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Biomedical subjects

A K Grover

Publications and source records attributed to A K Grover.

At least 55 records · Page 3Linked to original sources
PubMed1994

Free radicals uncouple the sodium pump in pig coronary artery.

Free radicals may impair vital functions of several types of tissues including coronary artery smooth muscle. Because the Na+ pump plays a key role in maintaining coronary tone, the effects of superoxide and peroxide on this protein were examined. Ouabain-sensitive Rb+ uptake by denuded coronary artery rings was used in lieu of K+ transport by this pump. It was inhibited by exposing the rings for 90 min either to peroxide [50% inhibitory concentration (IC50) = 0.56 +/- 0.18 mM] or to superoxide generated by xanthine oxidase (XO; 0.3 mM xanthine and xanthine oxidase, IC50 = 0.08 +/- 02 mU/ml). The effect of peroxide was not overcome by superoxide dismutase and that of superoxide was not prevented by catalase. K(+)-activated ouabain-sensitive hydrolysis of p-nitrophenyl phosphate in the plasma membrane-enriched fraction isolated from the coronary artery smooth muscle was monitored as the hydrolytic activity of the Na+ pump. It was inhibited by exposing the membranes only to very high concentrations of peroxide (IC50 = 9.85 +/- 3.5 mM) or XO (IC50 = 5 +/- 2 mU/ml). The exposure to 2.5 mM H2O2 or 0.5 mU/ml XO reduced the Na(+)-dependent acylphosphate levels only by 41 +/- 3 and 30 +/- 4%, respectively even though either inhibited the Rb+ uptake by > 80%. Thus superoxide and peroxide uncoupled the hydrolytic activity of the Na+ pump from Rb+ uptake. We speculate that such an uncoupling in ischemia and reperfusion would result in dual damage: ion imbalance and continuous hydrolysis of ATP in the cells that are already starved.

4-Nitrophenylphosphatase↗
PubMed1994

Coronary artery contractility, Na(+)-pump and oxygen radicals.

Oxygen radicals accumulated during ischemia and reperfusion may affect coronary contractility by endothelium dependent and independent pathways one of which may involve Na(+)-pump. Here we report a contractility assay for Na(+)-pump in pig coronary artery and use it to examine the effects of hydrogen peroxide and superoxide. Coronary artery rings contracted in a K(+)-free Krebs solution and relaxed upon subsequent exposure to K+. The relaxation approximated a single exponential decay whose rate constant depended on [K+]2. This K(+)-induced relaxation was abolished by ouabain and was attributed to Na(+)-pump. In tissues pretreated with peroxide, the rate of relaxation of the K(+)-free contracted arteries decreased with an IC50 = 1.6 +/- 0.6 mmol/l for peroxide. Another set of tissues was pretreated with the superoxide generating system containing 0.3 mmol/l xanthine + varying concentrations of xanthine oxidase (XO) and precontracted in K(+)-free Krebs solution. The rate of the K(+)-induced relaxation decreased with IC50 = 24 +/- 8 mU/ml for XO. Thus, using the relaxation assay we conclude that exposing coronary arteries to oxygen radicals can damage Na(+)-pumps.

Animals↗
PubMed1993

Effect of pH on stability of sarcoplasmic reticulum calcium pump in rabbit heart.

The sarcoplasmic reticulum (SR) membranes isolated from rabbit heart were preincubated at pH 6.8 or 7.8 and their Ca2+ pump properties were compared at pH 6.8. The ATP-dependent azide insensitive oxalate-stimulated Ca2+ uptake was reduced more rapidly from the membranes preincubated at 37 degrees C at pH 7.8 than from those preincubated at pH 6.8. The Ca(2+)-Mg(2+)-ATPase, and the Ca(2+)-dependent formation of 110 kDa acylphosphate were also inhibited by the preincubation at the higher pH. Including 1 mM DTT in the preincubation medium reduced the inactivation. The preincubation at 37 degrees C in the presence or absence of DTT caused membranes to become more leaky as the loss of Ca2+ uptake was more rapid than that of ATPase or the acylphosphate formation. The loss of these activities was not accompanied by a breakdown of the protein as monitored in Western blots. It is hypothesized that the SR Ca2+ pump inactivation involves a key-SH group and that the lower pH provides a compensatory protective mechanism for the SR during acidosis.

