Single Vascular Smooth Muscle Cells Research Articles

Emodin activates BK channel in vascular smooth muscle cells and relaxes the interlobar renal artery of rat

AimThe purpose of this study was to investigate the mechanical and electrophysiological effects of emodin on BK channels in the IRASMCs, of the rat. MethodsIsolated interlobar renal artery was used for vascular reactivity measurements using a pressure myograph system. Electrophysiological measurements of single vascular smooth muscle cells were conducted using whole-cell and cell-attached patch-clamp recording. Laser scanning confocal microscope technology was used to measure cytosolic calcium ion signals. Key resultsEmodin relaxed the interlobar renal artery and enhanced the outward currents amplitude of IRASMCs in a concentration-dependent manner, and IbTX inhibited these emodin-induced outward currents. Incubation of IRASMCs in a calcium ion free medium for 30 min decreased the observed effects of emodin on IRASMCs membrane currents. Furthermore, the application of nimodipine, an L-Type calcium ion channel blocker, ryanodine, a ryanodine receptor modifier, and heparin, an IP3 receptor blocker, decreased the emodin-induced BK channel currents, respectively. BAPTA-AM, a selective calcium ion chelator, abolished the emodin-induced BK channel currents. Emodin repolarized cytomembrane and enhanced BK channel open probabilities and elevated cytosolic calcium ion concentration. ConclusionThe vasorelaxant effect of emodin on vessels is mediated through the activation of BK channels.

Architecture-Dependent Mechano-Adaptation in Single Vascular Smooth Muscle Cells.

Arteries grow and remodel following mechanical perturbation. Vascular smooth muscle cells (VSMCs) within the artery undergo hyperplasia, hypertrophy, or change their contractility following sustained changes in loading. Experimental evidence in vivo and in vitro suggests that VSMCs grow and remodel to maintain a constant transmural stress, or "target" stress. This behavior is often described using a stress-dependent finite growth framework. Typically, computational models of arterial growth and remodeling account for VSMC behavior in a constrained mixture formulation that incorporates behavior of each component of the artery. However, these models do not account for differential VSMC architecture observed in situ, which may significantly influence growth and remodeling behavior. Here, we used cellular microbiaxial stretching (CμBS) to characterize how VSMCs with different cytoskeletal architectures respond to a sustained step change in strain. We find that VSMC F-actin architecture becomes more aligned following stretch and retains this alignment after 24 h. Further, we find that VSMC stress magnitude depends on cellular architecture. Qualitatively, however, stress behavior following stretch is consistent across cell architectures-stress increases following stretch and returns to prestretch magnitudes after 24 h. Finally, we formulated an architecture-dependent targeted growth law that accounts for experimentally measured cytoskeletal alignment and attributes stress evolution to individual fiber growth and find that this model robustly captures long-term stress evolution in single VSMCs. These results suggest that VSMC mechano-adaptation depends on cellular architecture, which has implications for growth and remodeling in regions of arteries with differential architecture, such as at bifurcations.

Probing Single-Cell Mechanical Allostasis Using Ultrasound Tweezers.

In response to external stress, cells alter their morphology, metabolic activity, and functions to mechanically adapt to the dynamic, local environment through cell allostasis. To explore mechanotransduction in cellular allostasis, we applied an integrated micromechanical system that combines an 'ultrasound tweezers'-based mechanical stressor and a Förster resonance energy transfer (FRET)-based molecular force biosensor, termed "actinin-sstFRET," to monitor in situ single-cell allostasis in response to transient stimulation in real time. The ultrasound tweezers utilize 1Hz, 10-s transient ultrasound pulses to acoustically excite a lipid-encapsulated microbubble, which is bound to the cell membrane, and apply a pico- to nano-Newton range of forces to cells through an RGD-integrin linkage. The actinin-sstFRET molecular sensor, which engages the actin stress fibers in live cells, is used to map real-time actomyosin force dynamics over time. Then, the mechanosensitive behaviors were examined by profiling the dynamics in Ca2+ influx, actomyosin cytoskeleton (CSK) activity, and GTPase RhoA signaling to define a single-cell mechanical allostasis. By subjecting a 1Hz, 10-s physical stress, single vascular smooth muscle cells (VSMCs) were observed to remodeled themselves in a biphasic mechanical allostatic manner within 30min that caused them to adjust their contractility and actomyosin activities. The cellular machinery that underscores the vital role of CSK equilibrium in cellular mechanical allostasis, includes Ca2+ influx, remodeling of actomyosin CSK and contraction, and GTPase RhoA signaling. Mechanical allostasis was observed to be compromised in VSMCs from patients with type II diabetes mellitus (T2DM), which could potentiate an allostatic maladaptation. By integrating tools that simultaneously permit localized mechanical perturbation and map actomyosin forces, we revealed distinct cellular mechanical allostasis profiles in our micromechanical system. Our findings of cell mechanical allostasis and maladaptation provide the potential for mechanophenotyping cells to reveal their pathogenic contexts and their biophysical mediators that underlie multi-etiological diseases such as diabetes, hypertension, or aging.

