Arteriolar Smooth Muscle Cells Research Articles

Pressor and renal regional hemodynamic effects of urotensin II in neonatal pigs

Renal expression of the peptide hormone urotensin II (UII) and its receptor (UTR) are dependent on kidney maturation and anatomical regions. However, renal regional hemodynamic effects of UII in neonates are unclear. Here, we investigated regional hemodynamic responses to acute intrarenal arterial administration of UII in newborn pigs. Western immunoblotting and immunofluorescence confirmed UTR expression and membrane localization in newborn pig renal afferent arterioles and afferent arteriolar smooth muscle cells respectively. Intrarenal arterial bolus injections of human UII (hUII; 1-100 ng/kg) resulted in a dose-dependent decrease in total renal blood flow (RBF) and an increase in mean arterial pressure (MAP) and renal vascular resistance (RVR) in newborn pigs. Moreover, hUII dose dependently reduced cortical blood flow (CBF) but increased medullary blood flow (MBF) in the piglets. hUII-induced MAP elevation and hemodynamic changes were inhibited by urantide, a UTR antagonist, but not losartan, a type 1 angiotensin II receptor antagonist. U-73122, a phospholipase C (PLC) inhibitor, and 2-aminoethoxydiphenyl borate, an inositol 1,4,5 trisphosphate (IP₃) receptor antagonist, attenuated hUII-induced MAP and RVR elevations, RBF and CBF reductions, but not MBF increase. These findings indicate that intrarenal arterial administration of hUII elevates blood pressure and induces region-selective renal hemodynamic changes in newborn pigs. Our data also suggest that the PLC/IP₃ signaling pathway contributes to hUII-induced alterations in MAP, RBF, RVR, and CBF but not MBF in newborn pigs.

Ischemic treatment hyperpolarizes cochlear arteriolar smooth muscle and endothelial cells via different channel mechanisms

Regional acute ischemia generally induces a compensatory vasodilation in the most vascular beds including the cochlear arterioles but the underlying cellular mechanism remains controversial. Using conventional intracellular and whole‐cell recording from smooth muscle (SMC) and endothelial cells (EC) in situ of and dispersed from segments of guinea pigs cochlear spiral modiolar artery, we analyzed the membrane actions of acute ischemic treatment (AIT, hypercapinic hypoxia, pO2 =0 mmHg, pH=6.2). The AIT for 2 to 20 min caused a reversible and partially repeatable vasodilation and a hyperpolarization in the majority of cells. ACh‐ and 10 mM K+‐induced dilation was significantly reduced, lost or reversed to a constriction during and after the AIT. Holding at −20 mV, AIT induced a reversible 10–65 pA outward current within 1–2 min in the majority of SMCs and ECs. During the AIT, the whole‐cell I/V curve became stronger outwardly rectifying at voltages >; −40 mV. The AIT‐induced net current in isolated SMCs exhibited a delayed outward rectification upon depolarization steps and partial blockade by 1–10 mM TEA or 4‐AP, suggesting an activation of both voltage‐gated (KV) and big conductance Ca2+‐activated (BK) K+‐channels. In contrast, the AIT‐induced current in isolated ECs was blocked by 30 μM niflumic acid but not by TEA, 4‐AP and linopirdine; the net current showed a voltage and time dependence typical of Ca2+‐activated Cl−‐channels (CaCC): slowly activating outwardly rectifying with a reversal potential near −30 mV. AIT (>;10 min) also reduced gap junction coupling current in in situ SMCs and ECs. We conclude that the acute ischemia in the cochlear arteriole induces hyperpolarization, dilation and loss of EDHF function via activation of KV and BK in smooth muscle cells and activation of Ca2+‐activated Cl−‐channels in endothelial cells, as well as an inhibition of gap junction coupling. Supported by NIH NIDCD DC 004716, P30 05983.

Microcirculatory anatomy can explain the illusory cooperativity seen in the flow response to exogenous adenosine

OBJECTIVETo demonstrate that illusory cooperativity can arise in the microcirculation as a result of competition and spatial localization.Adenosine in the myocardial interstitium binds to adenosine A2A receptors on vascular smooth muscle, leading to reduced tone and increased myocardial blood flow. The relationship between the concentration of supplied adenosine and the increase in flow is very steep (Hill coefficient 5–7), suggesting strong receptor binding site cooperativity. In fact, this apparent cooperativity may be illusory, and can arise solely from the anatomic geometry and cellular composition of myocardial tissue. Along the path from capillary to arteriolar smooth muscle cell, adenosine faces a permeability barrier (the endothelial clefts) and a diffusion distance (back from capillary to arteriole). Cellular transporters and metabolic enzymes compete for adenosine all along this path, influencing not only the apparent ED50 of arterially supplied adenosine, but also the shape of the dose‐response curve. Using a mathematical model, we demonstrate the independent influences of competition and access barriers (permeability barriers and diffusion distances), as well as how these effects can combine to make the dose‐response curve for a single binding site receptor (Hill coefficient 1) appear extremely cooperative.

