Differentiation of Ca2+ pumps linked to plasma membrane and endoplasmic reticulum in the microsomal fraction from intestinal smooth muscle

ATP promotes ⁴⁵Ca uptake by the microsomal fraction from the longitudinal smooth muscle of guinea-pig ileum and this uptake is stimulated by oxalate. As the microsomal fraction is made up of various subcellular entities, we examined the localization of the Ca²⁺-transport activity by density gradient centrifugation, taking advantage of the selective effect of digitonin (at low concentration) on the density of plasmalemmal elements. When the ⁴⁵Ca-uptake activity was measured in the absence of oxalate, its behavior in subfractionation experiments closely paralleled that of the plasmalemmal marker 5′-nucleotidase. In contrast, the additional Ca²⁺-transport activity elicited by oxalate behaved like NADH-cytochrome $\text{c}$ reductase, a putative endoplasmic reticulum marker. The endoplasmic reticulum vesicles constituted only a small part of the membranes in the microsomal fraction, which explains that their Ca²⁺-storage capacity was not detectable in the absence of Ca²⁺-trapping agent. Low digitonin concentrations selectively increased the Ca²⁺ permeability of the plasmalemmal vesicles. The two Ca²⁺-transport activities were further differentiated by their distinct sensitivities to K⁺, vanadate and calmodulin. In this respect, the oxalte-insensitive and oxalate-stimulated Ca²⁺-transport systems resembled, respectively, the sarcolemmal and sarcoplasmic reticulum Ca²⁺ pumps in cardiac and skeletal muscle, in accordance with the subcellular locations established by density gradient centrifugation.     

Differentiation of Ca pumps linked to plasma membrane and endoplasmic reticulum in the microsomal fraction from intestinal smooth muscle

ATP promotes ⁴⁵Ca uptake by the microsomal fraction from the longitudinal smooth muscle of guinea-pig ileum and this uptake is stimulated by oxalate. As the microsomal fraction is made up of various subcellular entities, we examined the localization of the Ca²⁺-transport activity by density gradient centrifugation, taking advantage of the selective effect of digitonin (at low concentration) on the density of plasmalemmal elements. When the ⁴⁵Ca-uptake activity was measured in the absence of oxalate, its behavior in subfractionation experiments closely paralleled that of the plasmalemmal marker 5′-nucleotidase. In contrast, the additional Ca²⁺-transport activity elicited by oxalate behaved like NADH-cytochrome c reductase, a putative endoplasmic reticulum marker. The endoplasmic reticulum vesicles constituted only a small part of the membranes in the microsomal fraction, which explains that their Ca²⁺-storage capacity was not detectable in the absence of Ca²⁺-trapping agent. Low digitonin concentrations selectively increased the Ca²⁺ permeability of the plasmalemmal vesicles. The two Ca²⁺-transport activities were further differentiated by their distinct sensitivities to K⁺, vanadate and calmodulin. In this respect, the oxalte-insensitive and oxalate-stimulated Ca²⁺-transport systems resembled, respectively, the sarcolemmal and sarcoplasmic reticulum Ca²⁺ pumps in cardiac and skeletal muscle, in accordance with the subcellular locations established by density gradient centrifugation.     

Calcium distribution and exchange in the rat uterus

The calcium content and distribution of the rat uterus were determined employing flame photometry and Ca⁴⁵ determinations. The total uterine calcium concentration was found to be 2.25 millimoles (mmoles) per kilogram wet weight, 0.45 of which was inexchangeable. The exchangeable Ca could be divided into 0.8 mmole/kg wet weight extracellular and 1.0 mmole/kg wet weight intracellular. The concentration of ionic Ca in rat serum was obtained by equilibrium dialysis as 1.5 mM or 53 % of the total serum Ca. The observed Ca distribution required that its active transport be postulated, since the membrane was shown to be permeable to Ca and the internal Ca concentration was far below its electrochemical equilibrium value. Metabolic inhibition by iodoacetate or dinitrophenol caused a net Ca uptake, but cooling to 4°C and ouabain did not. Iodoacetate did not affect the Ca⁴⁵ efflux, but did increase the influx, suggesting that active Ca transport is accomplished by an exclusion mechanism. In experiments with varied external sodium concentrations, no evidence was obtained that sodium competes with calcium for inward transport. Cellular Ca binding was measured under conditions of prolonged metabolic inhibition, which abolished both active transport and the membrane potential. The association constants obtained were compatible with intracellular Ca binding to proteins, but insufficient to account for the low level of intracellular ionic Ca believed essential for relaxation. Hence metabolically dependent intracellular Ca binding was postulated. The Ca⁴⁵ efflux was slowed down by Ca-free efflux media. The presence of Sr or EDTA could completely prevent this decrease in efflux rate, and Ba could partly prevent it. Changes in Mg and Na concentration did not affect the rate of Ca⁴⁵ efflux. A model to explain Ca exchange across smooth muscle membranes has been proposed.     