Disease-relevant transcriptional signatures identified in individual smooth muscle cells from healthy mouse vessels

Vascular smooth muscle cells (VSMCs) show pronounced heterogeneity across and within vascular beds, with direct implications for their function in injury response and atherosclerosis. Here we combine single-cell transcriptomics with lineage tracing to examine VSMC heterogeneity in healthy mouse vessels. The transcriptional profiles of single VSMCs consistently reflect their region-specific developmental history and show heterogeneous expression of vascular disease-associated genes involved in inflammation, adhesion and migration. We detect a rare population of VSMC-lineage cells that express the multipotent progenitor marker Sca1, progressively downregulate contractile VSMC genes and upregulate genes associated with VSMC response to inflammation and growth factors. We find that Sca1 upregulation is a hallmark of VSMCs undergoing phenotypic switching in vitro and in vivo, and reveal an equivalent population of Sca1-positive VSMC-lineage cells in atherosclerotic plaques. Together, our analyses identify disease-relevant transcriptional signatures in VSMC-lineage cells in healthy blood vessels, with implications for disease susceptibility, diagnosis and prevention.

Propofol-induced vasodilation of mesenteric arterioles via BKCa channel and gap junction.

The present study aimed to investigate the role of propofol in mediating the vasomotor activity of the mesenteric arteriole (MA) of Sprague Dawley (SD) rats, and to elucidate the underlying mechanisms. The pressure myograph technique was used to examine the effect of different concentrations of propofol on the relaxation of blood vessels in the 2–3 mm MA segments freshly separated from the SD rats. The whole-cell patch-clamp technique was applied to observe the outward current of single vascular smooth muscle cells (VSMCs) obtained from the MAs of the SD rats. Furthermore, immunofluorescence was utilized to assess the expression of connexin (Cx) in the MAs of SD rats. The results indicated the following: i) Propofol relaxed the MA of SD rats in a concentration-dependent manner from 1×10−7 to 3×10−4 mol/l; ii) in the acutely dissociated VSMCs, propofol (1×10−7 to 3×10−4 mol/l) enhanced the outward current of VSMCs in a concentration-dependent manner; iii) the enhanced outward currents induced by propofol (1×10−5 mol/l) may be reversed by tetraethylammonium (TEA; 1 mmol/l), a calcium-activated K+ channel inhibitor; iv) the effect of propofol on the relaxation of the vasculature wAS reduced after perfusion with 1 mmol/l TEA; v) Cx40, Cx43 and Cx45 were expressed on the MA; 6) 18β-glycyrrhetintic acid and 2-aminoethoxydiphenyl borate, two types of gap junction blocker, inhibited the propofol-induced relaxation. The present study provides evidence that propofol relaxes the MA, which may be associated with its effect of enhancing the channel current of large-conductance calcium voltage-activated potassium channels, contributing to the K+ outflow and leading to VSMC hyperpolarization; the gap junction may facilitate the hyperpolarization, which may lead to vascular synchronized relaxation and thereby reduce the blood pressure.

Peri/epicellular protein disulfide isomerase PDI acts as an organizer of cytoskeletal mechanoadaptation in vascular smooth muscle cells

