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Coordination of intercellular Ca(2+) signaling in endothelial cell tubes of mouse resistance arteries.

ABSTRACT: To test the hypothesis that Ca(2+) responses to GPCR activation are coordinated between neighboring ECs of resistance arteries.EC tubes were freshly isolated from superior epigastric arteries of C57BL/6 mice. Intercellular coupling was tested using microinjection of propidium iodide. Following loading with fluo-4 dye, intracellular Ca(2+) responses to ACh were imaged with confocal microscopy.Cell-to-cell transfer of propidium iodide confirmed functional GJCs. A 1 ?m ACh stimulus evoked Ca(2+) responses (9.8 ± 0.8/min, F/F(0) = 3.11 ± 0.2) which pseudo-line-scan analysis revealed as composed of Ca(2+) waves and spatially restricted Ca(2+) release events. A 100 nm ACh stimulus induced Ca(2+) responses of lower frequency (4.5 ± 0.7/min) and amplitude (F/F(0) = 1.95 ± 0.11) composed primarily of spatially restricted events. The time interval between Ca(2+) waves in adjacent cells (0.79 ± 0.12 s) was shorter (p < 0.05) than that between nonadjacent cells (1.56 ± 0.25 s). Spatially restricted Ca(2+) release events had similar frequencies and latencies between adjacent and nonadjacent cells. Inhibiting intracellular Ca(2+) release with 2-APB, Xestospongin C or thapsigargin eliminated Ca(2+) responses.With moderate GPCR stimulation, localized Ca(2+) release events predominate among cells. Greater GPCR stimulation evokes coordinated intercellular Ca(2+) waves via the ER. Calcium signaling during GPCR activation is complex among cells, varying with stimulus intensity and proximity to actively signaling cells.

SUBMITTER: Socha MJ

PROVIDER: S-EPMC3502682 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Coordination of intercellular Ca(2+) signaling in endothelial cell tubes of mouse resistance arteries.

Socha Matthew J MJ Domeier Timothy L TL Behringer Erik J EJ Segal Steven S SS

Microcirculation (New York, N.Y. : 1994) 20121101 8

<h4>Objective</h4>To test the hypothesis that Ca(2+) responses to GPCR activation are coordinated between neighboring ECs of resistance arteries.<h4>Methods</h4>EC tubes were freshly isolated from superior epigastric arteries of C57BL/6 mice. Intercellular coupling was tested using microinjection of propidium iodide. Following loading with fluo-4 dye, intracellular Ca(2+) responses to ACh were imaged with confocal microscopy.<h4>Results</h4>Cell-to-cell transfer of propidium iodide confirmed fun ...[more]

PMID: 22860994

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Project description:Background and purposeIn small arteries, small conductance Ca²?-activated K? channels (SK(Ca)) and intermediate conductance Ca²?-activated K? channels (IK(Ca)) restricted to the vascular endothelium generate hyperpolarization that underpins the NO- and PGI?-independent, endothelium-derived hyperpolarizing factor response that is the predominate endothelial mechanism for vasodilatation. As neuronal IK(Ca) channels can be negatively regulated by PKA, we investigated whether ?-adrenoceptor stimulation, which signals through cAMP/PKA, might influence endothelial cell hyperpolarization and as a result modify the associated vasodilatation.Experimental approachRat isolated small mesenteric arteries were pressurized to measure vasodilatation and endothelial cell [Ca²?]i , mounted in a wire myograph to measure smooth muscle membrane potential or dispersed into endothelial cell sheets for membrane potential recording.Key resultsIntraluminal perfusion of ?-adrenoceptor agonists inhibited endothelium-dependent dilatation to ACh (1 nM-10 ?M) without modifying the associated changes in endothelial cell [Ca²?]i . The inhibitory effect of ?-adrenoceptor agonists was mimicked by direct activation of adenylyl cyclase with forskolin, blocked by the ?-adrenoceptor antagonists propranolol (non-selective), atenolol (??) or the PKA inhibitor KT-5720, but remained unaffected by ICI 118 551 (??) or glibenclamide (ATP-sensitive K? channels channel blocker). Endothelium-dependent hyperpolarization to ACh was also inhibited by ?-adrenoceptor stimulation in both intact arteries and in endothelial cells sheets. Blocking IK(Ca) {with 1 ?M 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34)}, but not SK(Ca) (50 nM apamin) channels prevented ?-adrenoceptor agonists from suppressing either hyperpolarization or vasodilatation to ACh.Conclusions and implicationsIn resistance arteries, endothelial cell ??-adrenoceptors link to inhibit endothelium-dependent hyperpolarization and the resulting vasodilatation to ACh. This effect appears to reflect inhibition of endothelial IK(Ca) channels and may be one consequence of raised circulating catecholamines.

