Calcium Transport in Sealed Vesicles from Red Beet (Beta vulgaris L.) Storage Tissue : II. Characterization of Ca Uptake into Plasma Membrane Vesicles

Calcium uptake was examined in sealed plasma membrane vesicles isolated from red beet (Beta vulgaris L.) storage tissue using (45)Ca(2+). Uptake of (45)Ca(2+) by the vesicles was ATP-dependent and radiotracer accumulated by the vesicles could be released by the addition of the calcium ionophore A23187. The uptake was stimulated by gramicidin D but slightly inhibited by carbonylcyanide m-chlorophenylhydrazone. Although the latter result might suggest some degree of indirect coupling of (45)Ca(2+) uptake to ATP utilization via deltamuH(+), no evidence for a secondary H(+)/Ca(2+) antiport in this vesicle system could be found. Following the imposition of an acid-interior pH gradient, proton efflux from the vesicle was not enhanced by the addition of Ca(2+) and an imposed pH gradient could not drive (45)Ca(2+) uptake. Optimal uptake of (45)Ca(2+) occurred broadly between pH 7.0 and 7.5 and the transport was inhibited by orthovanadate, N,N'-dicyclohexylcarbodiimide, and diethylstilbestrol but insensitive to nitrate and azide. The dependence of (45)Ca(2+) uptake on both calcium and Mg:ATP concentration demonstrated saturation kinetics with K(m) values of 6 micromolar and 0.37 millimolar, respectively. While ATP was the preferred substrate for driving (45)Ca(2+) uptake, GTP could drive transport at about 50% of the level observed for ATP. The results of this study demonstrate the presence of a unique primary calcium transport system associated with the plasma membrane which could drive calcium efflux from the plant cell.     

Ca uptake by endoplasmic reticulum from zucchini hypocotyls : the use of chlorotetracycline as a probe for ca uptake

Ca(2+) uptake into microsomal vesicles was measured using the fluorescent probe chlorotetracycline. The Ca(2+) uptake was ATP-dependent and did not occur in the presence of the calcium ionophore A23187. There was a linear relationship between the rate of ATP-dependent fluorescence increase using chlorotetracycline and ATP-dependent (45)Ca(2+) uptake, indicating that chlorotetracycline can be used as a quantitative probe for Ca(2+) uptake. The fluorescent probe allows measurements to be made in real time, and avoids the use of radioisotopes. Ca(2+) transport was associated with endoplasmic reticulum on linear gradients when the endoplasmic reticulum was in either rough or smooth form. The Ca(2+) uptake had a pH optimum of 7.5, a K(m) for ATP of 0.1 millimolar, a K(m) for Ca(2+) of about 70 nanomolar, and was stimulated 2-fold by calmodulin. Vanadate inhibited uptake completely at a concentration of 50 micromolar, half-maximally at 5 micromolar. Carbonyl cyanide 4-(trifluoromethoxy)-phenyl-hydrazone, oligomycin, azide, and nitrate caused only slight inhibition. Dicyclohexylcarbodiimide (DCCD) stimulated slightly at concentrations as high as 400 micromolar. The hormones gibberellic acid, indoleacetic acid, and abscisic acid at 10 micromolar had no significant effect. Myo-inositol 1,4,5-trisphosphate did not cause release of Ca(2+) after uptake. The properties of the enzyme suggest that it has a functional role in regulating cytosolic Ca(2+) levels. Based on the lack of an effect by hormones, it may not act as a mediator of second messenger roles of Ca(2+). The inhibition by vanadate and slight stimulation by DCCD may be useful as a ;signature' for this endoplasmic reticulum Ca(2+) uptake system.     

Fast release of 45Ca2+ induced by inositol 1,4,5-trisphosphate and Ca2+ in the sarcoplasmic reticulum of rabbit skeletal muscle: evidence for two types of Ca2+ release channels.