Mechanobiologycal and redox processes sinergize to regulate physiological or pathological vascular conditions. More specifically, adaptations to cyclic stretch on vascular smooth muscle cells (VSMC) have been shown to be redox‐regulated. However, mechanisms connecting mechanoadaptation to oxidant generation are still unclear. Previously, we reported that protein disulfide isomerase A1 (PDI), a redox chaperone from endoplasmic reticulum, is targeted to peri/epicellular (pec) space and supports an anti‐constrictive remodeling effect in balloon‐injured arteries by means of cytoskeleton and extracellular matrix architecture organization. We hypothesized that pec PDI acts as a global redox adaptor by connecting oxidant generation with responses to extra‐ or intracellular forces. First, pecPDI inhibition through specific neutralizing antibody (PDI Ab) incubation, prevented stress fiber assembly in response to prolonged exposure to equibiaxial stretch (10–12% at 1 Hz for 24 h). In addition, uniaxial stretch promotes cell repositioning perpendicularly to stretch orientation. Such response was also regulated by pecPDI, as PDI Ab treatment attenuated cell alignment in stretched VSMC at 4h. Through biotinylation experiments followed by western detection, we showed that pecPDI sustains a pro‐oxidant effect on beta1 integrin thiols. To address whether pecPDI organizes intracellular force distribution in such events, we used traction force microscopy, which revealed a significantly decreased net contractile moment in PDGF‐exposed VSMC after pecPDI neutralization (0.70 ±0.08 AU vs control=0.9 ±0.09, p<0.05). Thus, pecPDI involvement in mechanoresponse is not only limited to external forces. The balance between intracellular traction forces and cell adhesion governs cell migration. Indeed, in a model of single VSMC migration, pecPDI impaired migration persistence without affecting total distance or velocity. Since the Rho GTPase RhoA is known to act as a master mechanoregulator, we addressed if downstream pecPDI‐related mechanisms involve RhoA. Neither RhoA expression nor total activity were affected by pecPDI inhibition. However, the polarized distribution of RhoA or caveolin‐3 clusters promoted by cyclic stretch was disrupted by pecPDI inhibition, which promoted a non‐polarized pattern of RhoA/caveolin‐3 cluster colocalization. Moreover, we searched for pecPDI effects on localized RhoA activity using a FRET biosensor. The higher local RhoA activity at cell protrusions compared to perinuclear regions was disrupted by pecPDI neutralization, confirming its regulation of localized RhoA activation. In conclusion, pecPDI acts as a redox organizer able to restrict the noise of cytoskeletal repositioning during mechanoresponses in VSMC. This effect may have several implications, including the role of redox processes and pecPDI on vascular remodeling.Support or Funding InformationSupported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), CEPID‐Redoxoma grant 2013/07937‐8 and scholarship grant 2013/17115‐5This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Proliferative Role of Kv11 Channels in Murine Arteries.

K+ channels encoded by the ether-a-go-go related gene (ERG1 or KCNH2) are important determinants of the cardiac action potential. Expression of both cardiac isoforms (ERG1a and ERG1b) were identified in murine portal vein and distinctive voltage-gated K+ currents were recorded from single myocytes. The aim of the present study was to ascertain the expression and functional impact of ERG channels in murine arteries.Methods: Quantitative RT-PCR was undertaken on RNA extracted from a number of murine arteries. Immunofluorescence was performed on single vascular smooth muscle cells using antibodies against the ERG1 expression product (Kv11.1). Single cell electrophysiology was performed on myocytes from portal vein and several different arteries, complimented by isometric tension recordings. Proliferation assays were undertaken on smooth muscle cells isolated from femoral arteries.Results: ERG1 transcripts were detected in all murine blood vessels, and Kv11.1 immunofluorescence was observed in all smooth muscle cells. However, K+ currents with properties consistent with ERG channels were only recorded in portal vein myocytes. Moreover, ERG channel blockers (E4031 or dofetilide, 1 μM) failed to depolarize carotid arteries or produce contraction. Proliferation of arterial smooth muscle cells was associated with a marked increase in ERG1 expression and ERG blockers suppressed proliferation significantly.Conclusions: These data reveal that arterial blood vessels express ERG channels that appear to be functional silent in contractile smooth muscle but contribute to proliferative response.

Reduction of Uterine Perfusion Pressure Induced Redistribution of Endothelin Receptor Type-B Between the Intima and Media Contributes to the Pathogenesis of Pregnancy-Induced Hypertension

Studies have shown that a change in endothelin receptor expression in the artery is related to pregnancy-induced hypertension (PIH). However, the mechanism underlying this change remains unclear. To test whether the distribution of endothelin receptor type-A (ETAR) and type-B (ETBR) plays an important role in PIH, a reduction of uterine perfusion pressure (RUPP) rat model was used to mimic some of the features of PIH; the resulting variable endothelin receptor expression was investigated in the media and intima of the aorta. Single vascular smooth muscle cells (VSMCs) were isolated from RUPP and normal pregnant (NP) rats to study the effect of ETAR and ETBR in smooth muscle cells. Compared with NP rats, RUPP rats had a significant redistribution of ETBR expression in the intima and media, while there was no significant difference in ETAR expression between the two groups. ETBR upregulation in VSMCs enhanced cellular contraction and contributed to PIH. The TNF-α plasma levels in RUPP rats were two-fold higher than those of NP rats, which upregulated the expression of ETBR in VSMCS through the NF-κB pathways in RUPP rats. Redistribution of ETBR between the media and intima played an important role in the pathogenesis of PIH.

