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Biomedical subjects

V Gopalakrishnan

Publications and source records attributed to V Gopalakrishnan.

At least 37 records · Page 2Linked to original sources
PubMed2000

Leptospirosis in Kuala Lumpur and the comparative evaluation of two rapid commercial diagnostic kits against the MAT test for the detection of antibodies to leptospira interrogans.

The aim of the study was to look into the epidemiology of serodiagnosed cases of leptospirosis at the University Hospital and compare two commercial ELISA Assays to the Microscopic Agglutination Test (MAT). Demographic data for all serodiagnosed cases for the years 1991-1997 were collected. From this data, 104 sera (n = 104) were selected as samples for comparative evaluation of the commercial ELISAs (INDX Dip-S-Ticks and PanBio ELISA) to the MAT test. Thirty two (n = 32) negative control sera were selected from serodiagnosed cases of other differential diagnosis of leptospira infection. The MAT test is a standard test that detects agglutination antibodies to leptospira biflexa, while the INDX Dip-S-Ticks is an ELISA dot test assaying for total anti-leptospira antibodies. The PanBio ELISA is a colorometric assay in test well strips to detect anti-leptospira IgM. The sensitivity, specificity, and efficiency of tests were calculated at a MAT cut-off value of 1:320. Demographic data showed that leptospirosis peaks during March-May and Aug-Nov coinciding with the inter-monsoon period with more men being infected than women and more adults than children. The sensitivity, specificity, and efficiency of test for the INDX Dip-S-Ticks were 83.3%, 93.8% and 87.5% while the values for the PanBio ELISA were 54.2%, 96.9% and 71.3%. The suboptimal PanBio result could be related to the blocking effect of high IgG titres or could be related to the diagnostic MAT cut-off values used in this study. The data hence reflects a pattern of transmission that is related to "wet" occupational risk factors. The commercial assays evaluated, are easier to perform but interpretation of results should be based on level of endemicity. The INDX Dip-S-Ticks allows this flexibility and is a practical alternative to the MAT test.

Adult↗
PubMed1999

Phosphatidic acid increases inositol-1,4,5,-trisphosphate and [Ca2+]i levels in neonatal rat cardiomyocytes.

Phosphatidic acid (PA), which can be synthesized de novo, or as a product of phosphatidylcholine hydrolysis and/or phosphorylation of 1,2-diacylglycerol (DAG), mediates diverse cellular functions in various cell types, including cardiomyocytes. We set out to characterize the effect of PA on intracellular free calcium ([Ca2+]i) and inositol-1,4,5-trisphosphate (IP(3)) levels in primary cultures of neonatal rat cardiomyocytes. Addition of PA led to rapid, concentration and time dependent increases in both IP(3) and [Ca2+]i levels in adherent cells. There was strong correlation in the concentration-response relationships between IP(3) and [Ca2+]i increases evoked by PA. Incubation with the sarcoplasmic reticulum (SR) Ca2+ pump inhibitor, cyclopiazonic acid (CPA), significantly attenuated the PA evoked [Ca2+]i increase but had no significant effect on IP(3) accumulation. The phospholipase C (PLC) inhibitor, D-609, attenuated both IP(3) and [Ca2+]i elevations evoked by PA whereas staurosporine (STS), a potent and non-selective PKC inhibitor, had no significant effect on either. Another PLC inhibitor, U73122, but not its inactive analog, U73343, also inhibited PA evoked increases in [Ca2+]i. Depletion of extracellular calcium attenuated both basal and PA evoked increases in [Ca2+]i. The PLA(2) inhibitors, bromophenylacyl-bromide (BPB) and CDP-choline, had no effect on PA evoked [Ca2+]i responses. Neither the DAG analog, dioctanoylglycerol, nor the DAG kinase inhibitor, R59949, affected PA evoked changes in [Ca2+]i. Taken together, these data indicate that PA, in a manner independent of PKC, DAG, or PLA(2), may enhance Ca2+ release from IP(3) sensitive SR Ca(2+) stores via activation of PLC in neonatal rat cardiomyocytes.

