Biomedical subjects

Srabani Mitra

Publications and source records attributed to Srabani Mitra.

6 recordsLinked to original sources

PubMed2006

Increased expression of cyclooxygenase-2 mediates enhanced contraction to endothelin ETA receptor stimulation in endothelial nitric oxide synthase knockout mice.

The aim of this study was to determine whether prolonged loss of NO activity, in endothelial NO synthase knockout (eNOS(-/-)) mice, influences endothelin (ET) ETA receptor-mediated smooth muscle contraction and, if so, to define the underlying mechanism(s). In isolated endothelium-denuded abdominal aortas, contractions to the selective ETA receptor agonist ET-1(1-31) were significantly increased in aortas from eNOS(-/-) compared with wild-type (WT) mice. In contrast, contractions to the alpha1-adrenergic agonist phenylephrine or the thromboxane (TX) A2 analog U-46619 were similar between eNOS(-/-) and WT mice. Immunofluorescent and Western blot analysis demonstrated that the aortic expression of ETA receptors was decreased in eNOS(-/-) compared with WT mice. Contractions evoked by ET-1(1-31), but not phenylephrine, were reduced by inhibition of cyclooxygenase-2 (COX-2) (indomethacin or celecoxib) or of TXA2/prostaglandin H2 receptors (SQ-29548). After COX inhibition, contractions to ET-1(1-31) were no longer increased and were actually decreased in eNOS(-/-) compared with WT aortas. Western blot analysis revealed that endothelium-denuded abdominal aortas express COX-2, but not COX-1, and that expression of COX-2 was significantly increased in eNOS(-/-) compared with WT mice. Contractions to the COX substrate arachidonic acid were also increased in eNOS(-/-) aortas. Furthermore, ET-1(1-31) but not phenylephrine stimulated production of the TXA2 metabolite TXB2, which was increased in eNOS(-/-) compared with WT aortas. Therefore, COX-2 plays a crucial and selective role in ETA-mediated smooth muscle contraction. Furthermore, COX-2 expression is increased in eNOS(-/-) mice, which overcomes a reduced expression of ETA receptors and enables a selective increase in contraction to ETA receptor stimulation.

Animals↗

PubMed2004

Imaging remodeling of the actin cytoskeleton in vascular smooth muscle cells after mechanosensitive arteriolar constriction.

Experiments were performed to determine whether remodeling of the actin cytoskeleton contributes to arteriolar constriction. Mouse tail arterioles were mounted on cannulae in a myograph and superfused with buffer solution. The alpha1-adrenergic agonist phenylephrine (0.1-1 micromol/l) caused constriction that was unaffected by cytochalasin D (300 nmol/l) or latrunculin A (100 nmol/l), inhibitors of actin polymerization. In contrast, each compound abolished the mechanosensitive constriction (myogenic response) evoked by elevation in transmural pressure (PTM; 10-60 or 90 mmHg). Arterioles were fixed, permeabilized, and stained with Alexa-568 phalloidin and Alexa-488 DNAse I to visualize F-actin and G-actin, respectively, using a Zeiss 510 laser scanning microscope. Elevation in PTM, but not phenylephrine (1 micromol/l), significantly increased the intensity of F-actin and significantly decreased the intensity of G-actin staining in arteriolar vascular smooth muscle cells (VSMCs). The increase in F-actin staining caused by an elevation in PTM was inhibited by cytochalasin D. In VSMCs at 10 mmHg, prominent F-actin staining was restricted to the cell periphery, whereas after elevation in PTM, transcytoplasmic F-actin fibers were localized through the cell interior, running parallel to the long axis of the cells. Phenylephrine (1 micromol/l) did not alter the architecture of the actin cytoskeleton. In contrast to VSMCs, the actin cytoskeleton of endothelial or adventitial cells was not altered by an elevation in PTM. Therefore, the actin cytoskeleton of VSMCs undergoes dramatic alteration after elevation in PTM of arterioles and plays a selective and essential role in mechanosensitive myogenic constriction.

Actin Cytoskeleton↗

PubMed2004

Distinct cAMP signaling pathways differentially regulate alpha2C-adrenoceptor expression: role in serum induction in human arteriolar smooth muscle cells.