Abstract 15: Increased Intrinsic Stiffness of Aortic Vascular Smooth Muscle Cells as a Mechanism for Increased Aortic Stiffness in Spontaneously Hypertensive Rats

An increase in vascular stiffness is a fundamental component of hypertension. Our hypothesis is that increased large artery stiffness in hypertension is due in part to intrinsic mechanical properties of vascular smooth muscle cells (VSMC). Adult spontaneously hypertensive rats (SHR) (16 weeks old) and age-matched Wistar-Kyoto normotensive (WKY) rats were studied. Aortic pressure, measured with a Millar catheter, was higher in SHR than WKY (MAP 123±4 versus 96±6 mmHg, p <0.05). Aortic stiffness, measured with pulse wave velocity, in vivo, was 2 to 2.5 fold higher in SHR rats (7.65 mm/ms) compared to normotensive rats(3.27 mm/ms, p<0.01 ). Aortic VSMCs were isolated from the SHR and WKY rats. Stiffness in single VSMC was continuously recorded in vitro using atomic force microscopy and temporal oscillations in VSMC elasticity, which represent the dynamic properties of elasticity, were observed and analyzed by Eigen decomposition. VSMC stiffness was consistently increased by 1.5 to 2 fold in SHR (24.0 kPa) versus WKY (11.8 kPa) ( p<0.05) . Moreover, the oscillations in elasticity were significantly different between the two groups in terms of frequency and amplitude ( p<0.05) . ML-7, a highly specific inhibitor of myosin light chain kinase (MLCK), produced a greater reduction in the VSMC elasticity from SHR aorta compared to WKY rats ( p<0.05 ), and also eliminated the difference between the SHR and WKY. Furthermore, ML-7 altered the oscillation in VSMC elasticity in SHR, but not WKY. These observations indicate that there is an inherently altered VSMC stiffness in hypertension that is associated with unique dynamic cytoskeletal behavior. These dynamic elements may be involved mechanistically in increasing aortic stiffness in SHR rats. MLCK participates in the regulation of VSMC stiffness in hypertension via altering both basal VSMC elasticity and temporal oscillations.

Vasoactive agents result in coordinated changes in vascular smooth muscle cell elasticity and extracellular matrix adhesion

Coordination of mechanotransduction and force transmission within the blood vessel wall requires a physical adhesion of vascular smooth muscle cells (VSMCs) to extracellular matrix proteins (ECM) and surrounding VSMCs. Our hypothesis was that integrin adhesion to the ECM is dynamically up‐regulated in VSMC during contractile activation and likewise down‐regulated during relaxation to support changes in vessel diameter and wall tension. Real‐time measurements of cell adhesion and cell elasticity were obtained with atomic force microscopy (AFM) in response to angiotensin II (Ang II) and adenosine (Ado). ECM adhesion behavior was evaluated with fibronectin (FN) coated microbead‐tipped (5 μm dia) AFM probes. Single VSMCs were isolated from rat cremaster skeletal muscle arterioles and cultured 3–8 days before AFM experiment. The results show that the administration of Ang II (10−6M) in cell bath immediately increased the strength of adhesion between the AFM probe and cell as well as probability of binding between FN and the cell. Cell stiffness also increased in parallel with adhesion upon Ang II stimulation. In contrast, Ado (10−4M) decreased cell elasticity and the adhesion of integrins to FN. These findings support the hypothesis that a vasoconstrictor can activate through inside‐out mechanisms both the contractile process and cell adhesion to the ECM in a coordinated fashion, while a vasodilator has the opposite effects. The role of the changes in adhesion may be related to altered mechanics or cell signaling involving the focal adhesion sites. (1P01HL095486 to G.A.M. and AHA 0835676N to Z.S.)

