Membrane Potential Of Smooth Muscle Cells Research Articles

Impaired intracellular Ca2+ signaling contributes to age-related cerebral small vessel disease in Col4a1 mutant mice.

Humans and mice with mutations in COL4A1 and COL4A2 manifest hallmarks of cerebral small vessel disease (cSVD). Mice with a missense mutation in Col4a1 at amino acid 1344 (Col4a1+/G1344D) exhibit age-dependent intracerebral hemorrhages (ICHs) and brain lesions. Here, we report that this pathology was associated with the loss of myogenic vasoconstriction, an intrinsic vascular response essential for the autoregulation of cerebral blood flow. Electrophysiological analyses showed that the loss of myogenic constriction resulted from blunted pressure-induced smooth muscle cell (SMC) membrane depolarization. Furthermore, we found that dysregulation of membrane potential was associated with impaired Ca2+-dependent activation of large-conductance Ca2+-activated K+ (BK) and transient receptor potential melastatin 4 (TRPM4) cation channels linked to disruptions in sarcoplasmic reticulum (SR) Ca2+ signaling. Col4a1 mutations impair protein folding, which can cause SR stress. Treating Col4a1+/G1344D mice with 4-phenylbutyrate, a compound that promotes the trafficking of misfolded proteins and alleviates SR stress, restored SR Ca2+ signaling, maintained BK and TRPM4 channel activity, prevented loss of myogenic tone, and reduced ICHs. We conclude that alterations in SR Ca2+ handling that impair ion channel activity result in dysregulation of SMC membrane potential and loss of myogenic tone and contribute to age-related cSVD in Col4a1+/G1344D mice.

Mechanisms of K ATP channel Activation by Metabolic Stress in Mediating Lymphatic Contractile Dysfunction

Lymphatic contractile dysfunction has been implicated in the sub-clinical edema observed in multiple disease states, including obesity, metabolic syndrome and inflammatory disease. The strong positive correlation between the upregulation or hyperactivation of KATP channels in lymphatic muscle and lymphatic contractile dysfunction suggests that KATP channel activation be a common cause of the contractile dysfunction. We investigated the role of KATP channels in lymphatic muscle in response to decreases in the intracellular ATP/ADP ratio and the increased generation of reactive oxygen species (ROS), both of which occur in metabolic diseases. We analyzed spontaneous lymphatic contractions and smooth muscle cell membrane potentials in isolated, pressurized popliteal lymphatic vessels from mice. Exposure of lymphatic vessels from WT mice to the mitochondrial complex inhibitor (antimycin, 30 nM) or the oxidative phosphorylation inhibitor (CCCP, 1 µM) led to an attenuated or complete block of lymphatic pumping, which was rescued by the KATP channel inhibitor glibenclamide (GLIB, 1 µM). In contrast, lymphatic vessels from Kir6.1-/- mice were resistant to antimycin and CCCP. Antimycin inhibited spontaneous action potentials in lymphatic muscle from WT vessels and treatment of GLIB rescued action potential generation. Antimycin increased DHR fluorescence in WT lymphatic vessels, confirming the production of ROS. The ROS scavengers, tiron (1 mM) and catalase (250 U/ml) blocked the increase in DHR fluorescence to antimycin. After pretreatment with tiron (1 mM), WT lymphatic vessels were resistant to the effects of antimycin, and there were no further changes in response to GLIB, suggesting that KATP channels were activated by ROS production. Similar results were found for catalase (250 U/ml) treatment. However, neither tiron nor catalase blocked the inhibitory effect of CCCP on lymphatic pumping. Based on these results, we propose that KATP channels in lymphatic muscle are activated by directly by metabolic stress or indirectly by ROS generation, leading to the inhibition of spontaneous lymphatic contractions and lymphatic contractile dysfunction.

A sex-specific, COX-derived/thromboxane receptor activator causes depolarization and vasoconstriction in male mice mesenteric resistance arteries.