Fenamates block gap junction coupling and potentiate BKCa channels in guinea pig arteriolar cells

We determined the actions of the fenamates, flufenamic acid (FFA) and niflumic acid (NFA), on gap junction-mediated intercellular coupling between vascular smooth muscle cells (VSMC) in situ of acutely isolated arteriole segments from the three vascular beds: the spiral modiolar artery (SMA), anterior inferior cerebellar artery (AICA) and mesenteric artery (MA), and on non-junctional membrane channels in dispersed VSMCs. Conventional whole-cell recording methods were used. FFA reversibly suppressed the input conductance (Ginput) or increased the input resistance (Rinput) in a concentration dependent manner, with slightly different IC50s for the SMA, AICA and MA segments (26, 33 and 56μM respectively, P>0.05). Complete electrical isolation of the recorded VSMC was normally reached at ≥300μM. NFA had a similar effect on gap junction among VSMCs with an IC50 of 40, 48 and 62μM in SMA, AICA and MA segments, respectively. In dispersed VSMCs, FFA and NFA increased outward rectifier K+-current mediated by the big conductance calcium-activated potassium channel (BKCa) in a concentration-dependent manner, with a similar EC50 of ∼300μM for both FFA and NFA in the three vessels. Iberiotoxin, a selective blocker of the BKCa, suppressed the enhancement of the BKCa by FFA and NFA. The KV blocker 4-AP had no effect on the fenamates-induced K+-current enhancement. We conclude that FFA and NFA blocked the vascular gap junction mediated electrical couplings uniformly in arterioles of the three vascular beds, and complete electrical isolation of the recorded VSMC is obtained at ≧300μM; FFA and NFA also activate BKCa channels in the arteriolar smooth muscle cells in addition to their known inhibitory effects on chloride channels.

Nitric Oxide Can Directly Mediate Renin Cell Recruitment

See related article, pp 400–407 In this issue of Hypertension , Neubauer et al1 fit missing pieces into the puzzle of how renin production is controlled, when the demand for renin is high. Our understanding of the acute release of renin is much more complete than what we know about the long-term adaptations to stimuli of renin. Our scarce knowledge of the mechanisms mediating long-term renin stimulation is remarkable with regard to the clinical importance of renin. Hypertension, fluid and electrolyte homeostasis, as well as inflammation and fibrosis all may involve chronic renin stimulation.2 More than 400 million years ago, renin first appeared in organisms,3 and renin is often seen as the first hormone discovered. Shortly before the 20th century, Tigerstedt and Bergman showed that kidney extracts can elevate blood pressure, when intravenously infused.4 Today, we recognize that the actions of renin go far beyond that of controlling blood pressure, and we have detailed insight into how renin is released into the circulation.5 The major stimuli of short-term renin release are blood pressure decreases, β1-adrenergic stimulation, and low salt, whereas …

Increased endothelin receptor B and G protein coupled kinase-2 in the mesentery of portal hypertensive rats

To elucidate the mechanisms of mesenteric vasodilation in portal hypertension (PHT), with a focus on endothelin signaling. PHT was induced in rats by common bile duct ligation (CBDL). Portal pressure (PP) was measured directly via catheters placed in the portal vein tract. The level of endothelin-1 (ET-1) in the mesenteric circulation was determined by radioimmunoassay, and the expression of the endothelin A receptor (ETAR) and endothelin B receptor (ETBR) was assessed by immunofluorescence and Western blot. Additionally, expression of G protein coupled kinase-2 (GRK2) and β-arrestin 2, which influence endothelin receptor sensitivity, were also studied by Western blot. PP of CBDL rats increased significantly (11.89 ± 1.38 mmHg vs 16.34 ± 1.63 mmHg). ET-1 expression decreased in the mesenteric circulation 2 and 4 wk after CBDL. ET-1 levels in the systemic circulation of CBDL rats were increased at 2 wk and decreased at 4 wk. There was no change in ETAR expression in response to CBDL; however, increased expression of ETBR in the endothelial cells of mesenteric arterioles and capillaries was observed. In sham-operated rats, ETBR was mainly expressed in the CD31⁺ endothelial cells of the arterioles. With development of PHT, in addition to the endothelial cells, ETBR expression was noticeably detectable in the SMA⁺ smooth muscle cells of arterioles and in the CD31⁺ capillaries. Following CBDL, increased expression of GRK2 was also found in mesenteric tissue, though there was no change in the level of β-arrestin 2. Decreased levels of ET-1 and increased ETBR expression in the mesenteric circulation following CBDL in rats may underlie mesenteric vasodilation in individuals with PHT. Mechanistically, increased GRK2 expression may lead to desensitization of ETAR, as well as other vasoconstrictors, promoting this vasodilatory effect.