Swelling rather than shrinkage precedes apoptosis in serum-deprived vascular smooth muscle cells

Contrasting cell volume behaviours (swelling vs. shrinkage) are considered as criteria to distinguish necrosis from apoptosis. In this study, we employed a time-lapse, dual-image surface reconstruction technique to assess the volume of single vascular smooth muscle cells transfected with E1A-adenoviral protein (E1A-VSMC) and undergoing rapid apoptosis in the absence of growth factors or in the presence of staurosporine. After 30- to 60-min lag-phase, serum-deprived E1A-VSMC volume was increased by ~40%, which preceded maximal increments of caspase-3 activity and chromatin cleavage. Swollen cells underwent rapid apoptotic collapse, documented by plasma membrane budding, and terminated in 10-15min by the formation of numerous apoptotic bodies. Suppression of apoptosis by inhibition of Na(+),K(+)-ATPase and activation of cAMP signalling with ouabain and forskolin, respectively, completely abolished the swelling of serum-deprived E1A-VSMC. In contrast to serum deprivation, apoptotic collapse of staurosporine-treated E1A-VSMC preceded attenuation of their volume by ~30%. Neither transient hyposmotic swelling nor isosmtotic shrinkage triggered apoptosis. Our results show that cell shrinkage can not be considered as ubiquitous hallmark of apoptosis. The involvement of stimulus-specific cell volume perturbations in initiation and progression of apoptosis in vascular smooth muscle cells should be examined further.

Increased Intrinsic Stiffness of Aortic Vascular Smooth Muscle Cells as a Mechanism for Increased Aortic Stiffness in Spontaneously Hypertensive Rats

An increase in vascular stiffness is a fundamental component of hypertension. Our hypothesis is that the increased large artery stiffness in hypertension is due in part to intrinsic properties of vascular smooth muscle cells (VSMCs). Adult spontaneously hypertensive rats (SHR) (16 weeks old) and age-matched Wistar-Kyoto normotensive (WKY) rats were studied. Aortic pressure, measured with a Millar catheter, was higher in SHR than WKY (mean arterial pressure 123±4 vs. 96±6 mmHg). Aortic stiffness, measured with Doppler imaging echocardiography, in vivo, was 2 to 2.5 fold higher, p<0.05, in hypertensive rats compared to normotensive rats. Aortic tissue rings and VSMCs were isolated from the SHR and WKY rats and VSMC stiffness was measured, in vitro, in both a reconstituted tissue model and with atomic force microscopy (AFM) in single VSMC's. The continuous force curves with the AFM in single VSMCs were collected and the temporal variation in VSMC elasticity was evaluated by Eigen decompensation. VSMC stiffness was consistently increased by 1.5 to 2 fold in SHR vs. WKY (p<0.05). Moreover, the oscillations in elasticity were significantly different between the two groups in terms of frequency and amplitude (p<0.05). These observations suggest that not only inherently altered VSMC stiffness, but also unique dynamic elements, are involved in the mechanism of increased aortic stiffness in hypertensive rats.

A silicone-based stretchable micropost array membrane for monitoring live-cell subcellular cytoskeletal response

External forces are increasingly recognized as major regulators of cellular structure and function, yet the underlying mechanism by which cells sense forces and transduce them into intracellular biochemical signals and behavioral responses ('mechanotransduction') is largely undetermined. To aid in the mechanistic study of mechanotransduction, herein we devised a cell stretching device that allowed for quantitative control and real-time measurement of mechanical stimuli and cellular biomechanical responses. Our strategy involved a microfabricated array of silicone elastomeric microposts integrated onto a stretchable elastomeric membrane. Using a computer-controlled vacuum, this micropost array membrane (mPAM) was activated to apply equibiaxial cell stretching forces to adherent cells attached to the microposts. Using the mPAM, we studied the live-cell subcellular dynamic responses of contractile forces in vascular smooth muscle cells (VSMCs) to a sustained static equibiaxial cell stretch. Our data showed that in response to a sustained cell stretch, VSMCs regulated their cytoskeletal (CSK) contractility in a biphasic manner: they first acutely enhanced their contraction to resist rapid cell deformation ('stiffening') before they allowed slow adaptive inelastic CSK reorganization to release their contractility ('softening'). The contractile response across entire single VSMCs was spatially inhomogeneous and force-dependent. Our mPAM device and live-cell subcellular contractile measurements will help elucidate the mechanotransductive system in VSMCs and thus contribute to our understanding of pressure-induced vascular disease processes.

