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Biomedical subjects

A K Grover

Publications and source records attributed to A K Grover.

At least 109 records · Page 6Linked to original sources
PubMed1985

Subcellular distribution of dihydropyridine isothiocyanate binding in guinea-pig ileal smooth muscle.

The subcellular distribution of the 3H--2,6-dimethyl-3,5-dicarbomethoxy-4(2-isothiocyano) phenyl-1,4-dihydropyridine (DPSCN) binding to guinea-pig ileal smooth muscle was studied by subcellular fractionation. Initial experiments on subcellular fractionation of 3H-DPSCN-labelled tissues by differential centrifugation showed that there was an excellent correlation between the levels of the label present in a fraction and the plasma membrane marker phosphodiesterase I (r = 0.98) but not between the label and the putative endoplasmic reticulum marker NADPH: cytochrome-c-reductase (r = 0.56) or the inner mitochondrial marker cytochrome-c-oxidase (r = 0.36). Centrifugation of the microsomes on a continuous sucrose density gradient showed an excellent correlation of the migration of the label with phosphodiesterase I activity (r = 0.93) but not with the activities of NADPH: cytochrome-c-reductase (r = 0.66) or cytochrome-c-oxidase (r = 0.44). Treatment of microsomes with digitonin (1 mg/ml) followed by centrifugation on continuous sucrose density gradients increased the weighted mean densities of the phosphodiesterase activity (plasma membrane marker) and the labelling by similar magnitudes (0.04 to 0.06 g/ml). The weighted mean densities of NADPH: cytochrome-c-reductase and the cytochrome-c-oxidase were not altered significantly. It is concluded that in the guinea-pig ileal smooth muscle, DPSCN labels the plasma membrane specifically.

Animals↗
PubMed1984

Subcellular distribution of [3H]nitrendipine binding in smooth muscle.

Distribution of specific binding sites for [3H]nitrendipine was studied in subcellular fractions isolated from rat gastric fundus smooth muscle and from rat myometrium. There was an excellent correlation between the distribution of [3H]nitrendipine binding determined at the nitrendipine concentrations of 0.138 and 1.38 nM, and the distribution of the plasma membrane markers K+-activated ouabain-sensitive p-nitrophenylphosphatase, 5'-nucleotidase, phosphodiesterase I, and Mg-ATPase, but not between the mitochondrial markers cytochrome c, oxidase, succinate-dependent cytochrome c reductase, or rotenone-insensitive NADH-dependent cytochrome c reductase or the putative endoplasmic reticulum marker NADPH-dependent cytochrome c reductase. The binding occurred with high affinity and with a similar (0.097-0.146 nM) equilibrium dissociation constant to all the fractions, even though the density of binding sites varied and was highest in the plasma membrane marker-enriched fractions. The maximal binding in the plasma membrane-enriched fraction from the rat gastric fundus smooth muscle was 0.43 +/- 0.04 pmol/mg, and in that from rat myometrium was 0.72 +/- 0.09 pmol/mg. Thus in the two smooth muscles studied the plasma membrane is the locus of the high affinity nitrendipine binding.

Animals↗
PubMed1984

Membrane fractionation of canine aortic smooth muscle: subcellular distribution of calcium transport activity.

Various subcellular membrane fractions were isolated from dog aortic smooth muscle by conventional differential centrifugation followed by isopycnic centrifugation on a sucrose density gradient. These subcellular fractions were characterized by membrane marker enzyme activities, morphological features and the electrophoretic patterns on a sodium dodecyl sulfate polyacrylamide gel. Our results showed that the microsomal membrane fraction isolated by differential centrifugation was very heterogeneous and contained substantial amount of plasma membranes which could be further enriched as a light density fraction on the sucrose density gradient. The subcellular distribution of Ca2+ binding in the absence of ATP and Ca2+ transport in the presence of ATP closely paralleled the distribution of plasma membrane markers. The ATP-supported Ca2+ transport was inhibited by several Ca2+ ionophores, enhanced by inorganic phosphate and oxalate ions and cosedimented toward higher density in a continuous source density gradient with plasma membrane marker enzyme activity in the presence of digitonin. Our present work strongly suggests that plasma membrane is the predominant component of microsomal fraction and responsible for most, if not all, of the azide-insensitive ATP-supported Ca2+ accumulation.

Adenosine Triphosphate↗
PubMed1984

Ca2+ handling properties of microsomal subfractions of rat vas deferens smooth muscle.

