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A K Grover

Publications and source records attributed to A K Grover.

At least 127 records · Page 7Linked to original sources
PubMed1983

High-affinity pH-dependent passive Ca binding by myometrial plasma membrane vesicles.

Rat myometrium plasma membrane-(PM) enriched fraction N1 binds calcium passively in a pH-dependent manner at a Ca2+ concentration of 1 microM. The Ca binding increases with increasing pH from 6.27 to 7.47 with a half maximum near 6.8. The difference between binding at 6.27 and 7.07 (the pH-dependent Ca binding) depends on the pH of the reaction medium rather than the pH of the medium in which the membranes had previously been suspended. The pH-dependent Ca binding is not an artifact due to EGTA, the pH buffer used, or soluble protein trapped inside the membrane vesicles. The pH-dependent Ca binding occurs with a dissociation constant value of 0.28 microM and Hill coefficient of 2.37 for Ca2+. The high affinity pH-dependent Ca uptake and the release of Ca2+ from the membranes is virtually complete in 10 s in the presence of 1 microM A23187 but not in its absence. The distribution of the pH-dependent Ca binding in the various rat myometrium subcellular fractions parallels the activity of 5'-nucleotidase in these fractions and not the activities of NADPH-dependent or succinate-dependent cytochrome c reductases. The high affinity and rapid binding and release of Ca at the pH-dependent Ca binding sites in the PM-enriched fraction suggests that the binding and release from these sites may play a key role in excitation-contraction coupling of the smooth muscle.

Animals↗
PubMed1983

Na-Ca exchange in a smooth muscle plasma membrane-enriched fraction.

Na-Ca exchange in the plasma membrane-enriched fraction N1 isolated from rat myometrium has been demonstrated as Ca gain or loss depending on the direction of the [Na+] gradient [Grover et al., Am. J. Physiol. 240 (Cell Physiol. 9): C175-C182, 1981]. In this study, the [Na+]gradient-dependent Ca uptake by this membrane fraction was demonstrated to occur optimally at pH 6.67; and 7.0 +/- 2.2 microMs Ca2+ was required for the half-maximal uptake. Such Ca uptake occurred in vesicles previously loaded with a [Ca] that was the same as or higher than the one present in the Ca-uptake medium, i.e., as an uphill Ca-transport process. The Na-Ca exchange-dependent Ca uptake was abolished by the Ca ionophore A23187 or by the monovalent cation (Na+) ionophore monensin. It was not influenced by the presence of KC1, KC1 plus valinomycin, or the proton uncouplers carbonyl cyanine p-trifluoromethoxyphenyl hydrazone or 5'-chloro-3-t-butyl-2'-chloronitrosalicylanilide in the Ca-uptake medium. The properties of the Na-Ca exchange are compared with those of passive and ATP-dependent Ca uptake by this membrane fraction, and physiological implications of this process are discussed.

Animals↗
PubMed1983

On the oxalate stimulation of ATP-dependent calcium accumulation by smooth muscle subcellular membranes.

Subcellular distribution of azide-insensitive, ATP-dependent Ca -accumulation in the presence and absence of 5 mM oxalate by fractions isolated from several different types of smooth muscle were studied. Although oxalate stimulated the Ca -accumulation to various degrees depending upon the source of smooth muscle used, there was no clear association between the stimulatory effect of oxalate and the distribution of any of the enzyme activities used as markers for various membranes. The distribution of ATP-dependent Ca -accumulation in the absence of oxalate, on the other hand, correlates relatively well with that of the plasma membrane marker in spite of reduced total recovery of the Ca -accumulation compared to those of the protein content and membrane markers. However, a greater reduction in the total percent recovery of the ATP-dependent Ca accumulation by fraction from three different types of smooth muscle was consistently observed when carried out in the presence compared to the absence of oxalate. The discrepancy in the total percent recovery of the Ca -accumulation in the presence and absence of oxalate poses a major concern on the uses of oxalate stimulated, ATP-dependent Ca accumulations as a subcellular membrane marker.

Adenosine Triphosphate↗
PubMed1982

Ocular motility in alternating squints: an electro-oculographic study.