Animals↗
PubMed1999

Pneumadin-evoked intracellular free Ca2+ responses in rat aortic smooth muscle cells: effect of dexamethasone.

The direct vascular effect of pneumadin (PN) was determined by studying the changes in intracellular free calcium ([Ca2+]i) levels in cultured rat aortic smooth muscle cells maintained between the second and fifth passages. PN evoked a rapid, concentration-dependent, biphasic increase in [Ca2+]i. The [Ca2+]i level rose from a basal value of 108 nM to a maximum increase in peak value of 170 nM. Although the level of maximal [Ca2+]i response evoked by PN was less than with other vasoactive agonists, it was more potent (EC50 0.5 nM) than even endothelin-1 (EC50 3.1 nM). At concentrations > 100 nM, [Ca2+]i elevations induced by PN above basal levels were no longer observed. Pretreatment with dexamethasone (100 nM for 24 hr) resulted in a significant increase (P < 0.01) in the peak [Ca2+]i response (310 nM) to PN. However, the biphasic pattern in the peak [Ca2+]i responses encountered with increasing concentrations of PN remained unaffected. The exaggerated [Ca2+]i response to PN was abolished by preincubation of cells with either the glucocorticoid antagonist mifepristone (RU 486) or the protein synthesis inhibitor cycloheximide. Inclusion of either an AT1 antagonist (losartan), a V1 selective vasopressin antagonist (d(Ch2)5 Tyr (Me) AVP), or an alpha-adrenoceptor antagonist (phentolamine) failed to affect the increases in [Ca2+]i induced by PN. PN-evoked increases in inositol 1,4,5-trisphosphate levels paralleled the [Ca2+]i changes. These data suggest that PN increases Ca2+ mobilization in rat aortic smooth muscle cells via activation of phospholipase C coupled receptors. This effect is up-regulated by dexamethasone.

Animals↗
PubMed1999

Plasma endothelin levels and vascular responses at different temporal stages of streptozotocin diabetes.

While alterations in the release and action of endothelial derived vasoactive factors such as endothelin- and endothelial derived relaxing factor (EDRF) occur in diabetes mellitus, the nature and direction of these changes is controversial. We examined the role of diabetes duration on endothelin- plasma levels and vasoconstrictor responses to endothelin-1 in aortic rings from streptozotocin-induced diabetic rats. Endothelin-1 plasma levels were attenuated at 2-weeks, but conversely elevated at 14-weeks, after diabetes induction. Similarly, maximal vasoconstriction to endothelin-1 in aorta was exaggerated in 2-week, but attenuated in 14-week diabetic rats. Also, sensitivity of aorta to endothelin-1 was enhanced in the 14-week group. Neither nitric oxide synthase inhibition with nitro-L-arginine-methyl-ester, nor endothelium removal affected alterations in maximal vasoconstriction, but both abolished changes in sensitivity to endothelin-1. Endothelium dependent (acetylcholine-evoked) vasorelaxation responses were attenuated in 14-week, but not 2-week, diabetic rats, while endothelium independent (sodium nitroprusside-evoked) responses remained unchanged. Together, these data indicate a deficiency in EDRF production in the 14-week group. Elevated plasma glucose and attenuated insulin levels were present in both groups, but plasma cholesterol and triglyceride levels were elevated only in 14-week rats. We conclude that differences in the pre-existing duration of diabetes differentially affect both plasma levels and action of endothelin-1. These changes might be linked to coincident diabetes duration dependent changes in EDRF production and plasma lipid levels. Such a shift in the production and action of endothelial derived relaxing and contracting factors might contribute to the characteristic early and late stage cardiovascular complications of diabetes mellitus.

Acetylcholine↗
PubMed1999

DNA replication specificity and functional E2 interaction of the E1 proteins of human papillomavirus types 1a and 18 are determined by their carboxyl-terminal halves.