Computer simulation and interpretation of45Ca efflux profile patterns

Summary Stimulations or inhibitions by various agents of⁴⁵Ca efflux from prelabeled cells or tissues display distinct and reproducible profile patterns when the results are plotted against time as fractional efflux ratios (FER). FER is the fractional efflux of⁴⁵Ca from stimulated cells divided by the fractional efflux from a control unstimulated group. These profile patterns fall into three categories: peak patterns, exponential patterns, and mixed patterns. Each category can be positive (stimulation) or negative (inhibition). The interpretation of these profiles is difficult because⁴⁵Ca efflux depends on three variables: the rate of calcium transport out of the cell, the specific activity of the cell compartment from which the calcium originates, and the concentration of free calcium in this compartment. A computer model based on data obtained by kinetic analyses of⁴⁵Ca desaturation curves and consisting of two distinct intracellular pools was designed to follow the concentration of the traced substance (⁴⁰Ca), the tracer (⁴⁵Ca), and the specific activity of each compartment before, during, and after the stimulation or the inhibition of calcium fluxes at various pool boundaries. The computer model can reproduce all the FER profiles obtained experimentally and bring information which may be helpful to the interpretation of this type of data. Some predictions of the model were tested experimentally, and the results support the views that a peak pattern may reflect a sustained change in calcium transport across the plasma membrane, that an exponential pattern arises from calcium mobilization from an internal subcellular pool, and that a mixed pattern may be caused by a simultaneous change in calcium fluxes at both compartment boundaries.     

Calcium incorporation by smooth muscle microsomes.

The purpose of the present work was to study the factors influencing calcium incorporation into a microsomal fraction prepared from the longitudinal smooth muscle of the guinea-pig ileum. Calcium incorporation required the presence of both ATP and Mg2+ and was unaffected by azide. It was enhanced by oxalate; this effect was pH dependent and it was maximal at pH 6.6. The relation between calcium uptake with oxalate and free Ca2+ concentration in the medium was represented by a curve with an optimum for Ca2+ equal to 3-10-5 M. The threshold concentration was comprised between 5-10-7 and 10-6 7. The optimum calcium uptake rate was 4.5 nmol Ca2+/mg protein per min. In the absence of oxalate, two distinct groups of binding sites were identified. Low affinity sites had a binding constant of 7-104 M-1 and a maximum binding capacity of 0.6-106 M-1 and a binding capacity of 33 nmol Ca2+/mg protein; their capacity was sensitive to pH changes. In the absence of oxalate, Ca2+ binding was depressed by Na+ with respect to K+ or choline. When the medium was supplemented with oxalate, the stimulation of 45Ca incorporation was barely detectable in the presence of choline+ and it was lower in a medium containing Na+ instead of K+. The subcellular distribution profiles of calcium incorporation with and without oxalate indicate the microsomal location of both activities. However, the oxalate-stimulated calcium uptake activity sedimented faster than the calcium binding activity. The subcellular distribution of marker enzyme actvities has been examined. The present results indicate that Ca2+ incorporations with and without oxalate are the result of two processes likely related to two different structures. The role of microsomal calcium uptake in excitation-contraction coupling and its modification by the activity of the sodium pump is discussed.     