We investigated whether sex differences exist in cyclooxygenase-dependent effects on membrane potential and relaxation in mice mesenteric resistance arteries. Mesenteric small arteries of 9- to 12-week-old, male and female, wild-type mice, db/+ mice and diabetic db/db mice were mounted in myographs for measurements of isobaric diameter and smooth muscle cell membrane potential. Acetylcholine caused smaller dilation of arteries from male db/+ mice compared to arteries from female db/+ mice. In the presence of the NO synthase inhibitor Nω -nitro-L-arginine methyl ester (L-NAME), acetylcholine-induced dilation of arteries from males increased in the presence of indomethacin and the COX-1-specific inhibitor FR122047. The presence of indomethacin was also associated with a more negative membrane potential in arteries from males. In arteries from db/db mice, no sex differences were seen. In arteries from male but not female wild-type mice, the thromboxane receptor blocker SQ29,548 increased relaxation to acetylcholine. In contrast to arteries from female mice, COX (most likely COX-1)-derived prostanoids and activation of thromboxane receptors counteract acetylcholine vasodilatation probably through increased smooth muscle depolarization in arteries from male mice. In mice with diabetes and pronounced endothelial dysfunction, inhibition of COX did not enhance acetylcholine vasodilatation.

Abstract TP404: A Novel Adipokine DPPIV Induces Inflammation and Apoptosis of Carotid Artery Smooth Muscle Cells: Implications for Atherosclerotic Plaque Instability

Background: Carotid Atherosclerotic (CA) plaque rupture is associated with ischemic stroke. Smooth muscle cells (SMC) play key role in plaque development. SMC also produce collagen fibers and maintain the fibrous cap thickness and strength, and SMC apoptosis and inflammation can lead to plaque rupture. Molecular mechanisms leading to plaque instability and stroke are not completely understood. Our study has shown that an adipokine dipeptidyl peptidase (DPPIV) is significantly upregulated in post-stroke rat brain, and in the plasma of over 75% of CA patients, suggesting a role for DPPIV in CA and stroke. In this study, we examined the effects of DPPIV on SMC viability, apoptosis, and identified responsible signaling pathways. Methods: Mouse carotid artery SMC were exposed to oxygen glucose deprivation (OGD). Cells were treated with either physiological (200-500 ng/ml) or high (1000-2000 ng/ml) dose of DPPIV. Using transcriptome array, we examined the expression profile of apoptotic and inflammatory pathway specific genes in these cells. DPPIV levels were examined by PCR and immunofluorescence staining. MTT assay was used to assess cell viability. Live/dead cell assay detected apoptosis. Mitochondrial membrane potential was detected using tetra-methyl-rhodamine ethyl ester (TMRE). Results: Ischemia/hypoxia upregulates DPPIV mRNA and protein levels in association with increased pro-apoptotic genes including caspase 7, Nod1, and Gadd45a. Transcriptome array data demonstrated that higher dose of exogenous DPPIV results in significant increase in same pro-apoptotic pathway genes including caspase 3,7, Nod1, Trp63, and Gadd45a. On the other hand, DPPIV decreased the expression of anti-apoptotic genes including Atf5 and Birc3. Levels of pro-inflammatory cytokines including IL1-beta and Ccl26/Eotaxin were increased in both DPPIV and ischemic SMCs. Higher dose of DPPIV increased apoptosis, decreased viability and mitochondrial membrane potential in SMCs. Conclusion: Higher levels of DPP4 activates the inflammatory and apoptotic signaling cascade resulting in the apoptosis of SMC. Further research in this area will enhance our understanding of the mechanism of CA progression and may provide clues to efficient therapeutic intervention for CA.

Inhibition of vascular smooth muscle inward-rectifier K+ channels restores myogenic tone in mouse urinary bladder arterioles.