Animals↗
PubMed1993

In situ hybridization and immunocytochemical localization of SERCA2 encoded Ca2+ pump in rabbit heart and stomach.

Heart tissue contains large amounts of the protein encoded by the Ca2+ pump gene SERCA2. The SERCA2 RNA can be spliced alternatively to produce mRNA encoding the proteins SERCA2a and SERCA2b which differ in their C-terminal sequences. In this study we report the tissue distribution of SERCA2a and SERCA2b isoforms by in situ hybridization to rabbit heart and stomach. The expression of SERCA2 mRNA was high in myocardial cells, being the highest in the atrial region. In contrast, there was more SERCA2 protein in Western blots in ventricles than in atria. Myocardial cells expressed predominantly the mRNA for the isoform SERCA2a. Whereas the stomach smooth muscle and the neuronal plexus expressed SERCA2 at levels much lower than myocardial cells, the expression was very high in the stomach mucosa. Mucosa contained mainly the mRNA for SERCA2b. From immunocytochemistry it was concluded that the anti-heart SR Ca2+ pump antibody IID8 reacted much better with heart and surface mucosal cells in the stomach than with the stomach smooth muscle, and that IID8 reactivity was intracellular. In contrast PM4A2B, an antibody against the plasma membrane Ca2+ pump, reacted well with heart and stomach smooth muscle, plexus and mucosa, and its localization appeared to be in the plasma membrane. Thus, stomach smooth muscle expressed SERCA2b mRNA and protein at low levels, mucosa expressed SERCA2b mRNA and protein at high levels, atria and ventricle expressed SERCA2a mRNA and protein at high levels, mRNA being more in atria, but protein being more in ventricles.

Animals↗
PubMed1993

Abundance of heteronuclear and messenger RNA for internal Ca pump in stomach smooth muscle and myocardium.

The sarcoplasmic reticulum Ca pump gene SERCA2 is alternatively spliced to express mRNA encoding the protein SERCA2a in heart and SERCA2b in stomach smooth muscle. The expression of SERCA2 protein in heart is 70 +/- 10 fold that in stomach smooth muscle. To understand the mechanism underlying this tissue difference in the expression level, a comparison is made of their mRNA and heteronuclear RNA (hn-RNA) contents. A 72 bp intron present in the gene encoding SERCA2a/b was cloned and sequenced. By reverse transcription of total RNA followed by PCR using primers based on the sequence of this intron and the cDNA sequences flanking it, a value of 0.37 +/- 0.03 was obtained for the ratio heart hn-RNA/mRNA:stomach hn-RNA/mRNA. Similarly, a ratio of 3.4 +/- 0.5 was obtained for heart:stomach mRNA/28S values. This value was slightly lower but statistically similar to a value of 5.7 +/- 1.8 obtained for the heart:stomach mRNA/poly A+ RNA obtained by Northern blot analysis using a conserved region SERCA2 cDNA probe. Based on mRNA/28S ratio of 3.4 +/- 0.5 and hn/mRNA ratio of 0.37 +/- 0.03, the ratio of heart:stomach for hn-RNA/28S was 1.2 +/- 0.2. Thus, heart which expresses SERCA2a contains 70 +/- 10 times more protein, 3.4 +/- 0.5 times more mRNA and only 1.2 +/- 0.2 times more hn-RNA for this message than the stomach smooth muscle which expresses SERCA2b.

Animals↗
PubMed1993

Expression of the internal calcium pump in pregnant rat uterus.

Uterine contraction to agents such as oxytocin during labour may utilize Ca2+ sequestered by Ca2+ pump into the sarcoplasmic reticulum (SR). Uterus expresses the SR Ca2+ pump gene SERCA2 as the mRNA splice which encodes the protein SERCA2b. The expression of SERCA2 mRNA and protein was monitored in uteri of day 15 pregnant and delivering rats. The ratio of SERCA2 mRNA to 28S RNA increased by 20% from day 15 to delivery. SERCA2 protein examined by two antibodies increased by 54-55%. Ca2+ dependent acylphosphate intermediate of 115 kD corresponding to SERCA2 also increased 72% during this period. Thus, there is an increase in the SERCA2 expression from day 15 to delivery.