Effects of adenosine 3′ : 5′-monophosphate and guanosine 3′ : 5′-monophosphate on calcium uptake and phosphorylation in membrane fractions of vascular smooth muscle

The effects of adenosine 3′ : 5′-monophosphate (cyclic AMP), guanosine 3′ : 5′-monophosphate (cyclic GMP) and exogenous protein kinase on Ca uptake and membrane phosphorylation were studied in subcellular fractions of vascular smooth muscle from rabbit aorta. Two functionally distinct fractions were separated on a continuous sucrose gradient: a light fraction enriched in endoplasmic reticulum (fraction E) and a heavier fraction containing mainly plasma membranes (fraction P).While cyclic AMP and cyclic GMP had no effect on Ca uptake in the absence of oxalate, both cyclic nucleotides inhibited the rate of oxalate-activated Ca uptake when used at concentrations higher than 10^?5 M. The addition of bovine heart protein kinase to either fraction produced an increase in the rate of oxalate-activated Ca uptake which was further augmented by cyclic AMP. Cyclic GMP caused smaller stimulations of protein kinase-catalyzed Ca uptake than cyclic AMP.Mg-dependent phosphorylation, attributable to endogenous protein kinase(s), was inhibited in fraction E by low concentrations (10^?8 M) of both cyclic AMP and cyclic GMP. In fraction P, an inhibition by cyclic AMP occurred also at a concentration of 10^?8 M, while with cyclic AMP a concentration of 10^?5 M was required for a similar inhibition. Bovine heart protein kinase stimulated the phosphorylation of the membrane fractions much more than Ca uptake. In fraction E, in the presence of bovine protein kinase, both cyclic AMP and cyclic GMP stimulated phosphorylation up to 200%. Under these conditions, no stimulation was observed in fraction P.These results are compatible with the hypothesis that in vascular smooth muscle soluble rather than particulate protein kinases are involved in the regulation of intracellular Ca concentration.     

Kinetics and Energetics of Mg2+,ATP-Dependent Ca2+ Transport in the Plasma Membrane of Smooth Muscle Cells

Calcium ions play a crucial role in the excitation/contraction coupling in smooth muscles. I would like to interpret the biochemical mechanisms underlying Ca²⁺ exchange and dynamics of such an exchange in the smooth muscles. Particular emphasis is laid on the examination of kinetic, energetic, and catalytic properties of the membrane-linked energy-dependent Ca²⁺-transporting systems involved in regulation of the intracellular Ca²⁺ concentration in smooth muscle cells (SMC). It was suggested that the Mg²⁺,ATP-dependent plasma membrane calcium pump (Ca²⁺,Mg²⁺-ATPase) plays a key role in regulation of the Ca²⁺ concentration in SMC. The purpose of this review is to analyze some of our own results concerning kinetic, energetic, and catalytic properties of the calcium pump of the SMC plasma membrane. In our experiments, we used different biochemical models (namely, fractions of the membrane subcellular structures, highly purified Ca²⁺,Mg²⁺-ATPase of the SMC plasma membrane solubilized and reconstituted in the lyposomes, and suspension of digitonin-treated SMC) and a number of methods (including preparative biochemistry, enzymology, membranology, tracer ⁴⁵Ca²⁺ flux analysis, and chemical and enzymological kinetics). We have shown that sodium azide-insensitive Mg²⁺,ATP-dependent Ca²⁺ accumulation in ureter smooth muscle microsomes is determined by two components. One component represents the Mg²⁺,ATP-dependent calcium pump of the sarcoplasmic reticulum functionally potentiated by Ca²⁺-precipitating permeating anions, oxalate or phosphate and inhibited by thapsigargin or cyclopiazonic acid, the highly selective inhibitors of the calcium pump of sarco(endo)plasmic rerticulum. Another component represents the Mg²⁺,ATP-dependent calcium pump of the plasma membrane functionally potentiated by phosphate. This pump is not inhibited by thapsigargin and cyclopiazonic acid. The effects of temperature, dielectric permeability (D), and ionic strength on the activity of purified Ca²⁺,Mg²⁺-ATPase solubilized from the myometrial sarcolemma were studied. The results suggest that changes in the polarity of the incubation medium markedly affect the activity of transport Ca²⁺,Mg²⁺-ATPase, and electrostatic interactions between the enzyme activity center and specific ligands (Mg·ADP^-, in particular) significantly contribute to the energetics of ATP hydrolysis. Therefore, our data show that changes in the incubation medium polarity significantly affects the ATP-hydrolase activity of Ca²⁺,Mg²⁺-ATPase solubilized from the SMC plasma membranes, and electrostatic interactions between the enzyme active sites and reactants (in particular, Mg·ADP^-) contribute to a significant extent to the energetics of ATP hydrolysis. We cannot rule out that under physiological conditions the local D values of the myoplasm may differ from that of water, and, moreover, may change (especially near the membrane surface) depending on the metabolic level of SMC. We suppose that local changes in the cytoplasmic D value will affect the plasma membrane calcium pump and, consequently, the efficiency of control of intracellular Ca²⁺ homeostasis in smooth muscle. So, our biochemical models are suitable experimental objects for studying the kinetic, energetic, and catalytic properties of the Mg²⁺,ATP-dependent calcium pump of the SMC plasma membrane. In addition, our data might be useful for screening of the mechanisms underlying the action of different physico-chemical factors involved in modulation of the contraction/relaxation cycle.     