Prolonged decreases in urinary bladder blood flow are linked to overactive and underactive bladder pathologies. However, the mechanisms regulating bladder vascular reactivity are largely unknown. To investigate these mechanisms, we examined myogenic and vasoactive properties of mouse bladder feed arterioles (BFAs). Unlike similar-sized arterioles from other vascular beds, BFAs failed to constrict in response to increases in intraluminal pressure (5-80 mmHg). Consistent with this lack of myogenic tone, arteriolar smooth muscle cell membrane potential was hyperpolarized (-72.8 ± 1.4 mV) at 20 mmHg and unaffected by increasing pressure to 80 mmHg (-74.3 ± 2.2 mV). In contrast, BFAs constricted to the thromboxane analog U-46619 (100 nM), the adrenergic agonist phenylephrine (10 µM), and KCl (60 mM). Inhibition of nitric oxide synthase or intermediate- and small-conductance Ca2+-activated K+ channels did not alter arteriolar diameter, indicating that the dilated state of BFAs is not attributable to overactive endothelium-dependent dilatory influences. Myocytes isolated from BFAs exhibited BaCl2 (100 µM)-sensitive K+ currents consistent with strong inward-rectifier K+ (KIR) channels. Notably, block of these KIR channels "restored" pressure-induced constriction and membrane depolarization. This suggests that these channels, in part, account for hyperpolarization and associated absence of tone in BFAs. Furthermore, smooth muscle-specific knockout of KIR2.1 caused significant myogenic tone to develop at physiological pressures. This suggests that 1) the regulation of vascular tone in the bladder is independent of pressure, insofar as pressure-induced depolarizing conductances cannot overcome KIR2.1-mediated hyperpolarization; and 2) maintenance of bladder blood flow during bladder filling is likely controlled by neurohumoral influences.

Inhibition Of Inward‐Rectifier K+ Channels In Vascular Smooth Muscle Restores Myogenic Tone Development In Mouse Bladder Arterioles

The urinary bladder is highly vascularized, and chronic decreases in blood flow are linked to bladder dysfunction. While increases in intraluminal pressure constrict most arterioles (“myogenic tone”), mouse bladder arterioles do not exhibit a myogenic response to pressure. We examined the electrophysiological and vasoactive properties of mouse bladder feed arterioles to determine why myogenic tone is absent. In isolated pressurized bladder arterioles, smooth muscle cell membrane potential was hyperpolarized at 20 mmHg (−72.8±1.4 mV), and did not change significantly when intraluminal pressure was increased to 80 mmHg (−74.3±2.2 mV). However, increasing extracellular K+ from 5.9 mM to 60 mM caused a significant depolarization (−22.7±0.2 mV) at 80 mmHg, which was accompanied by vessel constriction. Given that inward‐rectifier K+ (KIR) channels can have a strong influence on membrane potential in smooth muscle cells, membrane potential was again measured in the presence or absence of the KIR channel blocker BaCl2 (100 μM). In the absence of BaCl2, smooth muscle cell membrane potential remained hyperpolarized at both 20 to 80 mmHg (−70.4±1.9 mV and −72.2±3.1 mV, respectively). In the presence of BaCl2, membrane potential depolarized significantly at both 20 mmHg (−56.2±3.1 mV) and 80 mmHg (−43.3±2.0 mV). To determine if KIR channel inhibition could restore myogenic tone, diameter of isolated pressurized bladder arterioles was measured at increasing pressures (10–80 mmHg) in the presence and absence of 100 μM BaCl2. BaCl2 had no effect on vessel diameter at 20 and 40 mmHg, but resulted in the development of significant myogenic tone at both 60 and 80 mmHg (25.5±9.1% and 41.5±3.9% constriction, respectively). These data support the concept that, in urinary bladder arterioles, KIR channel conductance cannot be overcome by pressure‐induced depolarizing conductances, and thus smooth muscle cell membrane potential remains hyperpolarized and the arterioles do not constrict as intraluminal pressure increases. This represents a novel adaptation to maintain blood flow into the bladder during filling.Support or Funding InformationSupported by NIH R37DK053832 and K01DK103840.

Sympathetic denervation facilitates L-type Ca2+ channel activation in renal but not in mesenteric resistance arteries.