The kinetics of Ca2+ release induced by the second messenger D-myoinositol 1,4,5 trisphosphate (IP3), by the hydrolysis-resistant analogue D-myoinositol 1,4,5 trisphosphorothioate (IPS3), and by micromolar Ca2+ were resolved on a millisecond time scale in the junctional sarcoplasmic reticulum (SR) of rabbit skeletal muscle. The total Ca2+ mobilized by IP3 and IPS3 varied with concentration and with time of exposure. Approximately 5% of the 45Ca2+ passively loaded into the SR was released by 2 microM IPS3 in 150 ms, 10% was released by 10 microM IPS3 in 100 ms, and 20% was released by 50 microM IPS3 in 20 ms. Released 45Ca2+ reached a limiting value of approximately 30% of the original load at a concentration of 10 microM IP3 or 25-50 microM IPS3. Ca(2+)-induced Ca2+ release (CICR) was studied by elevating the extravesicular Ca2+ while maintaining a constant 5-mM intravesicular 45Ca2+. An increase in extravesicular Ca2+ from 7 nM to 10 microM resulted in a release of 55 +/- 7% of the passively loaded 45Ca2+ in 150 ms. CICR was blocked by 5 mM Mg2+ or by 10 microM ruthenium red, but was not blocked by heparin at concentrations as high as 2.5 mg/ml. In contrast, the release produced by IPS3 was not affected by Mg2+ or ruthenium red but was totally inhibited by heparin at concentrations of 2.5 mg/ml or lower. The release produced by 10 microM Ca2+ plus 25 microM IPS3 was similar to that produced by 10 microM Ca2+ alone and suggested that IP3-sensitive channels were present in SR vesicles also containing ruthenium red-sensitive Ca2+ release channels. The junctional SR of rabbit skeletal muscle may thus have two types of intracellular Ca2+ releasing channels displaying fast activation kinetics, namely, IP3-sensitive and Ca(2+)-sensitive channels.     

Effects of Cd2+ upon Ca2+ fluxes and proliferation in concanavalin A-stimulated lymphocytes

The mitogenic response of human peripheral blood lymphocytes to the lectin concanavalin A (conA) is inhibited by micromolar concentrations of CdCl2. This inhibition is partially relieved by an increase in the external Ca2+ concentration (from 0.6 to 2.2 mM). The initial rate of conA-induced 45Ca2+ influx is unaltered by CdCl2, although the level of 45Ca2+ accumulation increases. The basal rate of 45Ca2+ entry is not measurably disturbed by CdCl2 (100 microM). The steady-state efflux of 45Ca2+ and the calmodulin-activated (Ca2+ + Mg2+)-ATPase activity of erythrocyte ghosts are inhibited by CdCl2 (10 microM). Thus, the mechanism behind the Cd2+-induced suppression of the mitogenic response to conA is not due to alteration of mitogen-stimulated Ca2+ influx. We suggest that Cd2+ competes with Ca2+ for intracellular Ca2+-binding molecules, such as calmodulin, essential for the induction of cell proliferation.     

Plasma Membrane Ca-ATPase of Radish Seedlings : I. Biochemical Characterization Using ITP as a Substrate

In this work, we exploited the capability of the plasma membrane Ca-ATPase to utilize ITP as a substrate to study its characteristics in plasma membrane vesicles purified from radish (Raphanus sativus L.) seedlings. The majority of the ITPase activity of plasma membrane was Ca(2+)-dependent. The Ca(2+)-dependent ITPase activity was Mg(2+)-dependent and was stimulated by the calcium ionophore A23187. It was inhibited by erythrosin B (concentration giving 50% inhibition, 50 nanomolar) and by vanadate (concentration giving 50% inhibition, 3 micromolar) and displayed a broad pH optimum around pH 7.2 to 7.5. Both the hydrolytic and the transport activity of the plasma membrane Ca-ATPase were half-saturated by Ca(2+) in the micromolar concentration range. No major effect of EGTA on the saturation kinetics of the enzyme was observed. The affinity of the plasma membrane Ca-ATPase for Ca(2+) was about fourfold higher at pH 7.5 than at pH 6.9. The Ca(2+)-dependent ITPase activity was stimulated about twofold by polyoxyethylene 20 cetyl ether, although it was inhibited by Triton X-100 and by lysolecithin.     