Involvement of calcium in calcium-current inactivation in smooth muscle cells from rat vas deferens

Summary Ca-channel currents were recorded in Cs-loaded single smooth muscle cells from rat vas deferens to define the dependence of the inactivation time course on Ca concentration. The decay of Ca-channel current obtained in a Ba²⁺- or Sr²⁺-containing external solution during long voltage-clamp pulses was much slower than that in a Ca-containing solution. The difference was not due to a change in the surface potential of the membrane as judged from the steady-state activation and inactivation curves. When Ca was the charge carrier, increasing external Ca concentration slightly accelerated the rate of inactivation. In addition, the rate of inactivation of Ca-channel current in 10.8mm Ba was also accelerated by adding Ca to the external solution in a concentration-dependent manner. The time course of Ca-current inactivation was slowed when the cells were dialyzed with a high concentration of citrate, a Ca-chelating agent. From these results, we concluded that a mechanism regulated by intracellular Ca activity plays a role in the inactivation of Ca channels in smooth muscle. The Ca-dependent process may protect against Ca overload by regulating Ca entry in smooth muscle cells.     

Subcellular origin of the oxalate- or inorganic phosphate-stimulated Ca2+ transport by smooth muscle microsomes: revisitation of the old problem by a new approach using saponin

Saponin, a cell-skinning reagent which perforates the cell membrane via its specific interaction with plasmalemmal cholesterol, was used to identify the subcellular origin of ATP-dependent Ca2+ accumulation in the presence and absence of inorganic phosphate and oxalate by microsomal fractions isolated from rat vas deferens and dog aorta. The purified plasma membranes from rat gastric fundus muscle, which elicit the stimulation of ATP-dependent Ca2+ accumulation by inorganic phosphate but not by oxalate, were used as a control reference. Saponin at concentrations effective for skinning smooth muscle fibres (10-50 micrograms/ml) inhibited Ca2+ binding in the absence of ATP to a similar extent in all fractions, but the inhibition of ATP-dependent Ca2+ accumulation was more pronounced in dog aorta microsomes and rat gastric fundus muscle plasma membranes than in rat vas deferens microsomes. The resistance of phosphate- and oxalate-stimulated ATP-dependent Ca2+ accumulation to inhibition by saponin was much greater in rat vas deferens than in dog aorta microsomes. Our results suggest that phosphate- and oxalate-stimulated ATP-dependent Ca2+ accumulation also occurs in plasma membrane vesicles isolated from smooth muscle and is by no means an unique property of endoplasmic reticulum.     

Ca-pumps in smooth muscle: one in plasma membrane and another in endoplasmic reticulum

For several years it has been debated whether the Ca-pump in smooth muscle is located in the plasma membrane or in the endoplasmic reticulum (alias sarcoplasmic reticulum). Experimental evidence using skinned smooth muscle cells and subcellular membrane fractions isolated from a number of smooth muscles is reviewed here to hopefully resolve this issue. The inescapable conclusion is that there are two modes of nonmitochondrial ATP-dependent Ca-transport. The first one, unaffected by oxalate, is localized in the plasma membranes and the second, potentiated by oxalate, is localized in the endoplasmic reticulum. Clear experiments to delineate the roles of the two pumps in the excitation-contraction cycle of the smooth muscle remain to be conducted.     