Sympathetic denervation enhances agonist-induced vasoconstriction. This effect may involve altered function of signaling mechanisms such as Rho kinase (Rock) and L-type Ca channels downstream from vasoconstrictor receptors. We tested if enhanced Rock and L-type calcium channel activation contribute to exaggerated norepinephrine-induced vasoconstrictions in renal and mesenteric resistance arteries after sympathectomy. Rats underwent neonatal sympathectomy or sham sympathectomy. Resistance arteries were investigated by small vessel myography. Vascular Rock and L-type Ca channel expression as well as Rock activation were investigated by quantitative real-time PCR and Western blot. Vascular smooth muscle cell (VSMC) membrane potential was recorded with microelectrodes. Sympathetic denervation enhanced norepinephrine sensitivity in renal and mesenteric arteries. Both, Rock inhibition or L-type Ca inhibition shifted the norepinephrine concentration-response curve to the right. This effect was more pronounced in renal than in mesenteric arteries from sympathectomized vs. sham-sympathectomized animals. The L-type Ca channel activator S-(-)-BayK8644 elicited strong vasoconstrictions only in renal arteries from sympathectomized rats. Rock activity and L-type Ca channel α-subunit expression were similar in renal arteries from sympathectomized and sham-sympathectomized animals. VSMC membrane potential was -57.5 ± 2.0 and -64.3 ± 0.3 mV (P < 0.01), respectively, in renal arteries from sympathectomized and from sham-sympathectomized rats. Depolarization enhanced and KATP channel activation abolished S-(-)-BayK8644-induced contractions in renal arteries from sympathectomized rats. Sympathetic denervation enhances L-type Ca channel-dependent signaling in renal but not in mesenteric arteries. This effect may be partly explained by the decreased VSMC membrane potential in denervated renal arteries.

Caveolar disruption causes contraction of rat femoral arteries via reduced basal NO release and subsequent closure of BKCa channels.

Background and Purpose. Caveolae act as signalling hubs in endothelial and smooth muscle cells. Caveolar disruption by the membrane cholesterol depleting agent methyl-β-cyclodextrin (M-β-CD) has various functional effects on arteries including (i) impairment of endothelium-dependent relaxation, and (ii) alteration of smooth muscle cell (SMC) contraction independently of the endothelium. The aim of this study was to explore the effects of M-β-CD on rat femoral arteries.Methods. Isometric force was measured in rat femoral arteries stimulated to contract with a solution containing 20 mM K+ and 200 nM Bay K 8644 (20 K/Bay K) or with one containing 80 mM K+(80 K).Results. Incubation of arteries with M-β-CD (5 mM, 60 min) increased force in response to 20 K/Bay K but not that induced by 80 K. Application of cholesterol saturated M-β-CD (Ch-MCD, 5 mM, 50 min) reversed the effects of M-β-CD. After mechanical removal of endothelial cells M-β-CD caused only a small enhancement of contractions to 20 K/Bay K. This result suggests M-β-CD acts via altering release of an endothelial-derived vasodilator or vasoconstrictor. When nitric oxide synthase was blocked by pre-incubation of arteries with L-NAME (250 µM) the contraction of arteries to 20 K/Bay K was enhanced, and this effect was abolished by pre-treatment with M-β-CD. This suggests M-β-CD is inhibiting endothelial NO release. Inhibition of large conductance voltage- and Ca2+-activated (BKCa) channels with 2 mM TEA+ or 100 nM Iberiotoxin (IbTX) enhanced 20 K/Bay K contractions. L-NAME attenuated the contractile effect of IbTX, as did endothelial removal.Conclusions. Our results suggest caveolar disruption results in decreased release of endothelial-derived nitric oxide in rat femoral artery, resulting in a reduced contribution of BKCa channels to the smooth muscle cell membrane potential, causing depolarisation and contraction.

CO and CO‐releasing molecules (CO‐RMs) in acute gastrointestinal inflammation

Carbon monoxide (CO) is enzymatically generated in mammalian cells alongside the liberation of iron and the production of biliverdin and bilirubin. This occurs during the degradation of haem by haem oxygenase (HO) enzymes, a class of ubiquitous proteins consisting of constitutive and inducible isoforms. The constitutive HO2 is present in the gastrointestinal tract in neurons and interstitial cells of Cajal and CO released from these cells might contribute to intestinal inhibitory neurotransmission and/or to the control of intestinal smooth muscle cell membrane potential. On the other hand, increased expression of the inducible HO1 is now recognized as a beneficial response to oxidative stress and inflammation. Among the products of haem metabolism, CO appears to contribute primarily to the antioxidant and anti-inflammatory effects of the HO1 pathway explaining the studies conducted to exploit CO as a possible therapeutic agent. This article reviews the effects and, as far as known today, the mechanism(s) of action of CO administered either as CO gas or via CO-releasing molecules in acute gastrointestinal inflammation. We provide here a comprehensive overview on the effect of CO in experimental in vivo models of post-operative ileus, intestinal injury during sepsis and necrotizing enterocolitis. In addition, we will analyse the in vitro data obtained so far on the effect of CO on intestinal epithelial cell lines exposed to cytokines, considering the important role of the intestinal mucosa in the pathology of gastrointestinal inflammation.

