Rat Mesenteric Smooth Muscle Cells Research Articles

Selective Activation of Epac Increases the Frequency of Submembrane Calcium Sparks in Mesenteric Smooth Muscle Cells

The relaxation of vascular smooth muscle, which increases blood vessel diameter, is often mediated through vasodilator-induced elevations of intracellular cyclic AMP (cAMP) [1]. The vasculature expresses three distinct cAMP effectors: cAMP-dependent protein kinase (PKA), cyclic nucleotide-gated (CNG) ion channels and the more recently discovered exchange proteins directly activated by cAMP (Epacs) [2]. The mechanisms by which cAMP induces vasorelaxation are thus complex and diverse. Here we investigate the hypothesis that Epac activation increases the frequency of subsurface Ca2+ sparks within rat mesenteric smooth muscle cells, activating large-conductance Ca2+-activated potassium (BKCa) channels and inducing membrane hyperpolarization and vasorelaxation.In Fluo-4-AM-loaded mesenteric myocytes, application of the Epac-specific cAMP analogue 8-pCPT-2'-O-Me-cAMP-AM (10μM) increased spark frequency from 0.045 ± 0.008 sparks/s/μm under basal conditions to 0.103 ± 0.022 sparks/s/μm (p<0.05). Importantly this increase also occurred in the presence of myristoylated PKI amide (14-22), a potent and selective inhibitor of PKA.Application of 8-pCPT-2'-O-Me-cAMP-AM (5μM) reversibly increased both the frequency (0.94 ± 0.25 to 2.30 ± 0.72 s−1) and amplitude (23.9 ± 3.3 to 35.8 ± 7.7 pA) of spontaneous transient outward currents (STOCs) recorded in isolated mesenteric myocytes (n=7; p<0.05). These currents were sensitive to the selective BKCa channel blocker, iberiotoxin (100nM), and to ryanodine (30μM). In addition, current clamp recordings of isolated myocytes showed a 7.43 ± 0.96 mV (n=4) hyperpolarization in response to exposure to 8-pCPT-2'-O-Me-cAMP-AM (5μM).Our data suggest a novel cAMP-dependent mechanism in mesenteric smooth muscle cells whereby activation of Epac facilitates localized Ca2+ release which activates surface BKCa channels to modulate membrane potential and vascular tone.1. Morgado, M et al (2012). Cell. Mol. Life Sci. 69:2472. Bos, JL (2006). Trends in Biol. Sci. 31:680-686Supported by the British Heart Foundation

Intercellular calcium waves in primary cultured rat mesenteric smooth muscle cells are mediated by Connexin43

Intercellular Ca(2+) wave propagation between vascular smooth muscle cells (SMCs) is associated with the propagation of contraction along the vessel. Here, we characterize the involvement of gap junctions (GJs) in Ca(2+) wave propagation between SMCs at the cellular level. Gap junctional communication was assessed by the propagation of intercellular Ca(2+) waves and the transfer of Lucifer Yellow in A7r5 cells, primary rat mesenteric SMCs (pSMCs), and 6B5N cells, a clone of A7r5 cells expressing higher connexin43 (Cx43) to Cx40 ratio. Mechanical stimulation induced an intracellular Ca(2+) wave in pSMC and 6B5N cells that propagated to neighboring cells, whereas Ca(2+) waves in A7r5 cells failed to progress to neighboring cells. We demonstrate that Cx43 forms the functional GJs that are involved in mediating intercellular Ca(2+) waves and that co-expression of Cx40 with Cx43, depending on their expression ratio, may interfere with Cx43 GJ formation, thus altering junctional communication.

Propagation of fast and slow intercellular Ca 2+ waves in primary cultured arterial smooth muscle cells

Smooth muscle contraction is regulated by changes in cytosolic Ca 2+ concentration ([Ca 2+] i ). In response to stimulation, Ca 2+ increase in a single cell can propagate to neighbouring cells through gap junctions, as intercellular Ca 2+ waves. To investigate the mechanisms underlying Ca 2+ wave propagation between smooth muscle cells, we used primary cultured rat mesenteric smooth muscle cells (pSMCs). Cells were aligned with the microcontact printing technique and a single pSMC was locally stimulated by mechanical stimulation or by microejection of KCl. Mechanical stimulation evoked two distinct Ca 2+ waves: (1) a fast wave (2 mm/s) that propagated to all neighbouring cells, and (2) a slow wave (20 μm/s) that was spatially limited in propagation. KCl induced only fast Ca 2+ waves of the same velocity as the mechanically induced fast waves. Inhibition of gap junctions, voltage-operated calcium channels, inositol 1,4,5-trisphosphate (IP 3) and ryanodine receptors, shows that the fast wave was due to gap junction mediated membrane depolarization and subsequent Ca 2+ influx through voltage-operated Ca 2+ channels, whereas, the slow wave was due to Ca 2+ release primarily through IP 3 receptors. Altogether, these results indicate that temporally and spatially distinct mechanisms allow intercellular communication between SMCs. In intact arteries this may allow fine tuning of vessel tone.