Effects of vasoconstrictors on the biomechanical properties of single vascular smooth muscle cells

Vascular diameter is regulated by vasoconstrictors and vasodilators. Physical coupling of vascular smooth muscle cells (VSMC) to one another and to extracellular matrix (ECM) are necessary for a vessel to mechanically function. We hypothesized that VSMC adhesion to ECM may also be modulated by vasoactive agonists as one of the temporal events in the contractile process. Atomic force microscopy with fibronectin-functionalized probes was employed to investigate biomechanics of rat cremaster arterioles VSMC. Cellular responses were recorded by real time measurement of cell membrane displacement and cell elasticity in absence and presence of endothelin-1 (ET-1) or angiotensin II (AII). The results show that ET-1 (10−8M) or AII (10−9-10−6M) immediately cause vertical cell membrane displacement by 100–300 nm. An increase in cell elasticity followed at 20–30 min post ET-1 or AII treatment. These findings suggest that activation of the contractile apparatus rapidly causes cell shape change but other cytoskeletal changes occur more slowly. Further studies are required to clarify the molecular mechanisms linking cell contraction and elasticity with adhesion. (1P01HL095486 to G.A.M. and AHA 0835676N to Z.S.)

Atomic force microscope studies demonstrate increased vascular smooth muscle cell stiffness associated with aging

Vascular stiffness has been shown to increase with aging due largely to changes in the properties of the extracellular matrix (ECM). We hypothesized that increased stiffness of vascular smooth muscle cells (VSMC) could also play a role in vascular stiffness changes associated with aging. Studies were conducted on VSMCs from the thoracic aorta of old (30 mo) and young (6 mo) F344xBN rats and from old (24–25 yr) and young (5–8 yr) male monkeys. Stiffness of single VSMCs was measured using a nanoindentation technique with atomic force microscopy. The estimated elastic modulus was significantly increased (p&lt;0.001) in VSMCs from old rats (30.1±0.4 kPa; 57 cells) compared to young rats (14.±0.2 kPa; 140 cells) and similarly, was significantly higher (p&lt;0.001) in old monkeys (41.7±0.5 kPa; 76 cells) than in young monkeys (12.8±0.3 kPa; 40 cells). Measurements of stiffness for 30 min indicated that stiffness oscillates in VSMCs and that these oscillations are enhanced in older (period ~8 min) vs younger (period ~18 min) animals. These data support the concept that the increased vascular stiffness that occurs during aging includes mechanical contributions from an increased stiffness at the level of the single VSMC.

Daidzein relaxes rat cerebral basilar artery via activation of large-conductance Ca2+-activated K+ channels in vascular smooth muscle cells

Daidzein, a phytoestrogen, has been reported to produce vasodilation via inhibition of Ca(2+) inflow. However, the involvement of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels in the effect of daidzein is debated. Therefore, the present study was designed to investigate the effect of daidzein on the rat cerebral basilar artery and the underlying molecular mechanisms. Isolated cerebral basilar artery rings and single vascular smooth muscle cells (VSMCs) were used for vascular reactivity and electrophysiology measurements, to investigate the effect of daidzein on BK(Ca) channels in cerebral basilar artery smooth muscle. In addition, the human BK(Ca) channel alpha-subunit gene (hslo) was transfected into HEK293 cells, to directly assess whether daidzein activates BK(Ca) channels. The results showed that daidzein produced a concentration-dependent but endothelium-independent relaxation in rat cerebral basilar arteries. Paxilline, a selective BK(Ca) channel blocker, significantly inhibited the daidzein-induced vasodilation, whereas NS1619, a selective BK(Ca) channel opener, enhanced the vasodilation. In the whole-cell configuration, daidzein increased noisy oscillation currents in cerebral basilar artery VSMCs in a concentration-dependent manner, and washout of daidzein or blockade of BK(Ca) channels with paxilline fully reversed the increase. However, daidzein did not substantially affect hSlo currents in HEK293 cells when applied to the outside of the cell membrane. In conclusion, these results indicate that the activation of BK(Ca) channels in VSMCs at least partly contributes to the daidzein-induced vasodilation of the rat cerebral basilar artery. The beta1-subunit of BK(Ca) channels plays a critical role in the activation of BK(Ca) currents by daidzein.