A synthetic transmembrane segment derived from TRPV4 channel self-assembles into potassium-like channels to regulate vascular smooth muscle cell membrane potential.

Synthetic ion channels represent a new approach to mimicking natural ion channels and developing therapeutic drugs to restore ion channel dysfunction. The large superfamily of transient receptor potential (TRP) channels involved in numerous biological processes is an important and potent therapeutic target for various human diseases. In the present study, a synthetic peptide whose sequence is from the fourth transmembrane segment of TRPV4 is found that is capable of self-assembling into potassium (K+)-like ion channels designated as TRP-PK1 in the membranes of liposomes and live cells. TRP-PK1 effectively mediates K+ flow across the cell membrane to regulate the membrane potential. TRP-PK1 is also able to relax agonist-induced vessel contraction and regulate the resting blood pressure by hyperpolarizing the vascular smooth muscle cell membrane potential. TRP-PK1 represents a novel lead compound for mimicking K+ channels and treating hypertension, heart rate disorder and other K+ channel dysfunction-induced diseases. The present study also sheds new light onto the mimic ion channel function and the significant utilization of natural biological sources.

The influence of gap junction network complexity on pulmonary artery smooth muscle reactivity in normoxic and chronically hypoxic conditions

Experiments on intrapulmonary arteries (IPAs) isolated from rats maintained in normoxia and chronic hypobaric hypoxia showed that in normoxia, the IPA contractile sensitivity to KCl was not modified by gap junction inhibition. In contrast, chronic hypoxia induced an endothelium-independent hypersensitivity, which was suppressed by gap junction inhibition. For the theoretical analysis of these results, we developed a model of interconnected myocytes. Given that smooth muscle cells in IPAs are known to communicate via gap junctions, we regard the cytoarchitecture of the IPA as a spatial network, in which nodes represent individual smooth muscle cells and the links signify intercellular communication. A single-cell model that drives the dynamics of individual nodes includes the major elements of voltage-dependent Ca(2+) signalling. In addition, interindividual variability of SMCs is introduced by distributing the reversal potentials for K(+). Cell-to-cell connection consists of passive Ca(2+) diffusion and electrical coupling, and connection between cells is determined by the topology of the intercellular network. Model predictions indicate that the experimental results can be explained by topological modifications and not by changes in the number of gap junctions. According to the model, in normoxia the myocytes are connected in a complex network, whereas chronic hypoxia is related to loss of complexity, leading to hypersensitivity. Our results thus indicate that chronic hypoxia entails gap junction network rearrangements, leading to disturbances in the intercellular communication pathways.

Role of impaired endothelial cell Ca2+signaling in uteroplacental vascular dysfunction during diabetic rat pregnancy

Diabetes mellitus in pregnancy is associated with impaired endothelium-mediated dilatation of maternal arteries, although the underlying cellular mechanisms remain unknown. In this study, we hypothesized that diabetes during rat gestation attenuates agonist-induced uterine vasodilation through reduced endothelial cell (EC) Ca(2+) elevations and impaired smooth muscle cell (SMC) hyperpolarization and SMC intracellular Ca(2+) concentration ([Ca(2+)]i) responses. Diabetes was induced by an injection of streptozotocin to second-day pregnant rats and confirmed by the development of maternal hyperglycemia. Control rats were injected with a citrate buffer. Fura-2-based measurements of SMC [Ca(2+)]i or microelectrode recordings of SMC membrane potential were performed concurrently with dilator responses to ACh in uteroplacental arteries from control and diabetic pregnant rats. Basal levels of EC [Ca(2+)]i and ACh-induced EC [Ca(2+)]i elevations in pressurized vessels and small EC sheets were studied as well. Diabetes reduced ACh-induced vasodilation due to a markedly impaired EDHF-mediated response. Diminished vasodilation to ACh was associated with attenuated SMC hyperpolarization and [Ca(2+)]i responses. Basal levels of EC [Ca(2+)]i and ACh-induced EC [Ca(2+)]i elevations were significantly reduced by diabetes. In conclusion, these data demonstrate that reduced endothelium-mediated hyperpolarization contributes to attenuated uteroplacental vasodilation and SMC [Ca(2+)]i responses to ACh in diabetic pregnancy. Impaired endothelial Ca(2+) signaling is in part responsible for endothelial dysfunction in the uterine resistance vasculature of diabetic rats. Pharmacological improvement of EC Ca(2+) handling may provide an important strategy for the restoration of endothelial function and enhancement of maternal blood flow in human pregnancies complicated by diabetes.

