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Biomedical subjects

S Muallem

Publications and source records attributed to S Muallem.

At least 127 records · Page 7Linked to original sources
PubMed1987

Manoalide, a natural sesterterpenoid that inhibits calcium channels.

Manoalide is a marine natural product that has anti-inflammatory and anti-proliferative activities and is an irreversible inhibitor of phospholipase A2 and phospholipase C. It is now shown that the compound is a potent inhibitor of Ca2+ mobilization in several cell types. In A431 cells the increase in epidermal growth factor receptor-mediated Ca2+ entry and release from intracellular Ca2+ stores were blocked by manoalide in a time-dependent manner with an IC50 of 0.4 microM. The effect of manoalide on phosphoinositide metabolism, namely the production of inositol monophosphate, did not coincide with its effect on the epidermal growth factor response. In GH# cells, manoalide blocked the thyrotropin-releasing hormone-dependent release of Ca2+ from intracellular stores without inhibition of the formation of inositol phosphates from phosphatidylinositol 4,5-bisphosphate. Manoalide also blocked the K+ depolarization-activated Ca2+ channel in these cells as well as the activation of the channel by Bay K8644 with an IC50 of 1 microM. In addition, manoalide also inhibited the Ca2+ influx induced by concanavalin A in mouse spleen cells in a time- and temperature-sensitive manner with an IC50 of 0.07 microM. However, neither forskolin-activated adenylate cyclase in A431 cells nor the distribution of the potential sensitive dye, 3,3'-dipropylthiodicarbocyanide iodide in GH3 cells was affected by manoalide. Thus, manoalide acts as a Ca2+ channel inhibitor in all cells examined. This action may account for its effects on inflammation and proliferation and may be independent of its effect on phospholipases.

Animals↗
PubMed1987

Detection of a Na+-H+ antiporter in cultured rat renal papillary collecting duct cells.

To examine whether Na+-dependent H+ transport is present in the papillary collecting duct, changes in intracellular pH (pHi) were evaluated in cultured papillary collecting duct cells acidified to a pHi of 6.3 and then placed into Na+-free or Na+-containing solutions. pHi was determined from changes in the fluorescent signal of the pH-sensitive dye BCECF. pHi did not change significantly when cells were placed in tetramethylammonium chloride- or KCl-containing solutions; however, a significant rise in pHi occurred when acid-loaded cells were placed in solutions containing 140 mM NaCl. The Na+-dependent rise in pHi was blocked by high concentrations of amiloride, but was not affected by alterations in membrane potential across the cell. The rate of rise in pHi was a function of extracellular sodium concentration with a Km for Na+ of 30 +/- 12 mM (n = 6). The properties of this Na+-dependent H+ efflux supports the presence of a Na+-H+ antiporter in the papillary collecting duct.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1986

Regulation of free cytosolic Ca2+ in the peptic and parietal cells of the rabbit gastric gland.

Quin 2-loaded isolated rabbit gastric glands and purified peptic cells were used to measure free cytosolic Ca2+ ([Ca2+]i) during hormone stimulation. Rabbit gastric glands are composed of peptic and parietal cells with less than 1% endocrine cells. Although both cell types responded to the same hormones, they may be distinguished in terms of the source of Ca2+ bringing about the change in [Ca2+]i. Experiments were designed to assign changes in [Ca2+]i to either the peptic or parietal cells and to attempt to maintain these distinctions in the mixed cell population of gastric glands. It was shown that the peptide cholecystokinin octapeptide induced a rapid and transient increase in [Ca2+]i of isolated peptic cells. This signal was independent of medium Ca2+ and insensitive to the Ca2+ channel blockers La3+ and nifedipine. In gastric glands, the Ca2+ outdependent increase in (Ca2+)i (the secondary transient) was slower and dose dependently blocked by La3+ and nifedipine. This allowed [Ca2+]i levels in the physiologically more intact rabbit gastric glands to be dissected and correlated with fluorescence changes of quin 2 in either cell type. The transient increase in [Ca2+]i coincided with a burst of pepsin but not acid secretion. A subsequent slower phase of pepsin secretion took place while the cells restored near resting [Ca2+]i. Using a combination of the Ca2+ ionophore A23187 and the protein kinase C activating phorbol ester 12-O-tetra-decanoylphorbol 13-acetate, the hormone response pattern of pepsin secretion could be mimicked. The intracellular Ca2+ stores of the peptic cells in the gastric gland remained depleted of Ca2+ until specific antagonists were added. The reloading of intracellular stores required medium Ca2+ although [Ca2+]i was maintained at resting level during the entire reloading period. Hence, a specialized pathway of Ca2+ reloading is postulated.