Replication of most papillomaviruses (PVs) requires the viral-encoded E1 and E2 proteins that bind to the origin of replication (ori) containing the E1- and E2-binding sites and help recruit host replication factors during the initiation of DNA replication. We studied the ability of heterologous E1 and E2 proteins to interact in vivo and support replication, using the human papillomavirus (HPV) types 1a and 18 as model systems. The E1 protein of HPV-1a in combination with HPV-18 E2 supported high-level replication of various ori plasmids. In contrast, the HPV-18 E1 protein interacted weakly with HPV-1a E2 during the replication of ori plasmids. We have previously shown that the E1 protein of HPV-1a alone is sufficient for replication of HPV-1a ori plasmids, whereas HPV-18 replication requires both the E1 and E2 proteins. However, in the latter case, E2-binding sites alone in the absence of the E1-binding site can function as the minimal ori. Based on the above observations, we generated hybrids between HPV-1a and HPV-18 E1 proteins in an effort to identify their "replication specificity" domains using a transient replication assay. These hybrids were also used to localize the domains in the E1 proteins that are involved in their functional interaction with the E2 protein during replication. Our results suggest that the "replication specificity" and functional E2 interaction domains of the HPV-1a and HPV-18 E1 proteins are located in their carboxyl-terminal halves.

DNA Replication↗
PubMed1999

Endothelin: emerging role in diabetic vascular complications.

Since the discovery of endothelin-1 as the most potent endothelial-derived vasoconstrictor/mitogenic peptide a decade ago, considerable evidence has implicated this peptide in various cardiovascular disease states, including diabetes mellitus. Plasma and tissue concentrations of endothelin-1 as well as responses to the peptide are changed in various forms of the disease in humans and animals. Endothelin activity is also altered in atherosclerotic and ischaemic disease, nephropathy, retinopathy, erectile dysfunction, and neuropathy, many of the well-known complications of diabetes. Striking new evidence shows that antagonists of the endothelin system might beneficially affect and potentially overcome some of these complications. Despite this, lack of direct proof of causation makes this peptide's role in the disease uncertain. This review examines the current state of thought on the role of endothelin in diabetes and in the complications of the disease as well as the likely roles of altered metabolic variables in modulating endothelin-1 concentrations and its activity. It is concluded that although alterations in endothelin-1 release and action are clearly associated with the diabetic state, further studies using inhibitors of the endothelin system are warranted to determine its precise role in the complications of the disease.

Diabetic Angiopathies↗
PubMed1999

Total peripheral conductance mediates antihypertensive effect of nonpeptide mixed endothelin receptor antagonist in deoxycorticosterone acetate-salt hypertensive rats.

The relative contribution of cardiac output (CO) and total peripheral conductance (TPC) to the changes in blood pressure (BP) evoked by an intravenous injection of bosentan, a nonpeptide mixed endothelin (ET) antagonist, were investigated in conscious unrestrained deoxycorticosterone acetate (DOCA)-salt hypertensive rats and sham-control rats. Blood pressure was recorded by radiotelemetry devices and CO by ultrasonic transit-time flow probes. Bosentan significantly reduced BP (from 141 +/- 3 to 111 +/- 3 mm Hg) and increased TPC (from 1.19 +/-0.1 to 1.72 +/- 0.2 mL/min/kg/mm Hg) in DOCA-salt hypertensive rats, but not in sham rats. An increase in CO opposed the BP-lowering effect of the antagonist. The results demonstrate that the role of ET receptors in the maintenance of the hypertensive state in the DOCA-salt model of hypertension is exerted at the level of the resistance vessels and not on factors that regulate cardiac output.

Animals↗
PubMed1999

Vasopressin accelerates protein synthesis in neonatal rat cardiomyocytes.