The rat vas deferens smooth muscle microsomes on isopycnic centrifugation gave two fractions, namely F2 (15-30% sucrose) and F3 (30-40% sucrose), with comparable ATP-dependent azide-insensitive Ca2+-uptake capacities, although these fractions differed from each other in various enzyme marker activities. The fractions F2 and F3 also show similar pH profiles for the ATP-independent and ATP-dependent Ca2+ uptake, and similar ionized Ca2+-concentration dependence for the ATP-dependent Ca2+ uptake. However, the fractions F2 and F3 differ from each other in that: (a) F3 shows higher permeability to Ca2+, and (b) F3 shows higher stimulation of the ATP-dependent Ca2+ uptake by oxalate. The F3 fraction can also be used to obtain membrane vesicles loaded with Ca2+ oxalate in the presence of ATP. However, the yield of the Ca2+ oxalate enriched fraction is too low to permit their further characterization.

Adenosine Triphosphate↗
PubMed1984

Binding and degradation studies on angiotensin II.

125I-labelled angiotensin II (AII) and [3H]AII showed specific binding to rat mesenteric artery microsomes. The binding in either instance was inhibited by the AII analog saralasin. [3H]AII was not degraded by the microsomes but 125I-labelled AII was degraded. Autoradiography of thin layer chromatograms of 125I-labelled AII treated with microsomes showed the parent peak (Rf = 0.4-0.45) and a single major degradation product peak (Rf = 0.25-0.30), and [125I]NaI had an Rf value higher than both 125I-labelled AII and its degradation product. Chromatography of unlabelled AII or [3H]AII gave the same Rf value as 125I-labelled AII, but unlabelled AIII moved with Rf = 0.55-0.60. The formation of the degradation product was time and membrane concentration dependent. The degradation occurred at pH 6 and 7 but not at pH 8. However, specific binding of 125I-labelled AII was also lower at pH 8. The degradation could not be completely inhibited by the use of crude particulate fractions instead of microsomes, by preparing membranes in presence of protease inhibitors, or by including protease inhibitors and sulfhydryl agents in the assay medium. However, the degradation product neither showed specific binding to the microsomes nor interfered with the specific binding of 125I-labelled AII. Furthermore, the tightly bound material eluted from the microsomes in presence of 0.05 M acetic acid at 0 degree C consisted predominantly of the parent compound. The implications of these findings are discussed both in terms of validity of the binding experiments and possible relationship between the degradation and the receptor binding sites in the membrane.

Angiotensin II↗
PubMed1984

Effect of disulphonic stilbenes on Ca2+ transport in smooth muscle plasma membranes.

We studied the effects of two disulphonic stilbenes, 4',4'-diisothiocyano-2,2'-stilbene disulphonic acid (DIDS) and 4-acetamido-4'-isothiocyano-2,2'-stilbene disulphonic acid (SITS), on Ca2+ transport by plasma membrane vesicles from the circular muscle of the dog stomach. Both compounds inhibited ATP-dependent Ca2+ uptake and reduce the leak from loaded vesicles. The inhibition produced could not be significantly reduced by either permeant anions or by increasing the level of free Ca2+. The effects of DIDS could be rendered irreversible by incubating the membranes with this agent at 37 degrees C.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1984

Analysis of data on efflux of radioactive ions from isolated membrane vesicles.

Conventional semilogarithmic plots of radioisotope-retained [y(t)], by membrane vesicles during efflux, against time give a gross overestimate of the half-life (t 1/2) of the efflux, if a significant proportion of the radioisotope is retained at equilibrium. By using simulated efflux experiments, it is shown that apparent linearity of such plots evidenced by excellent linear correlation coefficients may not detect such error. 45Ca efflux from sarcolemma of rat myometrium further illustrates this point. Rat myometrium sarcolemmal vesicles were loaded at pH 6.27 and 7.07 with 1 mM CaCl2 and then diluted into solutions containing ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) to result in 1 microM Ca2+ at the above pH values. Analysis using log y(t) vs. t plots gave t 1/2 values of 93.32 +/- 8.64 and 28.63 +/- 1.22 min at pH 7.07 and 6.27, respectively. However, log [y(t) - yeq] vs. t plots yielded t 1/2 values of 28.28 +/- 2.15 and 24.88 +/- 1.00 min. The discrepancy results from a larger equilibrium binding at the higher pH. Therefore it is recommended that the quantity of the isotope retained at equilibrium (yeq) be determined experimentally and then that the data be analyzed using log [y(t) - yeq] vs. t plots to obtain true estimates of t 1/2. This recommendation would be applicable to a wide range of studies.

Animals↗
PubMed1984

On the ouabain-sensitive potassium activated p-nitrophenyl phosphatase activity of vascular muscle plasma membranes.