The role of caveolae and caveolin 1 in calcium handling in pacing and contraction of mouse intestine

In mouse intestine, caveolae and caveolin‐1 (Cav‐1) are present in smooth muscle (responsible for executing contractions) and in interstitial cells of Cajal (ICC; responsible for pacing contractions). We found that a number of calcium handling/dependent molecules are associated with caveolae, including L‐type Ca²⁺ channels, Na⁺‐Ca²⁺ exchanger type 1 (NCX1), plasma membrane Ca²⁺ pumps and neural nitric oxide synthase (nNOS), and that caveolae are close to the peripheral endo‐sarcoplasmic reticulum (ER‐SR). Also we found that this assemblage may account for recycling of calcium from caveolar domains to SR through L‐type Ca ⁺ channels to sustain pacing and contractions. Here we test this hypothesis further comparing pacing and contractions under various conditions in longitudinal muscle of Cav‐1 knockout mice (lacking caveolae) and in their genetic controls. We used a procedure in which pacing frequencies (indicative of functioning of ICC) and contraction amplitudes (indicative of functioning of smooth muscle) were studied in calcium‐free media with 100 mM ethylene glycol tetra‐acetic acid (EGTA). The absence of caveolae in ICC inhibited the ability of ICC to maintain frequencies of contraction in the calcium‐free medium by reducing recycling of calcium from caveolar plasma membrane to SR when the calcium stores were initially full. This recycling to ICC involved primarily L‐type Ca²⁺ channels; i.e. pacing frequencies were enhanced by opening and inhibited by closing these channels. However, when these stores were depleted by block of the sarco/endoplasmic reticulum Ca²⁺‐ATPase (SERCA) pump or calcium release was activated by carbachol, the absence of Cav‐1 or caveolae had little or no effect. The absence of caveolae had little impact on contraction amplitudes, indicative of recycling of calcium to SR in smooth muscle. However, the absence of caveolae slowed the rate of loss of calcium from SR under some conditions in both ICC and smooth muscle, which may reflect the loss of proximity to store operated Ca channels. We found evidence that these channels were associated with Cav‐1. These changes were all consistent with the hypothesis that a reduction of the extracellular calcium associated with caveolae in ICC of the myenteric plexus, the state of L‐type Ca²⁺ channels or an increase in the distance between caveolae and SR affected calcium handling.     

Mucilage Cells, Calcium Oxalate Crystals and Soluble Calcium in Opuntia ficus-indica

The subcellular location of soluble calcium in parenchymatousand mucilage cells of Opuntia ficus-indica was determined histochemically.Soluble calcium was observed in crystal chambers containingcalcium oxalate on the membrane of the vesicles. Calcium wasalso present in the plasmalemma, in plasmodesmata, in cell walls,in mitochondria and in the vacuoles. Especially marked was thepresence of soluble calcium in vesicles free or fused with theplasmalemma. Little free calcium was observed in other cellcompartments. In the calcium economy of tissues the location of soluble calciumand the transport of calcium to and from mucilage cells to parenchymatouscells and calcium oxalate idioblasts will play a role. Chelationof calcium by mucilage or oxalate, which depends on pH, ionicstrength, etc., will be important in this respect. Opuntia ficus-indica, calcium oxalate, mucilage cells, transport of calcium     

Regulation of calcium fluxes in rat pancreatic islets: Quinine mimics the dual effect of glucose on calcium movements

The effects of quinine and 9-aminoacridine, two blockers of potassium conductance in islet cells, on ⁴⁵Ca efflux and insulin release from perifused islets were investigated in order to elucidate the mechanisms by which glucose initially reduces ⁴⁵Ca efflux and later stimulates calcium inflow in islet cells. In the absence of glucose, 100 μM quinine stimulated ⁴⁵Ca net uptake, ⁴⁵Ca outflow rate and insulin release. Quinine also dramatically enhanced the cationic and the secretory response to intermediate concentrations of glucose, but had little effect on ⁴⁵Ca net uptake, ⁴⁵Ca fractional outflow rate and insulin release at a high glucose concentration (16.7 mM). The ability of quinine to stimulate ⁴⁵Ca efflux depended on the presence of extracellular calcium, suggesting that it reflects a stimulation of calcium entry in the islet cells. In the absence of extracellular calcium, quinine provoked a sustained decrease in ⁴⁵Ca efflux. Such an inhibitory effect was not additive to that of glucose, and was reduced at low extracellular Na⁺ concentration. At a low concentration (5 μM), quinine, although reducing ⁸⁶Rb efflux from the islets to the same extent as a non-insulinotropic glucose concentration (4.4 mM), failed to inhibit ⁴⁵Ca efflux. In the presence of extracellular calcium, 9-aminoacridine produced an important but transient increase in ⁴⁵Ca outflow rate and insulin release from islets perifused in the absence of glucose. In the absence of extracellular calcium, 9-aminoacridine, however, failed to reduced ⁴⁵Ca efflux from perifused islets. It is concluded that quinine, by reducing K⁺ conductance, reproduces the effect of glucose to activate voltage-sensitive calcium channels and to stimulate the entry of calcium into the B-cell. However, the glucose-induced inhibition of calcium outflow rate, which may also participate in the intracellular accumulation of calcium, does not appear to be mediated by changes in K⁺ conductance.     