Arginine vasopressin (AVP) has been shown to promote vascular smooth muscle cell hypertrophy and hyperplasia of fibroblasts. The present study examines the effect of AVP and endothelin-1 (ET-1) on protein, DNA, and RNA synthesis in primary cultures of serum deprived neonatal rat cardiomyocytes (RC) as assessed by changes in [3H] phenylalanine, [3H] thymidine, and [14C] uridine incorporation respectively. Both AVP and ET-1 evoked significant increases in protein synthesis in RC of 36 +/- 12% (p < 0.05) and 53 +/- 22% (p < 0.01) respectively. The stimulating action of AVP on [3H] phenylalanine incorporation was abolished by pretreatment with 2-nitro-4carboxyphenyl-N, N-diphenylcarbamate (NCDC), a phospholipase C (PLC) inhibitor. [14C] uridine incorporation was significantly higher in cells incubated with ET-1 (95 +/- 12%) but not AVP (9 +/- 11%). Neither AVP nor ET-1 significantly affected cell number or [3H]thymidine incorporation, suggesting a lack of a hyperplastic effect. AVP evoked an increase in [Ca2+]i levels (162 +/- 12 nmol/L from a basal value of 77 +/- 6 nmol/L) which was completely abolished by pretreatment with either NCDC or cyclopiazonic acid (sarcoplasmic reticulum (SR) Ca2+ pump inhibitor) but unaffected by ryanodine (ryanodine sensitive SR Ca2+ store depletor). Taken together, these data suggest that AVP, in a PLC dependent manner, both stimulates protein synthesis and augments [Ca2+]i release in RC from ryanodine insensitive (IP3 sensitive) Ca2+ stores. Thus, AVP may promote cardiac hypertrophy via direct effects on cardiomyocyte protein synthesis secondary to IP3 mediated [Ca2+]i release.

Animals↗
PubMed1999

Vasopressin-evoked [Ca2+]i responses in neonatal rat cardiomyocytes.

The presence of arginine vasopressin (AVP) V1 receptors on neonatal rat cardiomyocytes (NRCs) linked to processes capable of elevating intracellular free calcium ([Ca2+]i) is now firmly established. This study examined the sources and signaling involved in [Ca2+]i elevations evoked by AVP in NRCs. AVP promoted increases in both [Ca2+]i and 1,4,5-inositoltrisphosphate (IP3) levels in NRCs. The degree of [Ca2+]i elevation was less than that of angiotensin II, but greater than that of endothelin-1. Extracellular Mg2+ depletion led to diminution of the maximal [Ca2+]i response, with a rightward shift in the concentration-response curves to AVP. The phospholipase C inhibitors, D-609, NCDC, or U73122, and the IP3 receptor blocker, heparin, abolished the [Ca2+]i response to AVP. Neither cyclooxygenase inhibition with indomethacin nor PKC inhibition with staurosporine had any effect. Neither ryanodine nor caffeine, which deplete sarcoplasmic reticulum (SR) Ca2+ stores, nor ruthenium red, which inhibits both SR and mitochondrial Ca2+ stores, affected [Ca2+]i responses to AVP. The SR Ca2+ pump inhibitor, cyclopiazonic acid, abolished, and removal of extracellular Ca2+ attenuated, the response to AVP. These data indicate that activation of cardiac V1 receptors by AVP results in mobilization of Ca2+ from a distinct, non-SR, nonmitochondrial, intracellular Ca2+ pool that is Ca2+ pump replenished and IP3 sensitive. This process occurs secondary to phospholipase C (PLC)-mediated generation of IP3, requires the presence of Mg2+ and extracellular Ca2+, and occurs in a manner independent of PKC and cyclooxygenase activation. Such mechanisms of Ca2+ mobilization might indicate a distinct role for AVP in cardiac physiology and disease.

Angiotensin II↗
PubMed1999

Effect of sodium orthovanadate treatment on cardiovascular function in the hyperinsulinemic, insulin-resistant obese Zucker rat.