Mechanism of spironolactone-induced Ca<sup>2+</sup> increase in rat testicular arteriole smooth muscle cells revealed by real-time laser scanning confocal microscopy

Summary. We reported previously that spironolactone (SPL) induced an increase in intracellular Ca 2+ concentration ([Ca 2+ ]i) in rat testicular arteriole smooth muscle cells. In the present study, we further investigated the mechanism of SPL-induced [Ca 2+ ]i dynamics in rat arteriole smooth muscles. The increase in [Ca 2+ ]i induced by SPL (300 μM) was markedly inhibited in extracellular Ca 2+ -free conditions and in

English

Dyslipidemia and oxidative stress were evaluated in patients with polycystic ovarian syndrome. MATERIALS AND METHODS: Total cholesterol, Triglyceride, HDL cholesterol, LDL cholesterol, Malondialdehyde (MDA) and Total antioxidant capacity were measured in serum of PCOS subjects and age matched controls. RESULTS: Study group comprised of 31 women with PCOS and control group with 31 healthy volunteers. Mean serum levels of MDA, Cholesterol, Triglycerides and LDL cholesterol were significantly increased and TAC and HDL cholesterol were significantly decreased in PCOS subjects compared to controls. CONCLUSION: Our results revealed that PCOS is associated with dyslipidemia and altered oxidative status. Women with PCOS would be predicted to be at high risk for dyslipidemia because they have elevated androgen levels and are frequently obese. Moreover, since they are also often hyperinsulinemic and insulin resistant, they would also be expected to be at increased risk for the dyslipidemia associated with insulin resistance. Insulin, rather than androgen, levels correlate best with lipid abnormalities, and suppressing androgen levels does not alter lipid profiles in PCOS. Insulin resistance and hyperinsulinemia are also associated with an atherogenic plasma lipid profile. Elevated plasma insulin concentrations enhance very low density lipoprotein (VLDL) synthesis, leading to hypertriglyceridemia. Progressive elimination of lipid and apolipoproteins from the VLDL particle leads to an increased formation of intermediate-density and low-density lipoproteins, both of which are atherogenic. Last, insulin, independent of its effects on blood pressure and plasma lipids, is known to be atherogenic. The hormone enhances cholesterol transport into arteriolar smooth muscle cells and increases

P6 Hydrogen sulfide activates Ca2+ sparks to induce cerebral arteriole dilation

Hydrogen sulfide (H 2 S) is a gaseous vasodilator produced by endothelial cells. Mechanisms by which H 2 S induce vasodilation are unclear. We tested the hypothesis that H 2 S dilates piglet cerebral arterioles by modulating local and global intracellular Ca 2+ signals in smooth muscle cells. High-speed confocal imaging revealed that H 2 S increased Ca 2+ spark frequency ∼1.43-fold and decreased global intracellular Ca 2+ concentration ([Ca 2+ ] i ) by ∼37 nM in smooth muscle cells of intact cerebral arteries. In contrast, H 2 S did not alter Ca 2+ wave frequency. In voltage-clamped (−40 mV) cells, H 2 S increased the frequency of Ca 2+ spark-induced transient Ca 2+ -activated K + (K Ca ) currents ∼ 1.8-fold, but did not alter the amplitude of these events. H 2 S did not alter the activity of single K Ca channels recorded in the absence of Ca 2+ sparks in arteriole smooth muscle cells. H 2 S increased SR Ca 2+ load ([Ca 2+ ] SR ), measured as caffeine (10 mM)-induced [Ca 2+ ] i transients, ∼1.5-fold. H 2 S dilated pressurized cerebral arterioles. Iberiotoxin, a K Ca channel blocker, and ryanodine, a ryanodine receptor (RyR) channel inhibitor, reduced H 2 S-induced vasodilation by ∼38 and ∼37%, respectively. In summary, our data indicate that H 2 S elevates [Ca 2+ ] SR , leading to Ca 2+ spark activation in cerebral arteriole smooth muscle cells. The subsequent elevation in transient K Ca current frequency leads to a reduction in global [Ca 2+ ] i and vasodilation.