Ocular movements were studied electro-oculographically in 10 normal subjects and 10 patients with alternating strabismus. Fixation and pursuit movements and to a less extent the saccadic movements were observed to be abnormal in patients with alternating strabismus. The defect in pursuit movements was shown to be statistically significant. There was no correlation between the extent of ocular motor abnormalities and the angle of deviation or the size of facultative suppression scotoma. The possible common pathological mechanism leading to the ocular motor abnormalities qualitatively similar to those reported earlier in strabismus and anisometropic amblyopes are discussed.

Electrooculography↗
PubMed1982

Ca2+ dependence of calcium uptake by rat myometrium plasma membrane-enriched fraction.

ATP-dependent Ca uptake by rat myometrium plasma membrane fraction occurs only in the presence of magnesium. Studies of the membrane protein-concentration dependence of the ATP-dependent Ca uptake showed that at 33 micrograms protein/ml the uptake increased linearly with time up to 2 min and deviated from linearity at longer times. Studies of the Ca2+ concentration dependence of initial Ca-uptake velocity (reaction time 2 min) of the uptake reaction showed that 1) ATP-dependent Ca uptake increased with increasing Ca2+ concentration from 0.1 to 1 microM, and a further increase in Ca2+ concentration above 1 microM did not enhance the uptake further and 2) the Ca-uptake dependence on Ca2+ concentration was nonhyperbolic with a Hill coefficient of 1.3-1.7 and half saturation occurring at 0.4-0.5 microM Ca2+. These data suggest that the ATP-dependent Ca movement by rat myometrium plasma membranes may play a very important role in smooth muscle relaxation. Physiological implications of the nonhyperbolic kinetics are also discussed.

Adenosine Triphosphate↗
PubMed1982

Abnormal biochemistry of subcellular membranes isolated from nonvascular smooth muscles of spontaneously hypertensive rats.

Microsomal fractions enriched in plasma membranes and endoplasmic reticulum were isolated from stomach fundus and vas deferens from age-matched Okamoto spontaneously hypertensive (SHR) rats and corresponding Kyoto-Wistar normotensive rats (KWR). Alterations of several enzyme activities and Ca2+ accumulation of the isolated microsomal fraction from these nonvascular smooth muscles provide direct evidence of abnormal smooth muscle membrane biochemistry in SHR. Decreased Ca2+ accumulation in the presence of but not in the absence of adenosine triphosphate by the microsomal fractions of both fundus and vas deferens from SHR is consistent with previous findings using plasma membranes from vascular smooth muscles from SHR and cannot be explained in terms of adaptation induced by elevation of blood pressure in SHR. Defective Ca2+ handling now observed in both vascular and nonvascular smooth muscles from hypertensive animals not only provides a cellular basis for the altered reactivity and contractility of smooth muscles observed in SHR, but also supports the hypothesis that spontaneous hypertension is associated with a generalized widespread alteration in smooth muscle membrane fraction.

Animals↗
PubMed1982

Isolation and properties of plasma membrane from smooth muscle.

A generalized approach to obtain relatively pure fractions of plasma membrane from smooth muscle tissues for studying calcium transport is described. The use of various markers for cellular membranes to establish the purity of various fractions is critically considered. Plasma membranes from rat myometrium have been isolated in a purity estimated to be 95-99%. Plasma membrane purifications to 70-80% have been achieved from rat mesenteric arteries and veins, canine tracheal smooth muscle, rabbit intestinal muscle, rat vas deferens, rat fundus, and dog gastric corpus. The ATP-dependent transport of Ca is correlated with the distribution of plasma membrane markers. Ca gradient of greater than 1000-fold have been achieved. ATP-dependent active Ca transport by plasma membranes could sometimes be stimulated by oxalate or phosphate. Anion activation of Ca active transport is not a marker for endoplasmic reticulum. In some smooth muscles (e.g., rat vas deferens) ATP-dependent Ca uptake did not correlate exclusively with the distribution of plasma membrane markers. Instead, the correlation seemed to be with NADPH-cytochrome reductase EC 1.6.2.5 activity (putative endoplasmic reticulum marker) as well as with plasma membrane markers. In all smooth muscles, active Ca transport appears to be a property of the plasma membrane; in some it may also be a property of the endoplasmic reticulum. Mitochondria actively transport Ca, but in most systems studied to date, the Km for Ca2+ for this transport is higher than that for plasma membrane. Thus the plasma membrane may be the major physiological mechanism of active transport for Ca out of cytoplasm of smooth muscle cells. In two plasma membrane fractions (from rat myometrium and mesenteric arteries) it has been possible to demonstrate the existence of an Na-Ca exchange system. Its contribution to lowering cytoplasmic Ca is unknown.