Acylation↗
PubMed1993

Coronary artery acidosis: pH and calcium pump stability.

To study the effects of prolonged exposure to different pH, pig coronary artery smooth muscle subcellular fraction F3 enriched in sarcoplasmic reticulum was preincubated at 0 or 37 degrees C and pH 6.4-7.8 and then used for monitoring the rate of the oxalate-stimulated component of the ATP-dependent azide-insensitive Ca2+ uptake at constant pH (6.8) and temperature (37 degrees C). Ca2+ uptake by F3 decreased with the increasing preincubation pH. The loss of Ca2+ uptake was more rapid upon preincubation at 37 degrees C than at 0 degrees C. Dithiothreitol (DTT), when included in the preincubation solution, protected against the loss. Sucrose, KCl, ATP, and ATP plus CaCl2 protected slightly, and glutathione, catalase, superoxide dismutase, azide, ascorbate, CaCl2, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, mannitol, mercaptopropionylglycine, and FeSO4 had only marginal or no effects. Efflux of accumulated Ca2+ was more rapid from membranes that had been preincubated at 37 degrees C than from those preincubated at 0 degrees C, but it was not affected by the preincubation pH or by DTT. The loss of Ca2+ uptake due to incubation at pH 7.8 accompanied a decrease in the 115-kDa Ca(2+)-dependent acylphosphate formation due to the Ca2+ pump. The presence of DTT in the preincubation mixture increased the acylphosphate level in the control and protected against its loss at the preincubation pH 6.8 or 7.8. Thus, in the membranes isolated from coronary artery, acidosis may protect against damage to the sarcoplasmic reticulum Ca2+ pump by protonation of a key sulfhydryl group.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis↗
PubMed1992

Polymerase chain reaction assay of mRNA using 28S rRNA as internal standard.

A method is developed for PCR assay of mRNA using 28S rRNA as internal standard and its applicability is reported for 4 types of mRNA. It consists of reverse transcription of total RNA using primers selective for 28S rRNA and the message of interest. Routinely the 28S reaction mix is diluted 20,000-fold and the dilution error is minimized by adding 10 microCi of 3H2O before the dilution and determining the level of 3H before and after the dilution. Aliquots from the diluted 28S mix and the undiluted reverse transcripts are co-amplified by PCR in the presence of 32P-dATP. The samples are analyzed by electrophoresis, bands detected by autoradiography and in each lane the ratio is determined between cpm in the band of interest and cpm in the 28S band. The ratio is a measure of the level of mRNA in the sample and can be used to monitor changes in mRNA during treatments, development and pathogenesis.

Animals↗
PubMed1992

Calcium pump isoforms: diversity, selectivity and plasticity. Review article.

Ca2+ pumps are essential for removing cytosolic Ca2+ either across the plasma membrane (PM) or into internal organelles such as the sarcoplasmic reticulum (SR). Four genes (PMCA1, PMCA2, PMCA3 and PMCA4) have been reported to encode the PM Ca2+ pumps and three (SERCA1, SERCA2 and SERCA3) to encode the SR Ca2+ pumps. The PM Ca2+ pumps are stimulated by calmodulin, the SR Ca2+ pumps encoded by SERCA1 and SERCA2 are stimulated by phospholamban while the product of SERCA3 may be regulated directly by cAMP-dependent protein kinase. Alternative splicing of the primary transcripts of several of these genes has been reported to occur in a tissue selective manner and for others to alter during ontogeny. For the PM Ca2+ pump, alternative RNA splicing may result in isoforms with altered cyclic nucleotide dependent protein kinase sensitivity. The diversity in distribution of Ca2+ pump isoforms and their regulatory factors when coupled with different Ca2+ entry mechanisms allows for tissue selectivity and plasticity in stimulus-response coupling. The roles of various Ca2+ pump isoforms, the rationale behind their tissue selective expression and the plasticity in this expression are among the new challenges to researchers in this field.

Animals↗
PubMed1992

Peroxide inactivates calcium pumps in pig coronary artery.