Abstract 38: Rbp-j Regulates the Genetic Program of the Myo-epithelioid JG Cell

RBP-J , the major downstream effector of Notch signaling, is necessary to maintain the number of juxtaglomerular (JG) cells. In addition, RBP-J regulates the plasticity of arteriolar smooth muscle cells to adopt the renin cell phenotype when homeostasis is threatened. We hypothesized that RBP-J acts as an on/off switch controlling the expression of genes that determine the renin phenotype. To determine whether RBP-J directly affects renin gene expression, we generated mice harboring a bacterial artificial chromosome (BAC) transgene with green fluorescent protein (GFP) under the control of the renin gene carrying a mutation in its RBP-J- binding site (Mut-BAC). Mut-BAC mice had markedly reduced GFP expression to 12.9 % ±0.01 (n=3) of the control (Wt-BAC) and a diminished response to homeostatic challenges: mut-BAC mice had a reduced number of GFP positive JG areas per total number of glomeruli (Wt-BAC: 25.1 % ±3.0, n=3; Mut-BAC: 9.3 % ±1.4, n=2, p<0.02) and no GFP expression along the arterioles. To determine whether the decrease in the number of JG cells in mice lacking RBP-J (cKO) was due to a diminished endowment of renin progenitor cells, we traced the fate of cells derived from the renin lineage by generating mice ( RBP-J fl/fl ; Ren1d +/cre ; R26R +/- ) in which cells lacking RBP-J simultaneously expressed β-galactosidase (β-gal). The pattern of β-gal in cKO and control kidneys was identical, indicating that cells derived from the renin lineage did not die but instead changed their phenotype. Next we investigated the phenotype adopted by the cells derived from the renin lineage. Expression of α-smooth muscle actin and smoothelin (a marker of mature smooth muscle) was significantly decreased to 41 % ±7.0 (n=2) and 44 % ±8.8 (n=2) respectively with respect to controls (p<0.01). In addition, mutant JG cells in vivo did not express genes characteristic of the renin phenotype such as renin, calponin1, Nfat and Akr1b7 expressing instead fibroblast-specific protein 1 indicating the adoption of a fibroblast-like phenotype. Results indicate that RBP-J directly governs a genetic program that controls the dual endocrine-contractile phenotype of the JG cell, which is crucial to maintain blood pressure and fluid-electrolyte homeostasis.

Abstract 488: Arteriolar Smooth Muscle Calcium Waves Depend on Calcium Influx through Voltage-gated Calcium Channels

Smooth muscle cells (SMCs) in arterioles from striated muscle display IP 3 receptor-dependent Ca 2+ waves that contribute to global myoplasmic Ca 2+ concentration and myogenic tone. However, the contribution of voltage-gated Ca 2+ channels (VGCC) to these arteriolar Ca 2+ signals is unknown. We tested the hypothesis that Ca 2+ waves depend on Ca 2+ influx through VGCC in cremaster muscle arterioles loaded with Fluo-4 and imaged by confocal microscopy. At rest, with vessels pressurized to 80 cm H 2 O in 2 mM Ca 2+ , arteriolar diameter was 28 ± 2 μm (n = 5), and SMCs displayed Ca 2+ waves with frequency (FREQ) = 0.21 ± 0.06 Hz, occurrence (OCC) = 3.5 ± 1.0 waves/SMC and amplitude (AMP) = 1.7 ± 0.1 F/Fo. Removal of extracellular Ca 2+ dilated the arterioles to 39 ± 1 μm, and inhibited Ca 2+ waves (FREQ = 0.1 ± 0.03, OCC = 1.7 ± 0.5 waves/SMC and AMP = 1.4 ± 0.06 F/Fo; p < 0.05 vs. rest) indicating that Ca 2+ waves depended, in part, on influx of extracellular Ca 2+ . Similarly, the VGCC antagonist, nifedipine (1 μM), dilated the arterioles to 34 ± 1.3 μm and also inhibited Ca 2+ waves (FREQ = 0.07 ± 0.02 Hz, OCC = 1.1 ± 0.5 waves/SMC, AMP = 1.4 ± 0.05 F/Fo; p < 0.05 vs. rest). Hyperpolarization of SMCs with the K + channel agonist, cromakalim (10 μM), dilated arterioles from 49 ± 3 to 59 ± 4 μm (n = 4, p < 0.05) and also reduced Ca 2+ wave FREQ (0.1 ± 0.04 to 0.03 ± 0.003 Hz), OCC (1.7 ± 0.04 to 0.5 ± 0.05 waves/SMC) and AMP (1.5 ± 0.04 to 1.2 ± 0.004 F/Fo) (p < 0.05). Conversely, depolarization of SMCs with the BK Ca channel blocker, TEA (1 mM), constricted arterioles from 28 ± 2 to 16 ± 1 μm (n = 5, p < 0.05) and increased wave FREQ (0.2 ± 0.1 to 0.5 ± 0.1 Hz, p < 0.05) and OCC (4 ± 1 to 8 ± 2 waves/SMC, p < 0.05), effects blocked by nifedipine (1μM) (p < 0.05). Similarly, in arterioles pressurized to 20 cm H 2 O to eliminate myogenic tone and reduce basal VGCC activity, application of the VGCC agonist, BayK 8644 (5 nM) constricted the arterioles from 14 ± 1 to 8 ± 1 μm and increased wave FREQ (0.2 ± 0.1 to 0.6 ± 0.1 Hz) and OCC (3 ± 1 to 10 ± 1 waves/SMC) (n = 6; p < 0.05), effects that were independent of ryanodine receptors, as Ca 2+ waves were unaffected by ryanodine (50 μM) in the absence or presence of BayK 8644 (n = 6; p > 0.05). These data support the hypothesis that Ca 2+ waves in arteriolar SMCs depend, in part, on Ca 2+ influx through VGCC.

