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Thrombin-mediated increases in cytosolic [Ca2+] involve different mechanisms in human pulmonary artery smooth muscle and endothelial cells.

ABSTRACT: Thrombin is a procoagulant inflammatory agonist that can disrupt the endothelium-lumen barrier in the lung by causing contraction of endothelial cells and promote pulmonary cell proliferation. Both contraction and proliferation require increases in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)). In this study, we compared the effect of thrombin on Ca(2+) signaling in human pulmonary artery smooth muscle (PASMC) and endothelial (PAEC) cells. Thrombin increased the [Ca(2+)](cyt) in both cell types; however, the transient response was significantly higher and recovered quicker in the PASMC, suggesting different mechanisms may contribute to thrombin-mediated increases in [Ca(2+)](cyt) in these cell types. Depletion of intracellular stores with cyclopiazonic acid (CPA) in the absence of extracellular Ca(2+) induced calcium transients representative of those observed in response to thrombin in both cell types. Interestingly, CPA pretreatment significantly attenuated thrombin-induced Ca(2+) release in PASMC; this attenuation was not apparent in PAEC, indicating that a PAEC-specific mechanism was targeted by thrombin. Treatment with a combination of CPA, caffeine, and ryanodine also failed to abolish the thrombin-induced Ca(2+) transient in PAEC. Notably, thrombin-induced receptor-mediated calcium influx was still observed in PASMC after CPA pretreatment in the presence of extracellular Ca(2+). Ca(2+) oscillations were triggered by thrombin in PASMC resulting from a balance of extracellular Ca(2+) influx and Ca(2+) reuptake by the sarcoplasmic reticulum. The data show that thrombin induces increases in intracellular calcium in PASMC and PAEC with a distinct CPA-, caffeine-, and ryanodine-insensitive release existing only in PAEC. Furthermore, a dynamic balance between Ca(2+) influx, intracellular Ca(2+) release, and reuptake underlie the Ca(2+) transients evoked by thrombin in some PASMC. Understanding of such mechanisms will provide an important insight into thrombin-mediated vascular injury during hypertension.

SUBMITTER: Sacks RS

PROVIDER: S-EPMC2604792 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Thrombin-mediated increases in cytosolic [Ca2+] involve different mechanisms in human pulmonary artery smooth muscle and endothelial cells.

Sacks Richard S RS Firth Amy L AL Remillard Carmelle V CV Agange Negin N Yau Jocelyn J Ko Eun A EA Yuan Jason X-J JX

American journal of physiology. Lung cellular and molecular physiology 20081003 6

Thrombin is a procoagulant inflammatory agonist that can disrupt the endothelium-lumen barrier in the lung by causing contraction of endothelial cells and promote pulmonary cell proliferation. Both contraction and proliferation require increases in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)). In this study, we compared the effect of thrombin on Ca(2+) signaling in human pulmonary artery smooth muscle (PASMC) and endothelial (PAEC) cells. Thrombin increased the [Ca(2+)](cyt) in both cell types ...[more]