To study the effects of hydrogen peroxide, pig coronary artery smooth muscle subcellular fractions enriched in plasma membrane (F2) or sarcoplasmic reticulum (F3) were incubated in various concentrations of peroxide and 5 mM azide. ATP-dependent azide-insensitive oxalate-stimulated Ca2+ uptake was determined for F3 and phosphate-stimulated uptake for F2. Only 1.5-5 microM hydrogen peroxide was required for 50% inhibition of the Ca2+ uptake by F3, but the corresponding concentration for F2 was 10-50 microM. This effect was not prevented by superoxide dismutase. Hydrogen peroxide inhibited the Ca(2+)-dependent formation of a 115-kDa acylphosphate band in F3 and 140- and 115-kDa bands in F2. The inhibition of Ca2+ uptake in F3, however, exceeded the inhibition of the acylphosphate formation. Efflux of Ca2+ from F2 and F3 was enhanced by hydrogen peroxide but F3 was more sensitive than F2. We conclude that hydrogen peroxide has dual effect on Ca2+ dynamics in the coronary artery smooth muscle, i.e., it inactivates the Ca2+ pumps and increases membrane permeability to Ca2+. The effect is more pronounced on sarcoplasmic reticulum than on plasma membrane. Intrinsic catalase may, however, provide partial protection against such damage.

Animals↗
PubMed1992

Ca pump messenger RNA expression in pregnant rat uterus.

Rat uterus expresses transcripts for the plasma membrane Ca pump PMCA1 and for the sarcoplasmic reticulum Ca pump SERCA2. Uterus contains two mRNA isoforms for PMCA1, one encoding a cyclic nucleotide insensitive Ca pump (PMCA1a) and the other encoding a cyclic nucleotide sensitive pump (PMCA1b). The relative ratio of mRNA to 28S RNA for PMCA1a or PMCA1b and the amount of poly A+ RNA per microgram total RNA obtained from uteri of rats pregnant for 15 days and for parturient rats was similar but the ratio of SERCA2 mRNA to 28S RNA was significantly higher in the parturient rats.

Animals↗
PubMed1992

Adenosine metabolism in small coronary arteries of pig.

Adenosine preferentially relaxes small coronary arteries over large ones, and small bovine coronary arteries are also known to have a higher density of adenosine receptors. Here we report a possible role of adenosine metabolism in this process. Subcellular fractions, from right coronary artery (lumen diameter of 2-3 mm) of pig designated as large coronary artery and its subsequent branches (lumen diameter of 0.5-1 mm) as small coronary arteries, were prepared and characterized. In comparison to the various large artery subcellular fractions, the corresponding small artery fractions were richer in 5'-nucleotidase but poorer in adenosine deaminase. Thus a cascade of events may promote adenosine relaxation in small coronary arteries: higher activity of 5'-nucleotidase leads to production of more adenosine, larger number of receptors allows greater reactivity to adenosine, and lower adenosine deaminase level promotes prolonged action of adenosine.

5'-Nucleotidase↗
PubMed1991

Expression of cyclic-nucleotide-sensitive and -insensitive isoforms of the plasma membrane Ca2+ pump in smooth muscle and other tissues.

cDNA clones encoding the plasma membrane Ca2+ pump isoform PMCA1 were obtained from rabbit stomach smooth muscle. The PMCA1 gene has a 154 base exon which can be alternatively spliced. In splices containing 0, 87 or 114 bases of this exon, the mRNA downstream from this position encodes a protein containing the peptide sequence Lys-Arg-Asn-Ser-Ser (KRNSS), which can be phosphorylated by cyclic-nucleotide-sensitive protein kinase. However, in those splices containing 154 bases, the mRNA encodes a protein that does not contain this sequence. The cDNA clone obtained in this study did not contain the latter exon, and thus it coded for KRNSS. The presence of the various splices of PMCA1 was determined in stomach smooth muscle and other tissues by reverse transcription followed by a polymerase chain reaction. Percentage of transcripts encoding the potentially cyclic-nucleotide-sensitive isoform in various tissues were as follows: liver, 100%; stomach mucosa, 100%; heart, 100%; stomach smooth muscle, 86%; aorta, 83%; brain, 55%. Thus brain was the only tissue which expressed a very high proportion of the isoform of PMCA1 that is insensitive to cyclic-nucleotide-dependent protein kinases.

Amino Acid Sequence↗