We recently demonstrated that oral vanadate treatment ameliorates exaggerated vasoconstriction in aortic tissue from the hyperinsulinemic/insulin resistant obese Zucker rat. It has been suggested that changes in large artery contractility might contribute to the development of hypertension in this strain. Thus we examined the effect of vanadate treatment (0.5 mg/ml, p.o.) on conductance and resistance vessel function as well as blood pressure (BP) in Zucker rats. Vasoconstrictor responses to endothelin-1 (ET-1) and methoxamine and vasodilator responses to acetylcholine in the aorta and perfused mesenteric vascular bed served as indices of conductance and resistance function, respectively. Separate groups were treated with insulin (12 mU/kg/min, s.c.) to determine its role in the actions of vanadate. Vanadate treatment reduced (2.5-fold; p < 0.05) elevated plasma insulin levels and abolished exaggerated aortic vasoconstriction in obese rats. Vasoconstrictor responses in the mesenteric bed, however, were similar between obese and lean rats, and were unaffected by vanadate. Vanadate did not affect elevated BP in obese rats and actually increased BP in the lean group. Insulin treatment per se failed to affect vasomotor function or BP in either strain, and acetylcholine-evoked relaxation was similar in all groups. We conclude that whereas vanadate overcomes exaggerated central artery contractility in obese Zucker rats, it fails to affect resistance vessel function or BP in this strain, and might conversely elevate BP in normotensive lean control rats. The vascular actions of vanadate in obese rats appear to occur independent of changes in plasma insulin or endothelial function.

Adrenergic alpha-Antagonists↗
PubMed1998

Vanadate treatment normalizes exaggerated vascular smooth muscle responses in the obese Zucker rat.

The effect of oral vanadate treatment on isometric tension responses were examined in aortic rings isolated from obese and lean Zucker rats. Rats from both strains that were either maintained on food ad libitum or pair-fed were included to serve as controls. Higher plasma insulin and glucose levels and exaggerated aortic tension responses to endothelin-1, methoxamine, and KCl observed in obese Zucker rats were normalized in vanadate treated, but not pair-fed, rats. These data suggest that abnormal vascular responses in obese Zucker rats can be normalized by vanadate treatment in a manner at least partly independent of food intake.

Acetylcholine↗
PubMed1998

Insulin and vanadate restore decreased plasma endothelin concentrations and exaggerated vascular responses to normal in the streptozotocin diabetic rat.

Although insulin has been shown to raise plasma concentrations of endothelin (ET) and up regulate vascular smooth muscle ETA receptor expression, the interaction of vanadate, an insulinomimetic agent, with the vascular ET system has not been investigated. We compared the effects of oral vanadate treatment (0.5 mg/ml; p.o.) and insulin infusion (12 mU.kg-1.min-1 s.c.) for two weeks on plasma ET concentrations and vascular responses to endothelin-1 (ET-1) and the alpha-1 adrenoceptor agonist, methoxamine, in aortic ring preparations from streptozotocin (STZ) diabetic and non-diabetic adult male Sprague-Dawley rats. Plasma ET concentrations were lower (p < 0.01) in STZ diabetic rats compared with normal control rats. Insulin and vanadate treatment restored plasma ET to normal (p < 0.01) in STZ rats and increased ET concentrations in the control (p < 0.05) group. Higher maximal tension responses to both ET-1 (p < 0.01) and methoxamine (p < 0.05) were present in STZ rats in both endothelium intact and denuded aortic preparations compared with the control group. Both insulin and vanadate treatment returned these responses to normal. It is concluded that low plasma concentrations of insulin and high plasma glucose in STZ diabetic rats are accompanied by lower concentrations of plasma ET. Insulin and vanadate treatment restores diminished plasma ET to control concentrations and attenuates exaggerated agonist(s)-evoked vascular smooth muscle responses in STZ-induced diabetic rats. In addition to well known beneficial metabolic effects, insulin and vanadate may beneficially affect cardiovascular regulation in the STZ diabetic rat by correcting abnormal ET activity.

Animals↗
PubMed1998

Differential regulation of endothelin-1 action by insulin and vanadate in vascular smooth muscle.