PMID: 18836030

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Project description:The chronic phase of pulmonary arterial hypertension (PAH) is associated with vascular remodeling, especially thickening of the smooth muscle layer of large pulmonary arteries and muscularization of small pulmonary vessels, which normally have no associated smooth muscle. Serotonin (5-hydroxytryptamine, 5-HT) has been shown to induce proliferation and hypertrophy of pulmonary artery smooth muscle cells (PASMC), and may be important for in vivo pulmonary vascular remodeling. Here, we show that 5-HT stimulates migration of pulmonary artery PASMC. Treatment with 5-HT for 16h increased migration of PASMC up to four-fold as monitored in a modified Boyden chamber assay. Increased migratory responses were associated with cellular morphological changes and reorganization of the actin cytoskeleton. 5-HT-induced alterations in morphology were previously shown in our laboratory to require cAMP [Lee SL, Fanburg BL. Serotonin produces a configurational change of cultured smooth muscle cells that is associated with elevation of intracellular cAMP. J Cell Phys 1992;150(2):396-405], and the 5-HT4 receptor was pharmacologically determined to be the primary activator of cAMP in bovine PASMC [Becker BN, Gettys TW, Middleton JP, Olsen CL, Albers FJ, Lee SL, et al. 8-Hydroxy-2-(di-n-propylamino)tetralin-responsive 5-hydroxytryptamine4-like receptor expressed in bovine pulmonary artery smooth muscle cells. Mol Pharmacol 1992;42(5):817-25]. We examined the role of the 5-HT4 receptor and cAMP in 5-HT-induced bovine PASMC migration. PASMC express 5-HT4 receptor mRNA, and a 5-HT4 receptor antagonist and a cAMP antagonist completely blocked 5-HT-induced cellular migration. Consistent with our previous report that a cAMP-dependent Cl(-) channel is required for 5-HT-induced morphological changes in PASMC, phenylanthranilic acid, a Cl(-) channel blocker, inhibited actin cytoskeletal reorganization and migration produced by 5-HT. We conclude that 5-HT stimulates PASMC migration and associated cytoskeletal reorganization through the 5-HT4 receptor and cAMP activation of a chloride channel.

Project description:Background and purposealpha(1)-Adrenoceptor agonists induce Ca(2+)-transients in endothelial cells (ECs) of arterioles. However, the presence of alpha(1)-adrenoceptors on arteriolar ECs has not been excluded, and the identity of alpha(1)-adrenoceptor subtypes in arterioles only has been inferred from pharmacology. Therefore, we determined which subtypes were expressed by vascular smooth muscle cells (VSMCs) and ECs, and which subtype mediated alpha(1)-adrenoceptor-induced constriction.Experimental approachEC Ca(2+)-transients in isolated, cannulated hamster cremasteric arterioles or freshly isolated ECs were studied using Fura 2. Arteriolar diameter was measured by video microscopy. alpha(1)-Adrenoceptor expression was assessed by western blot of whole-arteriolar homogenates and real-time RT-PCR on enzymatically isolated VSMCs and ECs.Key resultsPhenylephrine-induced constriction and EC Ca(2+)-transients were abolished by the alpha(1)-adrenoceptor antagonist prazosin (30 nM) in arterioles. Phenylephrine-induced constriction was inhibited by the alpha(1D)-adrenoceptor antagonist BMY 7378 (K(B)=2.96 nM) and the alpha(1A)-adrenoceptor antagonist 5-methylurapidil (K(B)=4.08 nM), suggesting a significant role for alpha(1D)-adrenoceptors. Western blots confirmed alpha(1D)-adrenoceptor expression, but did not detect alpha(1A)-adrenoceptors. VSMCs expressed alpha(1D)- and alpha(1A)-, but not alpha(1B)-, adrenoceptor transcripts. No alpha(1)-adrenoceptor transcripts were detected in ECs. Neither phenylephrine (10 microM) nor noradrenaline (0.1-1 microM) elicited Ca(2+)-transients in freshly isolated ECs, whereas the endothelium-dependent vasodilators methacholine (1 microM) and substance P (100 nM) consistently increased Ca(2+).Conclusions and implicationsWe reject the hypothesis that hamster cremasteric arteriolar ECs express alpha(1)-adrenoceptors and conclude that alpha(1)-adrenoceptor agonists predominantly act on VSMC alpha(1D)-adrenoceptors to cause vasoconstriction and a subsequent rise in EC Ca(2+).

Dataset Information

Thrombin-mediated increases in cytosolic [Ca2+] involve different mechanisms in human pulmonary artery smooth muscle and endothelial cells.

Publications

Thrombin-mediated increases in cytosolic [Ca2+] involve different mechanisms in human pulmonary artery smooth muscle and endothelial cells.

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