Distribution of Adenylyl Cyclases in the Rat Ovary by Immunofluorescence Microscopy

In the mammalian ovary both gonadotropins and local cytokines, acting through G-protein coupled receptors, govern the physiology of the ovary in part by regulating the cyclic adenosine 3',5'-monophosphate via adenylyl cyclases. The nine transmembrane adenylyl cyclases and a soluble adenylyl cyclase are regulated by a diversity of ligands. In this study we have examined the rat ovaries, prior to and subsequent to gonadotropin treatment, for the presence of different transmembrane adenylyl cyclases by indirect immunofluorescence microscopy. Adenylyl cyclase I immunoreactivity was observed in the nuclei of oocytes in preantral and antral follicles along with some staining in granulosa cells. Equine chorionic gonadotropin injection increased adenylyl cyclase I staining in granulosa cells. Adenylyl cyclase I staining was also observed in luteal and endothelial cells. Adenylyl cyclase II was observed throughout the ovary, including granulosa cells and the ovarian surface epithelium. Adenylyl cyclase II staining was also found to increase in granulosa cells after equine chorionic gonadotropin injection. Adenylyl cyclase III was distributed primarily in theca and smooth muscle cells of arterioles, with faint staining in the oocytes of equine chorionic gonadotropin-injected ovaries. Adenylyl cyclase IV staining was present throughout the ovary, including the nuclei of oocytes. Adenylyl cyclase VIII staining in granulosa cells increased subsequent to equine chorionic gonadotropin injection and remained in luteal cells. Our study reveals the redundancy of adenylyl cyclases present in the rat ovary and, therefore, implies potential regulation of follicular and corpus luteum physiology by cyclic adenosine 3',5'-monophosphate generated through distinct adenylyl cyclases.