Sodium-pump ATPase is known to be a cell membrane enzyme being responsible for vectorial transport of Na+ against electrochemical gradient across the plasma membranes of most cell types. In smooth muscles, this Na+-pump ATPase activity is notoriously low and its measurement is complicated by remarkably high level of the basal ATPase activities. In this communication, the properties of ouabain-sensitive, potassium-activated p-nitrophenyl phosphatase (pNPPase) activity which is a partial reaction of the Na+-pump are reported and compared in plasma membrane enriched fractions of mesenteric arteries from hypertensive and normotensive rats. The pNPPase activity of isolated plasma membrane vesicles from this vascular muscle is optimally stimulated by low concentrations of KCl (5-25 mM) at pH 7.8. Ouabain at 1.0 mM inhibits up to 75% of the K+-activated pNPPase activity under the optimal assay condition. Osmotic shock of the plasma membrane vesicles resulted in about 30% enhancement of the ouabain-sensitive K+-pNPPase activity compared to the control values. Although apparent difference was observed in this enzyme activity using similar preparations obtained from rats of different strains, the relative enhancement of such ouabain-sensitive K+-pNPPase activities by osmotic shock was not significantly different between vascular membrane fractions from the spontaneously hypertensive rats and the Wistar-Kyoto normotensive rats. Our results suggest that the intrinsic Na+-pump activity of small blood vessels in genetic hypertension in rats is not altered and the isolation procedure does not have differential effect on the orientation of plasma membrane vesicles in fractions obtained from hypertensive and normotensive rats.

4-Nitrophenylphosphatase↗
PubMed1984

ATP-dependent Ca-uptake by rat vas deferens smooth muscle microsomes: properties of oxalate stimulated and oxalate-independent Ca-uptake.

The oxalate-stimulated and -independent components of the ATP-dependent azide insensitive Ca-uptake by rat vas deferens smooth muscle microsomes differ in the following properties: (a) 5, 5'-disulfonate stilbene (DIDS) and digitonin inhibited the oxalate-independent Ca-uptake more strongly than the oxalate-stimulated component; (b) phosphatidylserine inhibited the oxalate-stimulated Ca-uptake and had no significant effect on the oxalate-independent uptake and (c) digitonin treatment of microsomes changed the density distributions of the two modes differentially. The untreated microsomes gave two Ca-uptake peaks on sucrose density gradients: one at density 1.130 +/- 0.010 g/ml and the other at 1.214 +/- 0.005 g/ml. Digitonin treatment shifted the distribution of the lower density peak to slightly higher density for the oxalate-independent Ca-uptake but not for the oxalate-stimulated Ca-uptake. Distribution of phosphodiesterase I was also shifted to higher densities while distributions of the higher density Ca-uptake peak, NADPH: cytochrome c reductase and of cytochrome c oxidase were not significantly altered. Thus clearly the oxalate-stimulated and the oxalate-independent Ca-uptake in rat vas deferens smooth muscle microsomes show several differences but it is unknown whether the differences are due to the existence of two distinct Ca-pump proteins or due to different microenvironment of the same protein resulting from membrane heterogeneity.

Adenosine Triphosphate↗
PubMed1983

Oxalate-stimulation of ATP-dependent Ca-uptake is diminished during smooth muscle subcellular fractionation.

The ATP-dependent azide-insensitive Ca-uptake by the postnuclear supernatant from rat myometrium is stimulated more by 5 mM oxalate than by 25 mM phosphate. During subcellular fractionation, however, the percent recovery of the oxalate stimulated Ca-uptake diminishes more rapidly than that of the Ca-uptake without any added oxalate or phosphate. The percent recovery of the phosphate stimulated Ca-uptake also diminishes but not to as low levels as that of the oxalate stimulated Ca-uptake. The net result is higher stimulation of this uptake by 25 mM phosphate than by 5 mM oxalate in the various sucrose density gradient fractions. This discrepancy in percent recoveries presents a major concern about the use of oxalate or phosphate stimulated Ca-uptake as a marker for smooth muscle membranes.

Adenosine Triphosphate↗
PubMed1983

An analytical approach to the preparation and characterization of subcellular membranes from canine mesenteric arteries.

Subcellular membrane fractions were isolated from dog mesenteric arteries by differential and isopynic sucrose density gradient centrifugations. Isolated membrane fractions were characterized by marker enzyme activities, morphological features and sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns. Our results show that the microsomal fraction isolated by conventional differential centrifugation was highly heterogenous and contained substantial amount of plasma membranes which could be further enriched as a light density membrane fraction on a discontinuous sucrose density gradient. The microsomal fraction and its subfractions were vesicular in appearance under electron microscope and were capable of binding and actively transporting Ca2+. The binding of Ca2+ and ATP-supported Ca2+-transport in the presence or absence of oxalate paralleled the distribution of plasma membrane marker enzyme activities suggesting that plasma membranes in vascular smooth muscle may play a major role in handling Ca2+ and thus the control of contractile function.

Adenosine Triphosphate↗
PubMed1983

Membrane abnormalities occur in vascular smooth muscle but not in non-vascular smooth muscle from rats with deoxycorticosterone-salt induced hypertension.