Nitric oxide generated by the compound RuBPY promotes the vascular smooth cell membrane hyperpolarization

The cis-[Ru(bpy)2(py)NO2](PF6) specie (RuBPY) has been used as nitric oxide (NO) delivery agent. It is an NO reservoir and it is thermodynamically stable in aqueous solution. This study aimed to evaluate the NO specie generated by RuBPY as compared to NO released from sodium nitroprusside (SNP) and to study the cellular mechanisms specially focusing the activation of soluble guanylyl-cyclase (sGC), K+ channels and the cell membrane hyperpolarization, which are the main targets for NO-inducing vascular relaxation.NO generated by RuBPY and the vascular smooth muscle cell (VSMC) membrane potential were measured by confocal microscopy. The cellular mechanisms of aorta relaxation were investigated using K+ channel blockers and sGC inhibitor. NO released from RuBPY was higher than NO released from SNP. RuBPY released only radicalar NO0 and SNP released both NO− and NO0. The concentration–effect curves for RuBPY-induced relaxation was shifted to the right by inhibition of sGC with ODQ and by the non-selective blockade of K+ channels with TEA. The simultaneous combination of ODQ and TEA abolished the vasorelaxation induced by RuBPY. The membrane potential measured by the sensitive dye 4-Di-ANNEPS demonstrated that RuBPY induces cell membrane hyperpolarization. Taken together, our results indicate that the large amount of NO0 specie generated by RuBPY induces vasorelaxation due to activation of sGC, K+ channels sensitive to TEA, and cell membrane hyperpolarization. These results indicate that NO0 generated from RuBPY can also directly activate the K+ channels in an independent way of sGC.

Characteristics of ACh‐induced hyperpolarization and relaxation in rabbit jugular vein

The roles played by endothelium-derived NO and prostacyclin and by endothelial cell hyperpolarization in ACh-induced relaxation have been well characterized in arteries. However, the mechanisms underlying ACh-induced relaxation in veins remain to be fully clarified. ACh-induced smooth muscle cell (SMC) hyperpolarization and relaxation were measured in endothelium-intact and -denuded preparations of rabbit jugular vein. In endothelium-intact preparations, ACh (≤ 10⁻⁸ M) marginally increased the intracellular concentration of Ca²⁺ ([Ca²⁺](i)) in endothelial cells but did not alter the SMC membrane potential. However, ACh (10⁻¹⁰ -10⁻⁸ M) induced a concentration-dependent relaxation during the contraction induced by PGF(2α) and this relaxation was blocked by the NO synthase inhibitor N(ω) -nitro-l-arginine. ACh (10⁻⁸ -10⁻⁶ M) concentration-dependently increased endothelial [Ca²⁺](i) and induced SMC hyperpolarization and relaxation. These SMC responses were blocked in the combined presence of apamin [blocker of small-conductance Ca²⁺-activated K⁺ (SK(Ca) , K(Ca) 2.3) channel], TRAM 34 [blocker of intermediate-conductance Ca²⁺ -activated K⁺ (IK(Ca) , K(Ca) 3.1) channel] and margatoxin [blocker of subfamily of voltage-gated K⁺ (K(V) ) channel, K(V) 1]. In rabbit jugular vein, NO plays a primary role in endothelium-dependent relaxation at very low concentrations of ACh (10⁻¹⁰ -10⁻⁸ M). At higher concentrations, ACh (10⁻⁸ -3 × 10⁻⁶ M) induces SMC hyperpolarization through activation of endothelial IK(Ca) , K(V) 1 and (possibly) SK(Ca) channels and produces relaxation. These results imply that ACh regulates rabbit jugular vein tonus through activation of two endothelium-dependent regulatory mechanisms.