Project description:Arterial hyperpolarization to acetylcholine (ACh) reflects coactivation of K(Ca)3.1 (IK(Ca)) channels and K(Ca)2.3 (SK(Ca)) channels in the endothelium that transfers through myoendothelial gap junctions and diffusible factor(s) to affect smooth muscle relaxation (endothelium-derived hyperpolarizing factor [EDHF] response). However, ACh can differentially activate K(Ca)3.1 and K(Ca)2.3 channels, and we investigated the mechanisms responsible in rat mesenteric arteries. K(Ca)3.1 channel input to EDHF hyperpolarization was enhanced by reducing external [Ca(2+)](o) but blocked either with forskolin to activate protein kinase A or by limiting smooth muscle [Ca(2+)](i) increases stimulated by phenylephrine depolarization. Imaging [Ca(2+)](i) within the endothelial cell projections forming myoendothelial gap junctions revealed increases in cytoplasmic [Ca(2+)](i) during endothelial stimulation with ACh that were unaffected by simultaneous increases in muscle [Ca(2+)](i) evoked by phenylephrine. If gap junctions were uncoupled, K(Ca)3.1 channels became the predominant input to EDHF hyperpolarization, and relaxation was inhibited with ouabain, implicating a crucial link through Na(+)/K(+)-ATPase. There was no evidence for an equivalent link through K(Ca)2.3 channels nor between these channels and the putative EDHF pathway involving natriuretic peptide receptor-C. Reconstruction of confocal z-stack images from pressurized arteries revealed K(Ca)2.3 immunostain at endothelial cell borders, including endothelial cell projections, whereas K(Ca)3.1 channels and Na(+)/K(+)-ATPase alpha(2)/alpha(3) subunits were highly concentrated in endothelial cell projections and adjacent to myoendothelial gap junctions. Thus, extracellular [Ca(2+)](o) appears to modify K(Ca)3.1 channel activity through a protein kinase A-dependent mechanism independent of changes in endothelial [Ca(2+)](i). The resulting hyperpolarization links to arterial relaxation largely through Na(+)/K(+)-ATPase, possibly reflecting K(+) acting as an EDHF. In contrast, K(Ca)2.3 hyperpolarization appears mainly to affect relaxation through myoendothelial gap junctions. Overall, these data suggest that K(+) and myoendothelial coupling evoke EDHF-mediated relaxation through distinct, definable pathways.

Project description:BackgroundEndothelial dysfunction is an early pathogenic event in the progression of cardiovascular disease in patients with Type 2 Diabetes (T2D). Endothelial KCa2.3 and KCa3.1 K+ channels are important regulators of arterial diameter, and we thus hypothesized that SKA-31, a small molecule activator of KCa2.3 and KCa3.1, would positively influence agonist-evoked dilation in myogenically active resistance arteries in T2D.MethodologyArterial pressure myography was utilized to investigate endothelium-dependent vasodilation in isolated cremaster skeletal muscle resistance arteries from 22 to 24 week old T2D Goto-Kakizaki rats, age-matched Wistar controls, and small human intra-thoracic resistance arteries from T2D subjects. Agonist stimulated changes in cytosolic free Ca2+ in acutely isolated, single endothelial cells from Wistar and T2D Goto-Kakizaki cremaster and cerebral arteries were examined using Fura-2 fluorescence imaging.Main findingsEndothelium-dependent vasodilation in response to acetylcholine (ACh) or bradykinin (BK) was significantly impaired in isolated cremaster arteries from T2D Goto-Kakizaki rats compared with Wistar controls, and similar results were observed in human intra-thoracic arteries. In contrast, inhibition of myogenic tone by sodium nitroprusside, a direct smooth muscle relaxant, was unaltered in both rat and human T2D arteries. Treatment with a threshold concentration of SKA-31 (0.3 μM) significantly enhanced vasodilatory responses to ACh and BK in arteries from T2D Goto-Kakizaki rats and human subjects, whereas only modest effects were observed in non-diabetic arteries of both species. Mechanistically, SKA-31 enhancement of evoked dilation was independent of vascular NO synthase and COX activities. Remarkably, SKA-31 treatment improved agonist-stimulated Ca2+ elevation in acutely isolated endothelial cells from T2D Goto-Kakizaki cremaster and cerebral arteries, but not from Wistar control vessels. In contrast, SKA-31 treatment did not affect intracellular Ca2+ release by the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor cyclopiazonic acid.ConclusionsCollectively, our data demonstrate that KCa channel modulation can acutely restore endothelium-dependent vasodilatory responses in T2D resistance arteries from rats and humans, which appears to involve improved endothelial Ca2+ mobilization.

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Coordination of intercellular Ca(2+) signaling in endothelial cell tubes of mouse resistance arteries.

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Coordination of intercellular Ca(2+) signaling in endothelial cell tubes of mouse resistance arteries.

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