Aminopyrine↗
PubMed1985

The nature of the Ca2+-pump defect in the red blood cells of patients with cystic fibrosis.

The reduction in (Ca2+ + Mg2+)-ATPase activity in the cystic fibrosis red blood cells can be attributed to a reduction in the number of active Ca2+ pumps per red blood cell and an altered interaction of calcium ions with the pump. Despite this, the normal free intracellular [Ca2+] is preserved due to a lower rate of passive calcium entry.

Biological Transport, Active↗
PubMed1985

Role of free cytosolic calcium in secretagogue-stimulated amylase release from dispersed acini from guinea pig pancreas.

To determine the role of free cytosolic calcium ([Ca+2]i) in stimulated enzyme secretion from exocrine pancreas, we determined the effects of various pancreatic secretagogues on [Ca+2]i and amylase release in dispersed acini from the guinea pig pancreas. Cholecystokinin-octapeptide (CCK-OP), carbachol, and bombesin, but not vasoactive intestinal peptide, stimulated rapid increases in [Ca+2]i from 100 to 600-800 nM that were independent of extracellular calcium. The increases in [Ca+2]i were transient (lasting less than 5 min) and correlated with an initial rapid phase of amylase release. After 5 min, secretagogue-stimulated amylase release occurred at basal [Ca+2]i. Carbachol pretreatment of the acini abolished the effects of CCK-OP and bombesin on [Ca+2]i and the initial rapid phase of amylase release. 4 beta-phorbol 12-myristate 13-acetate (PMA) had no effect on [Ca+2]i but stimulated an increase in amylase release. The addition of CCK-OP or A23187 to PMA-stimulated acini caused an increase in [Ca+2]i and PMA-stimulated amylase release only during the first 5 min after addition of these agents. These results indicate that CCK-OP, carbachol, and bombesin release calcium from an intracellular pool, resulting in a transient increase in [Ca+2]i and that this increase in [Ca+2]i mediates enzyme secretion during the first few minutes of incubation. The results with PMA suggest that secretagogue-stimulated secretion not mediated by increased [Ca+2]i (sustained secretion) is mediated by 1,2-diacylglycerol.

Amylases↗
PubMed1985

Electrolyte transport across the basolateral membrane of the parietal cells.

The ion-transport properties of the basal lateral membranes of intact isolated parietal cells were studied at the cellular and subcellular level. The presence of an amiloride-sensitive Na+:H+ exchange was demonstrated in cells by proton gradient-driven Na+ uptake and by changes in cell pH as monitored by dimethylcarboxylfluorescein fluorescence both in a fluorimeter and on single isolated cells using a fluorescence microscope and an attached intensified photodiode array spectrophotometer. The presence of the Na+:H+ antiport in vesicles was shown both by intravesicular acidification monitored by acridine orange fluorescent quenching and by proton gradient-dependent Na+ uptake. The presence of Cl-:HCO-3 exchange was determined in intact cells by monitoring changes in cell pH due to Cl- uptake and was shown to be 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid- and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid-sensitive. In vesicles, Cl-:HCO-3 exchange was demonstrated by Cl- flux measurement. The apparent affinities for both Cl- and HCO-3 on either side of the membrane were determined to be Km Cli = 20 mM, Km Clout = 17.5 mM, Km HCO-3in = 2.5 mM, and Km HCO-3out = 7.5 mM. A K+ conductance in cells and vesicles was demonstrated by monitoring K+ gradient-dependent 86Rb uptake. No evidence was found for the presence of a Cl- conductance in either cells or vesicles but a H+ conductance was found to be present in vesicles but not in intact cells. In the latter, by determining the effect of either Na+ or Cl- gradients on cell pH and by flux calculations it was concluded that the Cl-:HCO-3 exchange was the major passive flux mechanism for pH regulation in this cell type.

Amiloride↗
PubMed1985

Inositol trisphosphate modification of ion transport in rough endoplasmic reticulum.

The ion transport properties of the rough endoplasmic reticulum (RER) from liver have been defined by using measurements of active and potential gradient-driven transport. The Ca2+ pump is shown to be electrogenic, and both ATP and potential difference is able to drive vanadate-inhibitable Ca2+ uptake into the RER. ATP-dependent Ca2+ transport into the RER depends on the presence of tetraethylammonium-sensitive cation conductance and a furosemide-inhibited cation/chloride cotransport pathway. Inositol trisphosphate does not affect either of the monovalent ion translocation systems but activates a Ca2+ conductance in the RER, allowing efflux of RER Ca2+ stores into the cytosol in exchange for K+ uptake.