Gender-Specific Reduction in Contraction is Associated with Increased Estrogen Receptor Expression in Single Vascular Smooth Muscle Cells of Female Rat

Gender differences in the incidence of cardiovascular disease have been related to plasma estrogen levels; however, the role of vascular estrogen receptor (ER) subtypes in these sex differences is less clear. We tested whether the gender differences in vascular smooth muscle (VSM) function reflect differential expression/activity of ERα, ERβ and the newly-identified GPR30. Single aortic VSM cells (VSMCs) were freshly isolated from male and female Sprague-Dawley rats, and their contraction to phenylephrine (PHE, 10^-5 M), AngII (10^-7 M) and membrane-depolarization by KCl (51 mM) was measured in the absence or presence of 10^-6 M 17β-estradiol (E2, stimulant of most ERs), PPT (ERα agonist), DPN (ERβ agonist), and ICI 182,780 (an ERα/ERβ antagonist with GPR30 agonistic properties). The cells were fixed and fluorescently labeled with ERα, ERβ or GPR30 antibody, and the subcellular distribution of ERs was examined using digital imaging microscopy. The mRNA expression and protein amount of aortic ER subtypes was examined using RT-PCR and Western blots. PHE, AngII, and KCl caused less contraction in VSMCs of females than males. Pretreatment of VSMCs with E2 reduced PHE-, AngII- and KCl-induced contraction in both males and females. PPT caused similar inhibition of PHE-, AngII- and KCl-induced contraction as E2, suggesting a role of ERα. DPN mainly inhibited PHE and KCl contraction, suggesting an interaction between ERβ and Ca²⁺ channels. ICI 182,780 did not reduce aortic VSMC contraction, suggesting little role for GPR30. RT-PCR and Western blots revealed greater expression of ERα and ERβ in VSMCs of females than males, but similar amounts of GPR30. The total immunofluorescence signal for ERα and ERβ was greater in VSMCs of females than males, and was largely localized in the nucleus. GPR30 fluorescence was similar in VSMCs of males and females, and was mainly in the cytosol. In PPT treated cells, nuclear ERα signal was enhanced. DPN did not affect the distribution of ERβ, and ICI 182,780 did not significantly increase GPR30 in the cell surface. Thus, ER subtypes demonstrate similar responsiveness to specific agonists in VSMCs of male and female rats. The reduced contraction in VSMCs of females could be due to gender-related increase in the expression of ERα and ERβ.

Elevations of intracellular calcium reflect normal voltage-dependent behavior, and not constitutive activity, of voltage-dependent calcium channels in gastrointestinal and vascular smooth muscle

In smooth muscle, the gating of dihydropyridine-sensitive Ca2+ channels may either be stochastic and voltage dependent or coordinated among channels and constitutively active. Each form of gating has been proposed to be largely responsible for Ca2+ influx and determining the bulk average cytoplasmic Ca2+ concentration. Here, the contribution of voltage-dependent and constitutively active channel behavior to Ca2+ signaling has been studied in voltage-clamped single vascular and gastrointestinal smooth muscle cells using wide-field epifluorescence with near simultaneous total internal reflection fluorescence microscopy. Depolarization (−70 to +10 mV) activated a dihydropyridine-sensitive voltage-dependent Ca2+ current (ICa) and evoked a rise in [Ca2+] in each of the subplasma membrane space and bulk cytoplasm. In various regions of the bulk cytoplasm the [Ca2+] increase ([Ca2+]c) was approximately uniform, whereas that of the subplasma membrane space ([Ca2+]PM) had a wide range of amplitudes and time courses. The variations that occurred in the subplasma membrane space presumably reflected an uneven distribution of active Ca2+ channels (clusters) across the sarcolemma, and their activation appeared consistent with normal voltage-dependent behavior. Indeed, in the present study, dihydropyridine-sensitive Ca2+ channels were not normally constitutively active. The repetitive localized [Ca2+]PM rises (“persistent Ca2+ sparklets”) that characterize constitutively active channels were observed rarely (2 of 306 cells). Neither did dihydropyridine-sensitive constitutively active Ca2+ channels regulate the bulk average [Ca2+]c. A dihydropyridine blocker of Ca2+ channels, nimodipine, which blocked ICa and accompanying [Ca2+]c rise, reduced neither the resting bulk average [Ca2+]c (at −70 mV) nor the rise in [Ca2+]c, which accompanied an increased electrochemical driving force on the ion by hyperpolarization (−130 mV). Activation of protein kinase C with indolactam-V did not induce constitutive channel activity. Thus, although voltage-dependent Ca2+ channels appear clustered in certain regions of the plasma membrane, constitutive activity is unlikely to play a major role in [Ca2+]c regulation. The stochastic, voltage-dependent activity of the channel provides the major mechanism to generate rises in [Ca2+].