Characteristics of the actions by which 5‐hydroxytryptamine affects electrical and mechanical activities in rabbit jugular vein graft

The vasomodulating actions of 5-HT in vein grafts, and the underlying mechanisms, remain to be fully clarified. Here, we characterized the actions by which 5-HT affects electrical and mechanical activities in rabbit autologous jugular vein grafts. Smooth muscle cell (SMC) membrane potential and isometric tension were measured in vein grafts 4 weeks after implantation into carotid arteries. Changes in the expression of 5-HT receptor subtypes and in myosin heavy chain isoforms (SM1, SM2 and SMemb) were examined by immunohistochemistry and Western blot analysis. The walls of grafted veins displayed massive increases in the number of SM1- and SM2-positive SMCs. 5-HT induced a large depolarization and contraction that were each reduced by both 5-HT(2A) - and 5-HT(1B/1D) -receptor antagonists. The 5-HT-induced contraction was not modified by a 5-HT₇ -receptor antagonist. The 5-HT₇ -receptor-selective agonist AS 19 did not induce relaxation during the contraction to prostaglandin F(2α) . Immunohistochemical and Western blot analyses revealed that immunoreactive responses against 5-HT(2A) and 5-HT(1B/1D) receptors were increased in the vein graft. 5-HT is able to induce a large contraction in rabbit autologous jugular vein grafts through (i) an increased number of differentiated contractile SMCs; (ii) an increased number of SMCs expressing contractile 5-HT(2A) - and 5-HT(1B/1D) receptors; and (iii) a down-regulation of the function of the relaxant SMC 5-HT₇ receptors. These changes in the vein graft may help it to resist the higher pressure present on the arterial side of the circulation.

Cyclic nucleotide-dependent relaxation pathways in vascular smooth muscle.

Vascular smooth muscle tone is controlled by a balance between the cellular signaling pathways that mediate the generation of force (vasoconstriction) and release of force (vasodilation). The initiation of force is associated with increases in intracellular calcium concentrations, activation of myosin light-chain kinase, increases in the phosphorylation of the regulatory myosin light chains, and actin-myosin crossbridge cycling. There are, however, several signaling pathways modulating Ca(2+) mobilization and Ca(2+) sensitivity of the contractile machinery that secondarily regulate the contractile response of vascular smooth muscle to receptor agonists. Among these regulatory mechanisms involved in the physiological regulation of vascular tone are the cyclic nucleotides (cAMP and cGMP), which are considered the main messengers that mediate vasodilation under physiological conditions. At least four distinct mechanisms are currently thought to be involved in the vasodilator effect of cyclic nucleotides and their dependent protein kinases: (1) the decrease in cytosolic calcium concentration ([Ca(2+)]c), (2) the hyperpolarization of the smooth muscle cell membrane potential, (3) the reduction in the sensitivity of the contractile machinery by decreasing the [Ca(2+)]c sensitivity of myosin light-chain phosphorylation, and (4) the reduction in the sensitivity of the contractile machinery by uncoupling contraction from myosin light-chain phosphorylation. This review focuses on each of these mechanisms involved in cyclic nucleotide-dependent relaxation of vascular smooth muscle under physiological conditions.

Models of neurovascular coupling via potassium and EET signalling

Functional hyperemia is an important metabolic autoregulation mechanism by which increased neuronal activity is matched by a rapid and regional increase in blood supply. This mechanism is facilitated by a process known as “neurovascular coupling”—the orchestrated communication system involving neurons, astrocytes and arterioles. Important steps in this process are the production of EETs in the astrocyte and the release of potassium, via two potassium channels (BK and KIR), into the perivascular space. We provide a model which successfully accounts for several observations seen in experiment. The model is capable of simulating the approximate 15% arteriolar dilation caused by a 60-s neuronal activation (modelled as a release of potassium and glutamate into the synaptic cleft). This model also successfully emulates the paradoxical experimental finding that vasoconstriction follows vasodilation when the astrocytic calcium concentration (or perivascular potassium concentration) is increased further. We suggest that the interaction of the changing smooth muscle cell membrane potential and the changing potassium-dependent resting potential of the KIR channel are responsible for this effect. Finally, we demonstrate that a well-controlled mechanism of potassium buffering is potentially important for successful neurovascular coupling.