Adenosine Triphosphate↗
PubMed1985

Ca2+ metabolism during cholinergic stimulation of acid secretion.

Carbachol-induced changes in 45Ca fluxes and cytosolic free Ca2+ were characterized in rabbit gastric glands and isolated dog parietal cells. Cholinergic stimulation was expressed as changes in the membrane permeability of the parietal cell to Ca2+ without Ca2+ release from intracellular stores. The changes in the membrane permeability to Ca2+ were sustained as long as the cells were exposed to carbachol, independent of extracellular Ca2+, and had the properties of a passive pathway across which Ca2+ distributed according to its chemical gradient. As a consequence of the changes in membrane permeability to Ca2+, carbachol caused a sustained increase in free cytosolic Ca2+ from a resting level of 134 +/- 11 to 533 +/- 81 nM (n = 14). After carbachol stimulation, calcium ions were sequestered in mitochondrial and probably nonmitochondrial compartments. When cell stimulation was terminated by atropine or La3+, the cells restored the resting level of the intracellular Ca2+ activity ([Ca2+]in), independent of uncoupling of mitochondrial oxidation phosphorylation. Reduction in [Ca2+]in was mainly through Ca2+ efflux across the plasma membrane of the parietal cell. Manipulation of intracellular Na+ ion activity in intact cells and studies with basal-lateral membrane vesicles from gastric mucosa indicated the absence of extracellular Na+-intracellular Ca2+ exchange activity in the plasma membrane of the parietal cell. A calmodulin-regulated, ATP-dependent Ca2+ pump that could maintain active Ca2+ extrusion from parietal cells was found in the basal-lateral membrane of the parietal cells.

Animals↗
PubMed1985

Role of calcium in cholecystokinin-stimulated phosphoinositide breakdown in exocrine pancreas.

In dispersed acini from guinea pig pancreas cholecystokinin octapeptide (CCK-OP) stimulated breakdown of the phosphoinositides phosphatidylinositol (PI) and its phosphorylated derivative, phosphatidylinositol 4,5-bisphosphate (PI-P2), as measured by a decrease in the mass of PI and decreases in the content of [3H]PI and [32P]PI-P2 in acini prelabeled with myo-[2-3H]inositol or H3(32)PO4. The breakdown occurred in the absence of extracellular Ca2+ and when the CCK-OP-induced rise in free intracellular Ca2+ ([Ca2+]i) was ablated by loading the acini with the Ca2+-selective indicator and chelator quin-2 in the absence of extracellular Ca2+. In contrast to CCK-OP, the calcium ionophore A23187 caused breakdown of PI and PI-P2 in the presence but not in the absence of extracellular Ca2+, although like CCK-OP A23187 stimulated 45Ca outflux, a measure of cellular Ca2+ mobilization, and amylase release during the first 5-10 min of incubation independent of extracellular Ca2+. In the absence of extracellular Ca2+ A23187 did not inhibit the ability of CCK-OP to cause PI breakdown. These results indicate that CCK-OP stimulates breakdown of PI and PI-P2 and that this breakdown is independent of extracellular Ca2+, mobilization of intracellular Ca2+, and the CCK-OP-induced rise in [Ca2+]i. These findings suggest that one of the initial events resulting from CCK-OP interaction with its receptor is phosphoinositide breakdown.

Aminoquinolines↗
PubMed1984

Changes in cytosolic free Ca2+ in isolated parietal cells. Differential effects of secretagogues.

The relationship of free cytosolic Ca2+ to secretagogue-dependent activation of acid secretion by the mammalian parietal cell was studied using quin 2 as an intracellular Ca2+ probe. The resting [Ca2+]in of isolated dog parietal cells was found to be 134 +/- 11 nM. Carbachol produced a steady-state increase of [Ca2+]in and its effect was blocked by atropine and Ca2+ -channel blocking agents. Gastrin transiently elevated [Ca2+]in and this was not affected by Ca2+ -channel blocking agents. Neither histamine nor dbcAMP changed resting [Ca2+]in in rabbit parietal cells.

Aminoquinolines↗
PubMed1984

Effects of affinity-purified antibodies on the Ca2+ pumping ATPase of erythrocyte membranes.