Adenosine Triphosphate↗
PubMed1981

Effect of digitonin on rat myometrium subcellular membrane fractions.

Isopycnic centrifugation experiments using sucrose density gradients showed that in digitonin-treated microsomes the distribution of the plasma membrane (PM) marker 5'-nucleotidase was shifted to higher densities. The treatment also caused similar but less pronounced changes in the distribution of protein, the putative endoplasmic reticulum (ER) marker NADPH-dependent cytochrome c reductase, and the inner mitochondrial marker cytochrome c oxidase. Similar experiments using more purified membrane fractions showed that the digitonin treatment led to a comparable increase in the densities of the fractions N1 and N2 previously described as subfractions of plasma membrane and to considerably less increase in the density of the fraction N3B which is enriched in the endoplasmic reticulum and the inner mitochondrial markers. Digitonin inhibited the ATP-dependent Ca uptake by the N1 fraction in a concentration-dependent manner (I50 = 0.3 mg/mL). Digitonin (0.5 mg/mL) inhibited the ATP-dependent azide-insensitive Ca uptake by all the fractions. The results support the hypothesis that (a) N1 and N2 are subfractions of plasma membrane, and (b) ATP-dependent azide-insensitive Ca uptake in rat myometrium is a property of plasma membranes.

5'-Nucleotidase↗
PubMed1981

Thallous ion activation of p-nitrophenylphosphatase of rat myometrium plasma membrane.

Addition of thallous ion (Tl+) inhibited the spontaneous mechanical activity of rat myometrium in K-free Krebs solutions with an I50 value of 30 microM. The corresponding value for I50 for similar inhibition by addition of K+ was 150 microM. Tl+ as well as K+-activated p-nitrophenylphosphatase (PNPPase) of isolated rat myometrium plasma membrane vesicles. Half maximal activation was caused by 0.47 mM Tl+ or 1.6 mM K+. Maximal enzyme activities obtained using Tl+ and K+ were comparable. The Km values for the substance p-nitrophenylphosphatase using Tl+ (1.24 mM) and K+ (1.46 mM) were also similar. Activation by either ion was inhibited by ouabain, Na+, inorganic phosphate, and vanadate (V +5). The results suggest that Tl+ can substitute for K+ for activation of the Na-K pump of rat myometrium plasma membrane.

4-Nitrophenylphosphatase↗
PubMed1981

Isolation and characterization of subcellular membranes from canine stomach smooth muscle.

Subcellular membrane fractions were isolated from the circular muscle of the corpus of canine stomach by differential and isopycnic sucrose density gradient centrifugation. Differential centrifugation gave a mitochondrial fraction enriched (fourfold) in cytochrome c oxidase and a microsomal fraction enriched (fourfold) in 5'-nucleotidase and NADPH-cytochrome c reductase over postnuclear supernatant. On the basis of a study using continuous gradient, a discontinuous sucrose density gradient was prepared to yield F1 to F5 fractions. The F3 fraction at the interface of 18-32% (w/w) sucrose was maximally enriched (13-fold) in 5'-nucleotidase. The fraction contained very low levels of cytochrome c oxidase but did contain NADPH-cytochrome c reductase (eightfold enrichment). The F4 fraction, at the interface of 32-40% (w/w) sucrose, was maximally enriched in NADPH-cytochrome c reductase (12-fold) and cytochrome c oxidase (6-fold). The distribution of the azide-insensitive. ATP-dependent Ca2+ uptake correlated very well with that of 5'-nucleotidase but less well with NADPH-cytochrome c reductase and not at all with cytochrome c oxidase. Sodium azide and ruthenium red inhibited the ATP-dependent Ca2+ uptake by the mitochondrial fraction and postnuclear supernatant, but not by the F3 fraction. ATP-dependent Ca2+ uptake by the F3 fraction was inhibited by calcium ionophores A23187 and ionomycin, but not by the sodium ionophore, monensin. These results are consistent with the hypothesis that the plasma membrane plays a major role ih regulating intracellular Ca2+ concentration in canine corpus circular muscle.

