction Cx37 in this vessel. Myoendothelial microdomain signaling sites are present in human mesenteric artery and are likely to contribute to endothelium-dependent vasodilation via a mechanism that is conserved between species.

The potential physiological role of plasmalemmal large-conductance calcium-activated potassium channels (BKCa) in vascular endothelial cells is controversial. Studies of freshly isolated and cultured vascular endothelial cells provide disparate results, both supporting and refuting a role for BKCain endothelial function. Most studies using freshly isolated, intact, healthy arteries provide little support for a physiological role for BKCain the endothelium, although recent work suggests that this may not be the case in diseased vessels. In isolated and cultured vascular endothelial cells, the autocrine action of growth factors, hormones, and vasoactive substances results in phenotypic drift. Such an induced heterogeneity is likely a primary factor accounting for the apparent differences, and often enhanced BKCaexpression and function, in isolated and cultured vascular endothelial cells. In a similar manner, heterogeneity in endothelial BKCaexpression and function in intact arteries may be representative of normal and disease states, BKCabeing absent in normal intact artery endothelium and upregulated in disease where dysfunction induces signals that alter channel expression and function. Indeed, in some intact vessels, there is evidence for the presence of BKCa, such as mRNA and/or specific BK subunits, an observation that is consistent with the potential for rapid upregulation, as may occur in disease. This perspective proposes that the disparity in the results obtained for BKCaexpression and function from freshly isolated and cultured vascular endothelial cells is largely due to variability in experimental conditions and, furthermore, that the expression of BKCain intact artery endothelium is primarily associated with disease. Although answers to physiologically relevant questions may only be available in atypical physiological conditions, such as those of isolation and culture, the limitations of these methods require open and objective recognition.