A gas that ignites a bright spark

Modulation of vascular tone has been subject to intense investigations for more than three decades. Numerous studies have looked into signalling pathways where interaction between endothelial cells (ECs) and smooth muscle cells (SMCs) regulates the vascular circulation. This signalling induces SMC contraction/relaxation which is dependent on cytosolic calcium ([Ca2+]i) concentrations and investigations on how this [Ca2+]i level is regulated has opened up myriad upstream signalling routes. Probably most importantly, the discovery of the endothelium-derived relaxing factor (EDRF) in 1980 (Furchgott & Zawadzki, 1980) and its identification as the gasotransmitter nitric oxide (NO) in 1987 (Palmer et al. 1987) broadened our understanding of regulation of vascular tone. Since then, other freely diffusible endogenous gasotransmitters have been identified, namely carbon monoxide (CO) and hydrogen sulfide (H2S). However, NO and CO (although less efficient than NO) can exert their effects though activation of soluble guanylyl cyclase and subsequent production of cGMP, whereas H2S does not. All of these gasotransmitters exhibit strong cytotoxic properties when reaching higher levels and are also involved in non-vascular signalling pathways, i.e. in nervous or immune systems, and are important signalling molecules in certain disease conditions (Steinert et al. 2010; Skovgaard et al. 2011). Work published by Liang et al. (2012) in a recent issue of The Journal of Physiology describes more evidence for the vascular actions of H2S. The study elegantly shows how H2S modulates Ca2+ signalling in pig cerebral arteriolar SMCs by increasing Ca2+ spark frequency, which ultimately decreases global [Ca2+]i concentrations. The authors suggest that H2S enhances levels of ryanodine receptor (RyR)-sensitive Ca2+ stores and increases the frequency of calcium sparks and transient Ca2+-activated K+ channel (BK) currents leading to SMC hyperpolarization and subsequent vessel relaxation. Single channel analyses revealed that the increased frequencies of transient BK currents are not matched by changes in single channel current amplitudes but are solely due to greater open probability. SR Ca2+ content depends on the fine balance between SR uptake and release (Bassani et al. 1995) and it would be interesting to continue this study to determine whether H2S can regulate Ca2+ uptake mechanisms, such as sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) activities or other store release pathways, such as inositol 1,4,5-trisphosphate (IP3) receptor signalling. As RyR-mediated Ca2+ release provides the main pathway for Ca2+ sparks, the questions now arises which of the reported Ca2+ release routes are affected by H2S, including single RyR within release clusters without activation of the remaining channels, isolated un-clustered RyRs or non-RyR leak pathways. H2S is produced in various tissues, including the nervous and cardiovascular systems, from l-cysteine, catalysed by cystathionine-β-synthase (CBS) and/or cystathionine-γ-lyase (CSE). More recently, additional pathways have been identified cysteine aminotransferase (CAT) and 3-mercaptopyrovate sulfuresterase (3MST) with CSE being expressed in the SMCs and ECs but CAT/3MST only localized in the endothelium (Fig. 1). Depending on its concentration, H2S induces variable vasoactive effects in different vascular beds. The liver has high H2S-generating capacity and may be responsible for maintaining the concentration of H2S in the blood, which has been reported to be in the range of 1–160 μmol l−1 although the detection methods do not always give consistent results. Figure 1 Schematic diagram of gasotransmitter signalling in the vasculature Similarly to NO, exogenous application of H2S donors leads to a peak release of H2S followed by rapid metabolism and drop of vasoactive concentrations thereby altering its potential to induce relaxation over time. The complexity and concentration dependency of H2S signalling becomes apparent when comparing vasorelaxing and vasoconstricting effects (reviewed by Skovgaard et al. 2011). In light of these difficulties, an approach where endogenously produced H2S is investigated might help to explain observed inconsistent vascular responses to different H2S concentrations. H2S donor application relaxes rat aortic rings or human mammary arteries via opening of ATP-sensitive K+ (KATP) channels (Zhao et al. 2001) and direct actions of H2S on KATP channels have been reported. In addition H2S modulates voltage-gated K+ (KCNQ) and BK currents in various preparations; however, the exact mechanisms by which H2S acts on BK have to be elucidated. Discrepancies in modulatory effects on BK are most likely due to different concentrations used or tissue variability, which accounts for differences in BK α- and β-subunit expression. The current paper by Liang et al. (2012) provides new insights into the mechanisms by which BK channels are regulated in response to H2S signalling, namely via enhancing Ca2+ spark frequency. Nevertheless, the ultimate response of SMCs is the reduction of voltage-gated Ca2+ channel open probability in order to achieve vascular relaxation and the study by Liang et al. (2012) greatly enhances our understanding of vascular signalling pathways downstream of H2S.

Arteriolar vascular smooth muscle cells: Mechanotransducers in a complex environment

Contraction of small artery (diameters typically less than 250μm) vascular smooth muscle cells (VSMCs) plays a critical role in local control of blood flow and arterial pressure through its affect on vascular caliber. Specifically, contraction of small arteries in response to increased intraluminal pressure is referred to as the myogenic response and represents an important role for mechanotransduction. Critical questions remain as to how changes in pressure are sensed by VSMCs and transduced across the cell membrane to tune the contractile state of the cell. Recent studies suggest a pivotal role for interactions between VSMCs and extracellular matrix (ECM) proteins. Thus, pressure-induced deformation of ECM proteins and their cell surface receptors (for example, integrins) may initiate contraction and cytoskeletal remodeling through modulation of ion channels, membrane depolarization, increased intracellular Ca2+ and actomyosin crossbridge cycling. Importantly, it is argued that the contractile properties of small artery VSMCs reflect an intimate and integrated interaction with their extracellular environment and the three-dimensional structure of the vessel wall.