Cyclic stretch decreases capacitative calcium entry in vascular smooth muscle cells from resistance and conduit vessels

An alteration in expression of TRPC channels may affect intracellular Ca2+ homeostasis and cell contractility. We previously showed that cyclic stretch, a model which simulates the elevated vessel strain in hypertension, decreased TRPC4 protein expression in cultured rat aortic smooth muscle cells (RASMC) and rat mesenteric smooth muscle cells (RMSMC). Since TRPC4 proteins have been shown to function as a store-operated channels, we hypothesized that cyclic stretch will also result in a decrease in capacitative Ca2+ entry (CCE). Cells were stretched for 6h using the Flexercell Strain Unit, loaded with fura-2-AM, and fluorescence ratio (340/380nm) was measured. Control cells were not stretched. Baseline Ca2+ levels in the absence of extracellular Ca2+ (0Ca2+) were higher in stretched RASMC (n = 88) than controls (n = 87, P = 0.009). The response to store depletion by cyclopiazonic acid (CPA) in 0Ca2+was decreased in stretched RASMC (P = 0.001). The peak fura-2 ratio and the rate of CCE after reintroduction of extracellular Ca2+ were both decreased by cyclic stretch (P = 0.002 and P < 0.0001). Likewise, stretched RMSMC showed a diminished CPA response (n = 66, P < 0.0001) and a decrease in CCE (P = 0.018) when compared to controls (n = 77). The stretch-induced decrease in TRPC4 expression and CCE may counteract the influx of Ca2+ resulting from increased strain in the vessel wall and prevent an exacerbated myogenic response.

Mechanism of rat mesenteric arterial KATP channel activation by 14,15-epoxyeicosatrienoic acid

Recently, we reported that 11,12-epoxyeicosatrienoic acid (11,12-EET) potently activates rat mesenteric arterial ATP-sensitive K(+) (K(ATP)) channels and produces significant vasodilation through protein kinase A-dependent mechanisms. In this study, we tried to further delineate the signaling steps involved in the activation of vascular K(ATP) channels by EETs. Whole cell patch-clamp recordings [0.1 mM ATP in the pipette, holding potential (HP) = 0 mV and testing potential (TP) = -100 mV] in freshly isolated rat mesenteric smooth muscle cells showed small glibenclamide-sensitive K(ATP) currents (19.0 +/- 7.9 pA, n = 5) that increased 6.9-fold on exposure to 5 microM 14,15-EET (132.0 +/- 29.0 pA, n = 7, P < 0.05 vs. control). With 1 mM ATP in the pipette solution, K(ATP) currents (HP = 0 mV and TP = -100 mV) were increased 3.5-fold on exposure to 1 microM 14,15-EET (57.5 +/- 14.3 pA, n = 9, P < 0.05 vs. baseline). In the presence of 100 nM iberiotoxin, 1 microM 14,15-EET hyperpolarized the membrane potential from -20.5 +/- 0.9 mV at baseline to -27.1 +/- 3.0 mV (n = 6 for both, P < 0.05 vs. baseline), and the EET effects were significantly reversed by 10 microM glibenclamide (-21.8 +/- 1.4 mV, n = 6, P < 0.05 vs. EET). Incubation with 5 microM 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE), a 14,15-EET antagonist, abolished the 14,15-EET effects (31.0 +/- 11.8 pA, n = 5, P < 0.05 vs. 14,15-EET, P = not significant vs. control). The 14,15-EET effects were inhibited by inclusion of anti-G(s)alpha antibody (1:500 dilution) but not by control IgG in the pipette solution. The effects of 14,15-EET were mimicked by cholera toxin (100 ng/ml), an exogenous ADP-ribosyltransferase. Treatment with the ADP-ribosyltransferase inhibitors 3-aminobenzamide (1 mM) or m-iodobenzylguanidine (100 microM) abrogated the effects of 14,15-EET on K(ATP) currents. These results were corroborated by vasodilation studies. 14,15-EET dose-dependently dilated isolated small mesenteric arteries, and this was significantly attenuated by treatment with 14,15-EEZE or 3-aminobenzamide. These results suggest that 14,15-EET activates vascular K(ATP) channels through ADP-ribosylation of G(s)alpha.