Comparison of the properties of active Ca2+ transport by the islet-cell endoplasmic reticulum and plasma membrane

The properties of active or ATP-dependent calcium transport by islet-cell endoplasmic reticulum and plasma membrane-enriched subcellular fractions were directly compared. These studies indicate that the active calcium transport systems of the two membranes are fundamentally distinct. In contrast to calcium uptake by the endoplasmic reticulum-enriched fraction, calcium uptake by islet-cell plasma membrane-enriched vesicles exhibited a different pH optimum, was not sustained by oxalate, and showed an approximate 30-fold greater affinity for ionized calcium. A similar difference in affinity for calcium was exhibited by the Ca²⁺-stimulated ATPase activities which are associated with these islet-cell subcellular fractions. Consistent with the effects of calmodulin on calcium transport, calmodulin stimulated Ca²⁺-ATPase in the plasma membranes, but did not increase calcium-stimulated ATPase activity in the endoplasmic reticulum membranes. The physiological significance of the differences observed in calcium transport by the endoplasmic reticulum and plasma membrane fractions relative to the regulation of insulin secretion by the islets of Langerhans is discussed.     

Dysfunction of calcium handling by smooth muscle in hypertension

Dysfunction of ion handling, including binding and fluxes (passive and active transport) of physiologically important ions such as potassium, sodium, calcium, and magnesium, by vascular smooth muscle cell membranes has repeatedly been reported to be associated with the pathophysiology of hypertension. The specific purpose of this review is to summarize and evaluate the evidence for alterations of calcium ion (Ca2+) handling by vascular smooth muscle in various forms of hypertension in the animal model on the basis that regulation of cytoplasmic Ca2+ concentration is a complex and yet vitally important process for a normal function of vascular smooth muscle and that derangement of such a regulation may result in excessive retention of cytoplasmic Ca2+, contribute toward increase of total peripheral resistance, and ultimately lead to elevation of blood pressure. Emphasis is placed upon the consideration of the usefulness of the subcellular membrane fractionation technique in studies of binding and transport of Ca2+ by vascular and nonvascular smooth muscle membranes from genetic as well as experimental hypertensive rats. The limitations of the interpretation of data using such an approach are also considered. Decreased active transport of Ca2+ across isolated plasma membrane vesicles from large and small arteries occurs in several but not all forms of hypertension. This membrane abnormality also occurs in nonvascular smooth muscles and other tissues or cells not confined to the cardiovascular system in genetic hypertension, but not in experimental hypertension. A hypothesis of general membrane defects in spontaneous hypertension is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Target size of Ca-pumps in pig coronary artery smooth muscle

The target sizes of the oxalate-independent Ca uptake by the plasma membrane enriched fraction F2, and the oxalate-stimulated Ca uptake by a fraction F3 slightly enriched in the endoplasmic reticulum were determined by radiation inactivation. The oxalate-independent Ca uptake was inactivated with a D37 value of 1.96 +/- 0.30 Mrad but the oxalate-stimulated Ca uptake had a D37 value of 0.45 +/- 0.07 Mrad. Thus, in the smooth muscle the oxalate-stimulated Ca uptake appeared to be due to a structure 3 to 6 times larger than was the oxalate-independent Ca uptake. The subcellular site of the ATP-dependent azide insensitive Ca uptake in the smooth muscle has been disputed in the past. It has been suggested to be plasma membrane (PM) by several workers, and endoplasmic reticulum (ER) by others. Recently, however, there has been substantial evidence to support the hypothesis that one Ca uptake system, unaffected by oxalate, resides in the PM and another, stimulated by oxalate, is located in the ER of the smooth muscle. The evidence has been reviewed recently. Here, we show that the two modes of Ca uptake differ in their target sizes as well. To our knowledge, this is the first report on the use of radiation inactivation to distinguish between the two modes of Ca uptake in any tissue.     