Synaptosomal plasma membrane Ca(2+) pump activity inhibition by repetitive micromolar ONOO(-) pulses.

A sustained increase of intracellular free [Ca(2+)] ([Ca(2+)](i)) has been shown to be an early event of neuronal cell death induced by peroxynitrite (ONOO(-)). In this paper, chronic exposure to ONOO(-) has been simulated by treatment of rat brain synaptosomes or plasma membrane vesicles with repetitive pulses of ONOO(-) during at most 50 min, which efficiently produced nitrotyrosine formation in several membrane proteins (including the Ca(2+)-ATPase). The plasma membrane Ca(2+)-ATPase activity at near-physiological conditions (pH 7, submicromolar Ca(2+), and millimolar Mg(2+)-ATP concentrations), which plays a major role in the control of synaptic [Ca(2+)](i), can be more than 75% inhibited by a sustained exposure to micromolar ONOO(-) (e.g., to 100 pulses of 10 microM ONOO(-)). This inhibition is irreversible and mostly due to a decreased V(max), and to the 2-fold increase of the K(0.5) for Ca(2+) stimulation and about 5-fold increase of the K(M) for Mg(2+)-ATP. [Ca(2+)](i) increases to >400 nM when synaptosomes are subjected to this treatment. Reduced glutathione can afford only partial protection against the inhibition produced by micromolar ONOO(-) pulses. Therefore, inhibition of the plasma membrane Ca(2+)-pump activity during chronic exposure to ONOO(-) may account by itself for a large and sustained increase of intracellular [Ca(2+)](i) in synaptic nerve terminals.     

Compartmental analysis of steady-state diaphragm Ca2+ kinetics in chronic congestive heart failure

An analytic method based on simulation and modeling of long-term 45Ca(2+) efflux data was used to estimate steady-state Ca(2+) contents (nmolCa(2+)g(-1)tissuewetwt.) and exchange fluxes (nmolCa(2+)min(-1)g(-1)tissuewetwt.) for extracellular and intracellular compartments in in vitro resting diaphragm from congestive heart failure (CHF, n=12) and sham-operated (SHAM, n=10) rats. Left hemidiaphragms were excised from experimental animals, loaded with 45Ca(2+) for 1h, and washed out with 45Ca(2+)-free perfusate for 8h. Tissue from the right hemidiaphragm was used to assess single-fiber cross-sectional area (CSA) as well as the relative proteolytic activity of Ca(2+)-dependent calpain. Kinetic analysis of 45Ca(2+) efflux data revealed that CHF was associated with increased Ca(2+) contents of extracellular and intracellular compartments as well as increased Ca(2+) exchange fluxes for all compartments. This accounted for the model prediction of a 250% increase in total diaphragm Ca(2+). Furthermore, single-fiber CSA was decreased 12% and proteolytic activity of calpain was increased twofold in CHF diaphragm relative to SHAM.CONCLUSIONS: The kinetic data are consistent with the hypothesis that diaphragm Ca(2+) overload in CHF required all intercompartmental Ca(2+) fluxes to increase. The potential relationships among Ca(2+) overload, increased activity of calpain, and wasting of the diaphragm in CHF are discussed.     