Hydrogen sulfide activates Ca2+ sparks to induce cerebral arteriole dilatation

Hydrogen sulfide (H₂S) is a gaseous vasodilator produced by endothelial cells. Mechanisms by which H₂S induces vasodilatation are unclear. We tested the hypothesis that H₂S dilates cerebral arterioles by modulating local and global intracellular Ca²⁺ signals in smooth muscle cells. High-speed confocal imaging revealed that Na₂S, an H₂S donor, increased Ca²⁺ spark frequency ∼1.43-fold and decreased global intracellular Ca²⁺ concentration ([Ca²⁺]i) by ∼37 nM in smooth muscle cells of intact piglet cerebral arterioles. In contrast, H₂S did not alter Ca²⁺ wave frequency. In voltage-clamped (-40 mV) cells, H₂S increased the frequency of iberiotoxin-sensitive, Ca²⁺ spark-induced transient Ca²⁺-activated K⁺ (KCa) currents ∼1.83-fold, but did not alter the amplitude of these events. H₂S did not alter the activity of single KCa channels recorded in the absence of Ca²⁺ sparks in arteriole smooth muscle cells. H₂S increased SR Ca²⁺ load ([Ca²⁺]SR), measured as caffeine (10 and 20mM)-induced [Ca²⁺]i transients, ∼1.5-fold. H₂S hyperpolarized (by ∼18 mV) and dilated pressurized (40 mmHg) cerebral arterioles. Iberiotoxin, a KCa channel blocker, reduced H₂S-induced hyperpolarization by ∼51%. Iberiotoxin and ryanodine, a ryanodine receptor channel inhibitor, reduced H₂S-induced vasodilatation by ∼38 and ∼37%, respectively. In summary, our data indicate that H₂S elevates [Ca²⁺]SR, leading to Ca²⁺ spark activation in cerebral arteriole smooth muscle cells. The subsequent elevation in transient KCa current frequency leads to membrane hyperpolarization, a reduction in global [Ca²⁺]i and vasodilatation.

Histone acetyl transferases CBP and p300 are necessary for maintenance of renin cell identity and transformation of smooth muscle cells to the renin phenotype.

In response to a homeostatic threat circulating renin increases by increasing the number of cells expressing renin by dedifferentiation and re-expression of renin in arteriolar smooth muscle cells (aSMCs) that descended from cells that expressed renin in early life. However, the mechanisms that govern the maintenance and reacquisition of the renin phenotype are not well understood. The cAMP pathway is important for renin synthesis and release: the transcriptional effects are mediated by binding of cAMP responsive element binding protein with its co-activators, CBP and p300, to the cAMP response element in the renin promoter. We have shown previously that mice with conditional deletion of CBP and p300 (cKO) in renin cells had severely reduced renin expression in adult life. In this study we investigated when the loss of renin-expressing cells in the cKO occurred and found that the loss of renin expression becomes evident after differentiation of the kidney is completed during postnatal life. To determine whether CBP/p300 is necessary for re-expression of renin we subjected cKO mice to low sodium diet + captopril to induce retransformation of aSMCs to the renin phenotype. The cKO mice did not increase circulating renin, their renin mRNA and protein expression were greatly diminished compared with controls, and only a few aSMCs re-expressed renin. These studies underline the crucial importance of the CREB/CBP/p300 complex for the ability of renin cells to retain their cellular memory and regain renin expression, a fundamental survival mechanism, in response to a threat to homeostasis.

Function and expression of ryanodine receptors and inositol 1,4,5‐trisphosphate receptors in smooth muscle cells of murine feed arteries and arterioles

We tested the hypothesis that vasomotor control is differentially regulated between feed arteries and downstream arterioles from the cremaster muscle of C57BL/6 mice. In isolated pressurized arteries, confocal Ca(2+) imaging of smooth muscle cells (SMCs) revealed Ca(2+) sparks and Ca(2+) waves. Ryanodine receptor (RyR) antagonists (ryanodine and tetracaine) inhibited both sparks and waves but increased global Ca(2+) and myogenic tone. In arterioles, SMCs exhibited only Ca(2+) waves that were insensitive to ryanodine or tetracaine. Pharmacological interventions indicated that RyRs are functionally coupled to large-conductance, Ca(2+)-activated K(+) channels (BK(Ca)) in SMCs of arteries, whereas BK(Ca) appear functionally coupled to voltage-gated Ca2+ channels in SMCs of arterioles. Inositol 1,4,5-trisphosphate receptor (IP3R) antagonists (xestospongin D or 2-aminoethoxydiphenyl borate) or a phospholipase C inhibitor (U73122) attenuated Ca(2+) waves, global Ca(2+) and myogenic tone in arteries and arterioles but had no effect on arterial sparks. Real-time PCR of isolated SMCs revealed RyR2 as the most abundant isoform transcript; arteries expressed twice the RyR2 but only 65% the RyR3 of arterioles and neither vessel expressed RyR1. Immunofluorescent localisation of RyR protein indicated bright, clustered staining of arterial SMCs in contrast to diffuse staining in arteriolar SMCs. Expression of IP(3)R transcripts and protein immunofluorescence were similar in SMCs of both vessels with IP(3)R1>>IP(3)R2>IP(3)R3. Despite similar expression of IP(3)Rs and dependence of Ca(2+) waves on IP(3)Rs, these data illustrate pronounced regional heterogeneity in function and expression of RyRs between SMCs of the same vascular resistance network. We conclude that vasomotor control is differentially regulated in feed arteries vs. downstream arterioles.