Regulation of calcium fluxes in rat pancreatic islets: Calcium extrusion by sodium-calcium countertransport

Summary The mechanisms by which glucose regulates calcium fluxes in pancreatic endocrine cells were investigated by monitoring the efflux of⁴⁵Ca from prelabeled and perifused rat pancreatic islets. In the absence of both extracellular calcium and glucose, partial or total removal of extracellular sodium decreases the efflux of⁴⁵Ca from prelabeled islets. Glucose also reduces the efflux of⁴⁵Ca from islets perifused in the absence of extracellular calcium. This inhibitory effect of glucose on⁴⁵Ca efflux is decreased by half when the extracellular concentration of sodium is lowered to 24mm. In the absence of extracellular calcium but presence of glucose, partial or even total removal of extracellular sodium fails to decrease the efflux of⁴⁵Ca. At normal extracellular calcium concentration (1mm) partial removal of extracellular sodium dramatically increases⁴⁵Ca efflux from pancreatic islets. This increase in⁴⁵Ca efflux is partially but not totally suppressed by either 16.7mm glucose or cobalt. It is totally suppressed by 4.4mm glucose or by the combination of 16.7mm glucose and cobalt. At normal extracellular calcium concentration, glucose initially reduces and subsequently increases⁴⁵Ca efflux. The initial fall is unaffected by tetrodotoxin but decreased by 50% at low extracellular sodium concentration (24mm). The present results suggest the existence in pancreatic endocrine cells of a glucose-sensitive process of sodium-calcium counter-transport. By inhibiting such a process, glucose may decrease the efflux of calcium from islet cells. The effect of glucose is not mediated by an increase in intracellular sodium concentration. It could contribute to the intracellular accumulation of calcium which is thought to trigger insulin release.This paper is the IVth in a series.     

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.     

Calcium pump of the plasma membrane is localized in caveolae

The Ca2+ pump in the plasma membrane plays a key role in the fine control of the cytoplasmic free Ca2+ concentration. In the present study, its subcellular localization was examined with immunocytochemical techniques using a specific antibody generated against the erythrocyte membrane Ca2+ pump ATPase. By immunofluorescence microscopy of cultured cells, the labeling with the antibody was seen as numerous small dots, often distributed in linear arrays or along cell edges. Immunogold EM of cryosections revealed that the dots correspond to caveolae, or smooth invaginations of the plasma membrane. The same technique applied to mouse tissues in vivo showed that the Ca2+ pump is similarly localized in caveolae of endothelial cells, smooth muscle cells, cardiac muscle cells, epidermal keratinocytes and mesothelial cells. By quantitative analysis of the immunogold labeling, the Ca2+ pump in capillary endothelial cells and visceral smooth muscle cells was found to be concentrated 18-25-fold in the caveolar membrane compared with the noncaveolar portion of the plasma membrane. In renal tubular and small intestinal epithelial cells, which have been known to contain the Ca2+ pump but do not have many caveolae, most of the labeling was randomly distributed in the basolateral plasma membrane, although caveolae were also positively labeled. The results demonstrate that the caveolae in various cells has the plasmalemmal Ca2+ pump as a common constituent. In conjunction with our recent finding that an inositol 1,4,5-trisphosphate receptor-like protein exists in the caveolae (Fujimoto, T., S. Nakade, A. Miyawaki, K. Mikoshiba, and K. Ogawa. 1992. J. Cell Biol. 119:1507-1513), it is inferred that the smooth plasmalemmal invagination is an apparatus specialized for Ca2+ intake and extrusion from the cytoplasm.     

Effects of calcium ionophores on the transport and distribution of calcium in isolated cells and in liver and kidney slices

Summary The effects of calcium ionophores on cellular calcium metabolism were studied in cultured kidney cells, in cells freshly isolated from rat kidney, and in liver and kidney slices. In isolated cells, these ionophores decreased the total cellular Ca content and the mitochondrial Ca.⁴⁵Ca efflux from prelabelled cells was also stimulated even in the absence of extracellular Ca. In slices, the ionophore A23187 increased the total slice Ca and the uptake of⁴⁵Ca. However, the mitochondria isolated from these slices treated with the ionophore had a lower total Ca and a depressed relative radioactivity. These results suggest that the increased cytosolic Ca produced by Ca ionophores may be due to mobilization of intracellular Ca stores rather than to a net shift of Ca from the extracellular fluids to the cell.