Microsomal fractions were isolated from the smooth muscle of gastric fundus, vasa deferentia and mesenteric arteries of rats made hypertensive by deoxycorticosterone-salt treatment. Several enzymatic activities, Ca2+ binding and ATP-dependent Ca2+ accumulation of the microsomal fractions from these hypertensive rats were compared with those from the control of rats which remained normotensive under similar treatment. Altered membrane properties were observed in microsomal fractions isolated from vascular smooth muscle but not in those isolated from non-vascular smooth muscles in this form of experimental hypertension. These alterations included decreased Mg2+ ATPase activity, enhanced alkaline phosphatase activity, decreased Ca2+ binding in the absence of ATP and decreased ATP-dependent Ca2+ accumulation. This result is in contrast to our previous findings that decreased ATP-dependent Ca2+ accumulation was observed in microsomal fraction isolated from non-vascular smooth muscles of rats with genetic hypertension. The present study, together with our previous findings, support the contention that altered Ca2+ handling by vascular smooth muscle is associated with the pathogenesis of hypertension, whereas altered Ca2+ handling by non-vascular smooth muscles previously observed in spontaneous hypertension may be associated with genetic factors not related to hypertension.

Animals↗
PubMed1983

Uptake and release of calcium by canine gastric corpus smooth muscle plasma membrane enriched fraction.

Calcium movements across plasma membrane enriched vesicles isolated from canine gastric corpus smooth muscle were investigated. The ATP-dependent Ca2+ uptake increased with time up to 10 min. The uptake for the initial 2-min period was approximately linear with time. The apparent initial velocity of the ATP-dependent Ca2+ uptake increased monotonically with free Ca2+ concentration from 0.1 to 2 microM, and further increases in free Ca2+ concentration did not increase the Ca2+ uptake. The free Ca2+ dependence curve could be described with a Hill coefficient of approximately 1.0 and Km of 0.85 +/- 0.01 microM for free Ca2+ concentration. Passive Ca2+ uptake (reaction time = 1 h) also increased with increasing free Ca2+ concentrations from 0.02 to 4.0 mM. Dilution of loaded vesicles in isotonic media containing EGTA led to initial rapid loss (less than 1 min) followed by a slower release which showed simple exponential decay. The t 1/2 values of the slower Ca2+ loss from these vesicles were 16.1 +/- 0.9 min (actively loaded n = 5) and 18.4 +/- 0.9 min (passively loaded n = 3), respectively. Dilution in isotonic medium containing both EGTA and A23187 released all the sequestered Ca2+ from these loaded vesicles.

Adenosine Triphosphate↗
PubMed1983

Studies on canine gastric antrum smooth muscle: preparation and characterization of a plasma membrane enriched fraction.

A method is described for preparation of large amounts of a plasma membrane (PM) enriched fraction from the smooth muscle of dog antrum. It consists of preparing microsomes, treating them with ATP + EGTA + Mg, centrifuging in 30% sucrose and then centrifuging the resulting supernatant in 15% sucrose to yield the plasma membrane enriched fraction P6. The subcellular fractions obtained at various steps during purification were characterized by: 5'-nucleotidase and phosphodiesterase I as plasma membrane markers; cytochrome c oxidase as an inner mitochondrial marker; NADPH-cytochrome c reductase as a putative endoplasmic reticulum marker; electron microscopy; polyacrylamide sodium dodecyl sulfate slab gel electrophoresis. The distribution of ATP-dependent and independent Ca uptake in presence and absence of azide and the effect of 5 mM oxalate or 25 mM phosphate on this uptake was also examined. The fraction P6 consists of mostly smooth surface vesicles 164.3 +/- 7.2 nm in diameter, has an exclusion volume of 9.7 microL/mg for [3H]inulin and 11.1 microL/mg for [3H]sucrose. P6 is maximally enriched in the ATP-dependent azide-insensitive Ca-uptake capacity and as compared with the postnuclear supernatant (S1) it shows a very small percent stimulation by oxalate and phosphate. The ATP-dependent Ca uptake by the P6 fraction occurs optimally at pH 7.0-7.4 and is much larger than the ATP-independent Ca uptake. At pH 7.1, the ATP-dependent Ca uptake occurs with a Km of 0.27 microM and a Hill coefficient greater than 2 for Ca2+. Half maximum binding of Ca2+ occurred at 300 microM Ca2+. Ca ionophores A23187 and ionomycin inhibited the ATP-dependent Ca uptake, and if added after the uptake, these caused a release of the accumulated Ca2+. From these and other data it is concluded that this PM preparation contains a Ca transport system which can lead to formation of greater than 1000-fold Ca2+ concentration gradient across the vesicle membrane in 1 min when extravesicular Ca2+ concentration is 0.3 microM. Thus this preparation is an extremely useful material for studying the mechanism of action of the Ca pump in smooth muscle plasma membrane.

Adenosine Triphosphate↗