Voltage-Dependent Ca2+ Channels Are Clustered But Not Constitutively-Active In Smooth Muscle

The organization and distribution of Ca2+ signals derived from depolarization-evoked Ca2+ entry has been studied in voltage-clamped single vascular and gastrointestinal smooth muscle cells using widefield epi-fluorescence with near simultaneous (2 ms) total internal reflection fluorescence microscopy. Depolarization activated a voltage-dependent Ca2+ current (ICa) and evoked a rise in [Ca2+] in the subplasma membrane space and bulk cytoplasm. The rise which occurred in various regions of the bulk cytoplasm ([Ca2+]c) was approximately uniform; that of the subplasma membrane space ([Ca2+]PM) had a wide range of amplitudes and time courses. The [Ca2+]PM variations presumably reflected an uneven distribution of active Ca2+ channels (clusters) across the sarcolemma. Constitutive activity in clusters of voltage-dependent Ca2+ channels has been proposed to determine bulk average [Ca2+]c. In the present study, channels are not constitutively active. The repetitive localized [Ca2+]PM rises (‘Ca2+ sparklets’) which characterize constitutively-active channels were observed rarely (<1 in 50 cells). Nor did constitutively-active voltage-dependent Ca2+ channels regulate the bulk average [Ca2+]c. A dihydropyridine blocker of voltage-dependent Ca2+ channels, nimodipine, which blocked ICa and accompanying [Ca2+]c rise, reduced neither the resting bulk average [Ca2+]c (at -70 mV) or the rise in [Ca2+]c which accompanied an increased electrochemical driving force on the ion by hyperpolarization (-130 mV). Activation of protein kinase C with indolactam-V did not induce constitutive channel activity. Thus while voltage-dependent Ca2+ channels appear clustered on the plasma membrane, constitutive activity in the channel is unlikely to play a major role in regulation [Ca2+]c. The voltage-dependent activity of the clustered channels may enable selective activation of various cellular processes by generating a localized rises in subplasma membrane [Ca2+].Supported by the Wellcome Trust and British Heart Foundation

Sensing tension: epithelial sodium channel/acid-sensing ion channel proteins in cardiovascular homeostasis.

The epithelial sodium (Na+) channel (ENaC) plays a critical role in blood pressure regulation by controlling renal salt and water reabsorption. Channel overactivity can lead to severe hypertension and underactivity to salt wasting and hypotension.1 In addition to their role in salt/water homeostasis, recent studies suggest that ENaC proteins, and their relatives, the acid-sensing ion channel (ASIC) proteins, may play more ubiquitous roles in cardiovascular regulation than considered previously. Recent evidence suggests that ENaC/ASIC proteins may act as mechanosensors and chemosensors in the cardiovascular system. ENaC/ASIC proteins are expressed in mechanosensing and chemosensing tissues, such as vascular smooth muscle cells (VSMCs), carotid body glomus cells, and sensory neurons innervating arterial baroreceptors, heart, and skeletal muscle. Disruption of the ENaC/ASIC channels alters myogenic constriction, arterial chemoreceptor and baroreceptor responses, and acid-induced responses in heart and skeletal muscle. This brief review summarizes the evidence supporting a role for ENaC and ASIC proteins in diverse systems of cardiovascular mechanosensing and chemosensing. Together, these studies suggest that ENaC/ASIC proteins contribute to cardiovascular homeostasis by mediating neural and local regulatory mechanisms. ENaC and ASIC proteins are members of a protein family termed the degenerin (DEG)/ENaC/ASIC family. Members of this family are expressed in a wide range of species (nematode Caenorhabditis elegans , Drosophila , and mammals) and participate in diverse biological functions, including neurodegeneration, acid sensation, taste, learning and memory, proprioception, Na+/water transport, and mechanosensation. All of the members of the DEG/ENaC/ASIC family share a highly conserved structure: intracellular N and C termini and 2 membrane-spanning domains separated by a large extracellular domain. Most DEG/ENaC/ASIC proteins form amiloride sensitive, nonvoltage, gated cation channels.1,2 ### C elegans DEGs Members were first identified in the nematode, where a chemically induced mutation caused a subset of neurons to swell and lyse. This phenotype led to the first …