The effects of insulin (10 nM) and sodium orthovanadate (SOV, 25 microM) pretreatment for 24 h on endothelin-1 (ET-1) and IRL-1620 (ETB agonist)-evoked increases in intra-cellular free calcium levels [Ca2+]i were determined by fura-2 fluorescence methodology using cultured rat aortic smooth-muscle cells (ASMCs). Whereas insulin preincubation led to an increase (p < 0.05) in [Ca2+]i responses to ET-1, SOV enhanced [Ca2+]i responses to both ET-1 (p < 0.05) and IRL-1620 (p < 0.01). Saturation binding studies revealed that insulin pretreatment led to a 2.2-fold (p < 0.01) increase in [125I]ET-1 binding sites, and SOV pretreatment led to a 2.4-fold (p < 0.01) and 6.6-fold (p < 0.01) increase in the maximal number of binding sites labeled by [125I]ET-1 and [125I]IRL-1620, respectively. Northern blot analysis revealed that insulin upregulated only ETA mRNA levels, and SOV enhanced both ETA and ETB mRNA levels. Pretreatment with the tyrosine kinase inhibitor genistein abolished these effects. These data suggest that insulin pretreatment of ASMCs selectively upregulates ETA receptor expression and ETA-mediated [Ca2+]i, signaling, and SOV upregulates both ETA and ETB receptor expression and exaggerates [Ca2+]i responses mediated by both ETA and ETB in rat ASMCs.

Animals↗
PubMed1998

Nuclear Ca2+ signaling to endothelin-1 in rat aortic smooth-muscle cells.

Vascular smooth-muscle cells (VSMCs) isolated from genetically hypertensive animals show increased intracellular free calcium levels ([Ca2+]i) in response to endothelin-1 (ET-1). The differences in time course and distribution of Ca2+ increase after addition of ET-1 within the VSMCs are unknown. Therefore, ET-1-evoked changes in fluo-3 fluorescence were determined using a confocal laser scanning microscope in primary cultures of aortic smooth-muscle cells (ASMCs) from 12-week-old male Sprague-Dawley (SD) rats, Wistar-Kyoto (WKY) rats, and spontaneously hypertensive rats (SHR). Syto-11 staining enabled the assessment of intracellular free [Ca2+]i changes in the cytosolic ([Ca2+]c), perinuclear ([Ca2+]p), and nuclear ([Ca2+]n) regions. In the basal state, [Ca2+] was evenly distributed throughout the rat ASMCs. There were no significant differences in basal fluorescence values among the three strains. ET-1 evoked a concentration-dependent increase in fluo-3 intensity. The peak [Ca2+]i rise to ET-1 was much more rapid in ASMCs from SHR and WKY strains. The changes in [Ca2+]n were greater than in [Ca2+]c. Pretreatment of rat ASMCs with BQ-123 (an ETA antagonist) and BQ-788 (an ETB antagonist) abolished the rapid peak rise and the slow sustained elevation in [Ca2+]i, respectively. The nonselective antagonist bosentan attenuated both phases of the ET-1 response in all three strains. The ETB-selective agonist IRL 1620 evoked a significant elevation in [Ca2+]n values at 2 min in the ASMCs of SHR. These data suggest that ETA activation is linked to initial rapid increases in [Ca2+]c and [Ca2+]n, whereas ETB activation promotes slow [Ca2+]n signaling, particularly in ASMCs of SHR.

Aniline Compounds↗
PubMed1998

Hemodynamic effects of a selective endothelin--a receptor antagonist in deoxycorticosterone acetate-salt hypertensive rats.

The time-course changes in blood pressure (BP), cardiac output (CO), and total peripheral conductance (TPC) were investigated during a bolus i.v. infusion of BMS-182874, a selective ETA receptor antagonist, in conscious, unrestrained DOCA-salt hypertensive rats and Sham-operated control rats (SHAM). BP was recorded with radiotelemetry devices and CO with ultrasonic transit-time flow probes. In contrast to vehicle-treated controls, BMS-182874 reduced BP and increased TPC in DOCA-salt hypertensive rats but not in SHAM-rats. A small increase in CO tended to oppose the BP-lowering effect of the antagonist. The results suggest that the role of ETA receptors in the maintenance of the hypertensive state in the DOCA-salt model of hypertension is exerted at the level of the resistance vessels and not on factors that regulate CO.

Animals↗