The physiological role of alpha(2)-adrenoceptors (alpha(2)-ARs) in cutaneous, arteriolar, vascular smooth muscle cells (VSMs) is to mediate cold-induced constriction. In VSMs cultured from human cutaneous arterioles, there is a selective increase in alpha(2C)-AR expression after serum stimulation. In the present study, we examined the cellular mechanisms contributing to this response. Serum induction of alpha(2C)-ARs was paralleled by increased expression of cyclooxygenase-2 (COX-2), increased release of prostaglandins, and increased intracellular concentration of cAMP. Inhibition of COX-2 by acetyl salicylic acid (1 mM), NS-398 (5 microM), or celecoxib (3 microM) abolished the increase in cAMP and markedly reduced alpha(2C)-AR induction in response to serum stimulation. The cAMP agonists, forskolin (10 microM), isoproterenol (10 microM), and cholera toxin (0.1 microg/ml) each dramatically increased expression of alpha(2C)-ARs in human cutaneous VSMs. The A-kinase inhibitor H-89 (2 microM) inhibited phosphorylation of cAMP response element binding protein, but not the increase in alpha(2C)-AR expression in response to these agonists. cAMP-dependent but A-kinase independent signaling can involve activation of guanine nucleotide exchange factors for the GTP-binding protein, Rap. Indeed, pull-down assays demonstrated Rap1 activation by serum and forskolin in VSM. Transient transfections using alpha(2C)-AR promoter-luciferase reporter construct demonstrated that Rap1 increased reporter activity, whereas the A-kinase catalytic subunit decreased reporter activity. These results indicate that cAMP signaling can have dual effects in cutaneous VSMs:activation of alpha(2C)-AR transcription mediated by Rap1 GTPase and suppression mediated by A-kinase. The former effect predominates in serum-stimulated VSMs leading to a COX-2, cAMP, and Rap 1-dependent increase in alpha(2C)-AR expression. Such increased expression of alpha(2C)-ARs may contribute to enhanced cold-induced vasoconstriction and Raynaud's phenomenon.

Arterioles↗

PubMed2004

The impact of cyclooxygenase-2 mediated inflammation on scarless fetal wound healing.

Cyclooxygenase-2 (COX-2) and the prostaglandin products generated as a result of COX-2 activity mediate a variety of biological and pathological processes. Scarless healing occurs in fetal skin in the first and second trimesters of development. This scarless healing process is known to proceed without a significant inflammatory response, which appears to be important for the lack of scarring. Because the COX-2 pathway is an integral component of inflammation, we investigated its role in the fetal repair process using a mouse model of scarless fetal wound healing. COX-2 expression in scarless and fibrotic fetal wounds was examined. In addition, the ability of exogenous prostaglandin E(2) to alter scarless fetal healing was evaluated. The results suggest that the COX-2 pathway is involved in scar production in fetal skin and that targeting COX-2 may be useful for limiting scar formation in adult skin.

Animals↗

PubMed2003

Regulation of alpha(2)-adrenoceptors in human vascular smooth muscle cells.

This study analyzed the regulation of alpha2-adrenoceptors (alpha2-ARs) in human vascular smooth muscle cells (VSMs). Saphenous veins and dermal arterioles or VSMs cultured from them expressed high levels of alpha2-ARs (alpha2C > alpha2A, via RNase protection assay) and responded to alpha2-AR stimulation [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK-14,304, 1 microM)] with constriction or calcium mobilization. In contrast, VSMs cultured from aorta did not express alpha2-ARs and neither cultured cells nor intact aorta responded to UK-14,304. Although alpha2-ARs (alpha2C >> alpha2A) were detected in aortas, alpha2C-ARs were localized by immunohistochemistry to VSMs of adventitial arterioles and not aortic media. In contrast with aortas, aortic arterioles constricted in response to alpha2-AR stimulation. Reporter constructs demonstrated higher activities for alpha2A- and alpha2C-AR gene promoters in arteriolar compared with aortic VSMs. In arteriolar VSMs, serum increased expression of alpha2C-AR mRNA and protein but decreased expression of alpha2A-ARs. Serum induction of alpha2C-ARs was reduced by inhibition of p38 mitogen-activated protein kinase (MAPK) with 2 microM SB-202190 or dominant-negative p38 MAPK. UK-14,304 (1 microM) caused calcium mobilization in control and serum-stimulated cells: in control VSMs, the response was inhibited by the alpha2A-AR antagonist BRL-44408 (100 nM) but not by the alpha2C-AR antagonist MK-912 (1 nM), whereas after serum stimulation, MK-912 (1 nM) but not BRL-44408 (100 nM) inhibited the response. These results demonstrate site-specific expression of alpha2-ARs in human VSMs that reflects differential activity of alpha2-AR gene promoters; namely, high expression and function in venous and arteriolar VSMs but no detectable expression or function in aortic VSMs. We found that alpha2C-ARs can be dramatically and selectively induced via a p38 MAPK-dependent pathway. Therefore, altered expression of alpha2C-ARs may contribute to pathological changes in vascular function.

Adolescent↗

PubMed2003

The vasculopathy of Raynaud's phenomenon and scleroderma.

The scleroderma (SSc) disease process involves dramatic dysfunction in acute and chronic vascular regulatory mechanisms; it presents initially with heightened vasoconstrictor or vasospastic activity and progresses to structural derangement or vasculopathy of the microcirculation. This article discusses the regulatory mechanisms that contribute to this dysfunction and the vascular changes in the context of the other aspects of the SSc disease process in a novel attempt to integrate the individual pathologies of the disease process.

Humans↗