Autoregulation and conduction of vasomotor responses in a mathematical model of the rat afferent arteriole

We have formulated a mathematical model for the rat afferent arteriole (AA). Our model consists of a series of arteriolar smooth muscle cells and endothelial cells, each of which represents ion transport, cell membrane potential, and gap junction coupling. Cellular contraction and wall mechanics are also represented for the smooth muscle cells. Blood flow through the AA lumen is described by Poiseuille flow. The AA model's representation of the myogenic response is based on the hypothesis that changes in hydrostatic pressure induce changes in the activity of nonselective cation channels. The resulting changes in membrane potential then affect calcium influx through changes in the activity of the voltage-gated calcium channels, so that vessel diameter decreases with increasing pressure values. With this configuration, the model AA maintains roughly stable renal blood flow within a physiologic range of blood flow pressure. Model simulation of vasoconstriction initiated from local stimulation also agrees well with findings in the experimental literature, notably those of Steinhausen et al. (Steinhausen M, Endlich K, Nobiling R, Rarekh N, Schütt F. J Physiol 505: 493-501, 1997), which indicated that conduction of vasoconstrictive response decays more rapidly in the upstream flow direction than downstream. The model can be incorporated into models of integrated renal hemodynamic regulation.

Hydrogen sulfide activates Ca2+ sparks to induce cerebral arteriole dilation

Hydrogen sulfide (H2S) is a gaseous vasodilator produced by endothelial cells. Mechanisms by which H2S induce vasodilation are unclear. We tested the hypothesis that H2S dilates piglet cerebral arterioles by modulating local and global intracellular Ca2+ signals in smooth muscle cells. High‐speed confocal imaging revealed that H2S increased Ca2+ spark frequency ~1.43 fold and decreased global intracellular Ca2+ concentration ([Ca2+]i) by ~37 nM in smooth muscle cells of intact cerebral arteries. In contrast, H2S did not alter Ca2+ wave frequency. In voltage‐clamped (–40 mV) cells, H2S increased the frequency of Ca2+ spark‐induced transient Ca2+‐activated K+ (KCa) currents ~1.8 fold, but did not alter the amplitude of these events. H2S did not alter the activity of single KCa channels recorded in the absence of Ca2+ sparks in arteriole smooth muscle cells. H2S increased SR Ca2+ load ([Ca2+]SR), measured as caffeine (10 mM)‐induced [Ca2+]i transients, ~1.5 fold. H2S dilated pressurized cerebral arterioles. Iberiotoxin, a KCa channel blocker, and ryanodine, a ryanodine receptor (RyR) channel inhibitor, reduced H2S‐induced vasodilation by ~38 and ~37%, respectively. In summary, our data indicate that H2S elevates [Ca2+]SR, leading to Ca2+ spark activation in cerebral arteriole smooth muscle cells. The subsequent elevation in transient KCa current frequency leads to a reduction in global [Ca2+]i and vasodilation.NIH/NHLBI

A Functional Role for N‐cadherin in Mediating Mechanotransduction in Arteriolar Vascular Smooth Muscle Cells

A hybrid atomic force (AFM)‐confocal microscopy system was used to examine whether mechanical forces applied through N‐cadherin adhesions on vascular smooth muscle cells (VSMC) could induce i) cellular contraction and ii) signaling events. AFM probes were modified by attaching a borosilicate microsphere (5 μm) to the tip and coating with either 1) a recombinant N‐cadherin chimera fusion protein (rhNcad), or 2) an N‐cadherin antibody; or 3) attaching a live VSMC to the end of the AFM cantilever. In all 3 cases, a strong adhesion was formed between the AFM probe and cell membrane; and AFM pulling forces (~1000 pN) induced a strong VSMC contractile response that pulled the probe oppositely. In addition, clustering of N‐cadherin‐GFP and RhoA‐GFP was observed when an rhNcad coated AFM probe was engaged on the VSMC membrane, and application of AFM pulling force enhanced the clustering. Immuno‐fluorescence labeling showed a dramatic increase of N‐cadherin clustering in the vessel wall of pressurized arterioles that developed spontaneous myogenic tone compared to unpressurized vessels, as well as spatial correlations between the N‐cadherin and the clusters of active RhoA, myosin II and the phosphorylated myosin phosphatase targeting subunit. Collectively, these results support an important role for N‐cadherin in mediating the myogenic mechanotransduction in VSMC. (AHA 0835676N to Z.S. and NIH P01HL095486 to G.A.M.)