Adenosine Triphosphate↗
PubMed1981

Na--Ca exchange in rat myometrium membrane vesicles highly enriched in plasma membranes.

When isolated rat myometrium vesicles highly enriched in plasma membranes were preincubated with 100 mM NaCl and then diluted 21-fold in Na-free media, an ATP-independent Ca uptake value of 4.10 +/- 0.23 mumol/g protein occurred, compared to a value of 2.87 +/- 0.16 for a similar uptake by vesicles preincubated in Na-free media. Brief (less than 10 s) exposure of the membrane vesicles to 5 mM ethyleneglycol-bis(beta-aminoethyl)-N,N'-tetraacetic acid (EGTA) after the Ca uptake showed that the NaCl preincubated vesicles retained more Ca than the sucrose or KCl preincubated vesicles. A NaCl concentration in the membrane fractions identical to its concentration in the Ca uptake medium did not enhance the Ca uptake by the vesicles did not show an increased Ca uptake. NaCl added to plasma membrane vesicles actively loaded with Ca caused retention of less Ca than the control. NaCl added to actively loaded vesicles along with EGTA also enhanced calcium efflux compared to EGTA alone. Sucrose, K+, Rb+, or Cs+ could not replace Na+ for the Na+-dependent Ca uptake or release, while Li+ was a poor substitute in both the instances. Na+-dependent Ca-uptake distribution in the various fractions correlated very well with their 5'-nucleotidase activity but not with their NADPH- or succinate-dependent cytochrome c reductase activities. The results have been discussed using a Na--Ca exchange model as well as by a model in which Na+ competes for calcium binding to the membranes.

Adenosine Triphosphate↗
PubMed1980

13C and 31P nuclear magnetic resonance of lipid dispersions from human aorta.

Coarse ("multilamellar") and sonicated ("unilamellar") dispersions of lipid constituents of human aorta have been prepared. 31P nuclear magnetic resonance (nmr) spectra of the coarse dispersions show an unusual pattern compared with pure phospholipid bilayers. The reconstituted membrane is suggested to have considerable amounts of nonbilayer phase, possibly in the form of regions of hexagonal phase with a short major axis, or regions of cubic or rhombic phase admixed with the bilayer phase. High resolution 13C nmr spectra of the sonicated dispersions give narrow signals, indicative of lipids in a fluid environment, and are due mostly to cholesteryl esters and triglycerides.

Aorta↗
PubMed1980

Smooth muscle membrane vesicle orientation: a study on intactness and sidedness of rat myometrium plasma membrane vesicles.

Plasma membrane vesicles of rat myometrium were prepared in media containing 240 mM sucrose. The vesicles were exposed to isotonic, hypertonic, and hypotonic sucrose concentrations, fixed, sectioned, and studied using the electron microscope. The vesicles fixed in isotonic media were circular in appearance. Vesicles fixed in hypertonic media were distorted and showed a reduced volume to surface ratio consistent with the hypothesis that greater than 80% of the vesicles were osmotically active to sucrose. Cationized ferritin binding studies and Ca binding and release studies were also consistent with this finding. Exposure to hypotonic media also yielded membranes with distorted profiles indicating that they had been ruptured. [3H]Sucrose trapping experiments revealed that the vesicles had an internal volume of 1.20-1.44 mL/g protein. Hypotonic shock treatment reduced this intravesicular volume to 0.20-0.28 mL/g protein. The hypotonic shock treatment also led to enhanced galactose oxidase catalyzed Na3B3H4 labelling of the membranes and to increased K+-activated ouabain-sensitive p-nitrophenyl phosphatase activity. The enhancement was the same (55 +/- 10%) in the various membrane preparations for both the parameters. The data are interpreted to conclude that the rat myometrium plasma membrane vesicles consisted of 20% broken vesicles and equal proportions of intact vesicles of inside-out and rightside-out orientations.