Basal protein kinase Cδ activity is required for membrane localization and activity of TRPM4 channels in cerebral artery smooth muscle cells

The melastatin (M) transient receptor potential channel (TRP) channel TRPM4 is a critical regulator of vascular smooth muscle cell membrane potential and contractility. We recently reported that PKCδ activity influences smooth muscle cell excitability by promoting translocation of TRPM4 channel protein to the plasma membrane. Here we further investigate the relationship between membrane localization of TRPM4 protein and channel activity in native cerebral arterial myocytes. We find that TRPM4 immunolabeling is primarily located at or near the plasma membrane of freshly isolated cerebral artery smooth muscle cells. However, siRNA mediated downregulation of PKCδ or brief (15 min) inhibition of PKCδ activity with rottlerin causes TRPM4 protein to move away from the plasma membrane and into the cytosol. In addition, we find that PKCδ inhibition diminishes TRPM4-dependent currents in smooth muscle cells patch clamped in the amphotericin B perforated patch configuration. We conclude that TRPM4 channels are mobile in native cerebral myocytes and that basal PKCδ activity supports excitability of these cells by maintaining localization TRPM4 protein at the plasma membrane.

Small and intermediate conductance Ca2+‐activated K+channels regulate spontaneous contraction of mouse portal vein myocytes

Ca2+‐activated K+ channels (KCa) are key components of vascular myocytes EC coupling through their control of smooth muscle cell membrane potential. Whilst BKCa is expressed ubiquitously in smooth muscle cells, only proliferative VSMCs express KCa3.1 channels. However, mouse portal vein (mPV) is an atypical blood vessel showing spontaneous and rhythmic contractions. Since KCa channels are involved in the regulation of excitability of other cell types like neurons, the contribution of KCa2.x and KCa3.1 channels to the control of mPV excitability was investigated. Expression of KCa2.x and KCa3.1 was detected in mPV by PCR and immunohistochemistry. In single cell patch clamp studies, the RMP of isolated mPV myocytes was hyperpolarized (≈15 mV) by exposure to NS309 (10 μM), a KCa2.x and KCa3.1 channel activator. Inhibition of KCa2.x channels with UCL1689 (3 μM) reversed the effect of NS309 whilst inhibition of KCa3.1 channels (TRAM‐34; 10 μM) had no effect. NS309 significantly decreased the amplitude and increased the frequency of spontaneous contractions of intact mPV. These effects were reversed by application of UCL1689, suggesting that the NS309‐induced changes in spontaneous mPV contractions is carried out by KCa2.x channels. This study suggest that KCa2.x and KCa3.1 channels are expressed in mouse portal vein myocytes and play an important role in regulation of spontaneous contractions.Supported by FICM

Role of ryanodine receptors in acidic pH‐induced dilation of brain parenchymal arterioles

Ryanodine receptors (RyRs) are Ca2+ permeable channels in the sarcoplasmic reticulum. RyRs regulate vascular smooth muscle cell (VSMC) membrane potential through their localized Ca2+‐release events, termed Ca2+ sparks, which activate hyperpolarizing BK currents leading to vasodilation. This mechanism is clearly demonstrated by the non‐additive constrictions induced by RyR or BK channel blockers in pressurized pial arteries. However these blockers have little effect on the diameter of pressurized parenchymal arterioles (PAs) from the brain, even though functional BK channels and RyRs are present. At normal pH (7.4), VSMCs in pressurized PAs exhibit largely Ca2+ waves but not Ca2+ sparks. Alkaline pH has been shown to shift Ca2+ sparks to Ca2+ waves in pial arteries. Therefore, we hypothesized that acidic pH might dilate PAs by reshaping the intracellular Ca2+ dynamic from Ca2+ waves to Ca2+ sparks, thereby activating BK channels. As predicted, reducing extracellular pH from 7.4 to 7.0 dramatically decreased Ca2+ wave activity, and increased spark activity. Acidic pH caused a dilation of up to 70% of the maximal diameter, and this dilation was inhibited by about 60% by BK or RyR blockers. These findings suggest that RyRs play a major role in the acidic pH‐induced dilation of PAs through Ca2+ spark‐mediated activation of BK channels. Supported by AHA 09POST2290090 (FD) and the NIH HL095488, HL44455 and HL58231.