Antibodies raised in rabbits against the purified erythrocyte membrane Ca2+ pumping ATPase were affinity-purified using an ATPase-Sepharose column. Addition of a few molecules of the purified antibody per molecule of ATPase was sufficient to inhibit the ATPase activity. Extensively washed ghosts or preincubated pure ATPase sometimes develop an appreciable Mg2+-ATPase activity. In such cases, the antibodies inhibited the Mg2+-ATPase as well as the Ca2+-ATPase. This is consistent with the hypothesis that a portion of the Mg2+-ATPase activity of ghosts is derived from the Ca2+-ATPase. When nitrophenylphosphatase activity was observed, both Mg2+- and Ca2+-stimulated activities were observed. Only the Ca2+ activity was inhibited by the antibodies, confirming that this activity is due to the Ca2+ pump, and suggesting that the Mg2+-nitrophenylphosphatase is due to a separate enzyme. Amounts of antibody comparable to those which inhibited the Ca2+-ATPases had no effect on the Na+-K+-ATPase; 4-fold higher amounts of antibody significantly stimulated the Na+-K+-ATPase, but this effect of the antibody was not specific: Immunoglobulins from the nonimmune serum also significantly stimulated the Na+-K+-ATPase. In resealed erythrocyte membranes, antibodies incorporated into the ghosts inactivated the Ca2+-ATPase, while antibodies added to the outside had no significant effect.

Adenosine Triphosphatases↗
PubMed1983

Catalytic and regulatory ATP-binding sites of the red cell Ca2+ pump studied by irreversible modification with fluorescein isothiocyanate.

Catalytic and regulatory binding sites for ATP on the red cell Ca2+ pump have been investigated using fluorescein isothiocyanate (FITC). Both (Ca2+ + Mg2+)-ATPase activity and ATP-dependent Ca2+ flux are selectively and irreversibly inactivated by FITC and the pump is protected from FITC by the presence of ATP. The time course of inactivation by FITC is characteristically biphasic. Analysis of the kinetics of inactivation by FITC and protection by ATP reveals the participation of both high and low affinity binding sites for ATP and FITC. The sites binding ATP or reacting with FITC do not, however, appear to co-exist on the same enzyme molecules. Thus, "flip-flop" mechanisms for (Ca2+ + Mg2+)-ATPase, involving negative interactions between high and low affinity ATP sites, are considered unlikely. The two affinities for ATP are most simply explained by assuming that the Ca2+ pump protein exists in alternative conformational forms, E1 having a high affinity for ATP and E2 having a low affinity for ATP. Ca2+ pumping and (Ca2+ + Mg2+)-ATPase involve interconversion between these forms. It is suggested that regulation of Ca2+ pump activity by Mg-ATP reflects acceleration of the conformational transition between the E1 and E2 forms, as well as a previously described acceleration of phosphoenzyme hydrolysis (Muallem, S., and Karlish, S. J. D. (1981) Biochim. Biophys. Acta 647, 73-86; Garrahan, P. J., and Rega, A. F. (1978) Biochim. Biophys. Acta 513, 59-65).

Adenosine Triphosphate↗
PubMed1982

Regulation of the Ca2+-pump by calmodulin in intact cells.

ATP-enriched human red cells display high rates of Ca2+-dependent ATP hydrolysis (16 mmol . litre cells-1 . h-1) with a high Ca2+ affinity (K0.5 approximately 0.2 microM). The finding suggests a mechanism for regulation of cell Ca2+ levels, involving highly-cooperative stimulation of active Ca2+ extrusion following binding of calmodulin to the (Ca2+ +Mg2+)-ATPase.

Adenosine Triphosphate↗
PubMed1980

Regulatory interaction between calmodulin and ATP on the red cell Ca2+ pump.

The interactions between calmodulin, ATP and Ca2+ on the red cell Ca2+ pump have been studied in membranes stripped of native calmodulin or rebound with purified red cell calmodulin. Calmodulin stimulates the maximal rate of (Ca2+ + Mg2+)-ATPase by 5-10-fold and the rate of Ca2+-dependent phosphorylation by at least 10-fold. In calmodulin-bound membranes ATP activates (Ca2+ + Mg2+)-ATPase along a biphasic concentration curve (Km1 approximately 1.4 microM, Km2 approximately 330 microM), but in stripped membranes the curve is essentially hyperbolic (Km approximately 7 microM). In calmodulin-bound membranes Ca2+ activates (Ca2+ + Mg2+)-ATPase at low concentrations (Km less than 0.28 microM) in stripped membranes the apparent Ca2+ affinities are at least 10-fold lower. The results suggest that calmodulin (and perhaps ATP) affect a conformational equilibrium between E2 and E1 forms of the Ca2+ pump protein.

Adenosine Triphosphate↗