4-Nitrophenylphosphatase↗
PubMed1980

Effect of vanadate on rat myometrium plasma membrane enzyme activities.

Vanadate inhibited K+-activated and K+-activated ouabain-sensitive p-nitrophenyl phosphatases of rat myometrium at nanomolar concentrations. The vanadate concentrations required for 50% inhibition were 220 +/- 30 nM for the K+-activated component of the enzyme and 200 +/- 30 nM for the K+-activated ouabain-sensitive component. Micromolar concentrations of vanadate inhibited acid and alkaline p-nitrophenyl phosphatases. ATP-dependent Ca uptake by the plasma membrane vesicles was not inhibited by 10nM - 1 mM vanadate. Mg2+-ATPase was also not affected. Thus K+-activated and K+-activated ouabain-sensitive p-nitrophenyl phosphatase activities of the plasma membrane were most sensitive to inhibition by vanadate. Preliminary experiments demonstrated that similar to ouabain, vanadate inhibited potassium-induced abolition of spontaneous contractile activity of isolated rat myometrium in K-free Krebs. This effect of vanadate is consistent with vanadate inhibition of K+-activated ouabain-sensitive p-nitrophenyl phosphatase.

Animals↗
PubMed1980

Characteristics of calcium transport and binding by rat myometrium plasma membrane subfractions.

A gradient has been designed to yield two subfractions of plasma membrane vesicles from rat myometrium, a low buoyant density (8-24% sucrose) fraction N1 richer in 5'-nucleotidase and a higher buoyant density (24-30% sucrose) fraction N2, instead of a previously described fraction F1. Both N1 and N2 had very low activities of NADPH-cytochrome c reductase and succinate-cytochrome c reductase. Electron micrographs of thin sections of N1 showed clear vesicles, whereas N2 consisted of vesicles with electron-dense bodies attached to them. These plasma membrane vesicles can actively take up Ca. The active uptake of Ca was potentiated by oxalate and phosphate and abolished by the Ca ionophore A23187. Dilution of actively loaded vesicles in isotonic media containing EGTA led to loss of a small proportion of the stored Ca instantaneously and the remainder more slowly in a biphasic manner. Dilution in hypotonic media with EGTA led to a release of a much larger proportion of the accumulated Ca. A23187 at high concentrations (10 microM) caused a release of all the sequestered Ca whether the active Ca uptake had been carried out in the presence or in the absence of oxalate. A23187, 0.5 microM, released all the sequestered Ca from the vesicles that were actively loaded in the absence of oxalate, but only 37% when the vesicles were actively loaded with Ca in the presence of oxalate. Comparison of the composite plasma membrane fraction F1 (8-30% sucrose) and the subfractions N1 and N2 showed that they had different capacities for Ca uptake in the presence and absence of ATP. An attempt has been made to analyze the active Ca-uptake data in terms of various Ca pools.

Adenosine Triphosphatases↗
PubMed1980

Gap junction formation and regulation in myometrium.

Myometrial tissues from pregnant rats were examined by electron microscopy for the presence of gap junctions after incubation in vitro with a variety of agents. Gap junctions were present in low frequency or absent prior to incubation in vitro. The junctions were present in control tissues in high frequency after 48 h incubation. The addition of cycloheximide or actinomycin D inhibited the incorporation of [3H]leucine into TCA-precipitable proteins and prevented gap junction formation. A prostacyclin analog (carbacyclin), a thromboxane synthesis inhibitor, and indomethacin also prevented gap junction formation. Oxytocin had no effect on gap junction formation but isoxsuprine decreased their number and increased their size. Isoxsuprine and isoproterenol also produced electron opaque crystals associated with the gap junctions. Dibutyryl cAMP treatment but not monobutyryl cGMP also increased the size of gap junctions. Based upon this and previous studies, we propose at least four sites for regulation of gap junctions and possible control of labor.

Animals↗