Micromolar Ca2+ concentrations are essential for Mg2+-dependent binding of collagen by the integrin alpha 2beta 1 in human platelets

Integrin receptor alpha(2)beta(1) requires micromolar Ca(2+) to bind to collagen and to the peptide GPC(GPP)(5)GFOGER(GPP)(5)GPC (denoted GFOGER-GPP, where O represents hydroxyproline), which contains the minimum recognition sequence for the collagen-binding alpha(2) I-domain (Knight, C. G., Morton, L. F., Peachey, A. R., Tuckwell, D. S., Farndale, R. W., and Barnes, M. J. (2000) J. Biol. Chem. 275, 35-40). Platelet adhesion to these ligands is completely dependent on alpha(2)beta(1) in the presence of 2 mm Mg(2+). However, we show here that this interaction was abolished in the presence of 25 microm EGTA. Adhesion of Glanzmann's thrombasthenic platelets, which lack the fibrinogen receptor alpha(IIb)beta(3), was also inhibited by micromolar EGTA. Mg(2+)-dependent adhesion of platelets was restored by the addition of 10 microm Ca(2+), but millimolar Ca(2+) was inhibitory. Binding of isolated alpha(2)beta(1) to GFOGER-GPP was 70% inhibited by 50 microm EGTA but, as with intact platelets, was fully restored by the addition of micromolar Ca(2+). 2 mm Ca(2+) did not inhibit binding of isolated alpha(2)beta(1) to collagen or to GFOGER-GPP. Binding of recombinant alpha(2) I-domain was not inhibited by EGTA, nor did millimolar Ca(2+) inhibit binding. Our data suggest that high affinity Ca(2+) binding to alpha(2)beta(1), outside the I-domain, is essential for adhesion to collagen. This is the first demonstration of a Ca(2+) requirement in alpha(2)beta(1) function.     

Characterization of CCK(A) receptor affinity states and Ca(2+) signal transduction in vagal nodose ganglia

CCK(A) receptors are present on vagal afferent fibers. The objectives of this study were to identify the presence of high- and low-affinity CCK(A) receptors on nodose ganglia and to characterize the intracellular calcium signal transduction activated by CCK. Stimulation of acutely isolated nodose ganglion cells from rats with 1 nM CCK-8 primarily evoked a Ca(2+) transient followed by a sustained Ca(2+) plateau (45% of cells responded), whereas 10 pM CCK-8 evoked Ca(2+) oscillations (37% of cells responded). CCK-OPE, a high-affinity agonist and low-affinity antagonist of CCK(A) receptors, primarily elicited Ca(2+) oscillations (29% of cells responded). CCK-OPE inhibited the Ca(2+) transient induced by 1 nM CCK-8 but not by carbachol and high K(+). This result suggests the presence of high- and low-affinity states of CCK(A) receptors on nodose ganglia. We further demonstrated that nicardipine (10 microM) but not omega-conotoxins GVIA and MVIIC (10-100 nM) abolished Ca(2+) signaling induced by CCK-8, indicating that an L-type voltage-dependent Ca(2+) channel and not an N- or Q-type Ca(2+) channel is coupled to CCK(A) receptors. In a separate study, we showed that the G protein activator NaF (10 mM) elicited a Ca(2+) transient and inhibited CCK-8-evoked Ca(2+) signaling, indicative of G protein(s) involvement in CCK(A) receptor activity. The G(q) protein antagonist Gp antagonist-2A (10 microM) also abolished the action of CCK-8. This study indicates that CCK(A) receptors exist in both high- and low-affinity states in the nodose ganglia. Activation of high-affinity CCK(A) receptors elicits Ca(2+) oscillations, whereas stimulation of low-affinity CCK(A) receptors evokes a sustained Ca(2+) plateau. These Ca(2+)-signaling modes are mediated through the L-type Ca(2+) channel and involve the participation of G(q) protein.     

Ca2+ regulation of gelsolin by its C-terminal tail

Gelsolin is activated by Ca(2+) to sever actin filaments. Ca(2+) regulation is conferred on the N-terminal half by the C-terminal half. This paper seeks to understand how Ca(2+) regulates gelsolin by testing the "tail helix latch hypothesis," which is based on the structural data showing that gelsolin has a C-terminal tail helix that contacts the N-terminal half in the absence of Ca(2+). Ca(2+) activation of gelsolin at 37 degrees C occurs in three steps, with apparent K(d) for Ca(2+) of 0.1, 0.3, and 6.4 x 10(-6) m. Tail helix truncation decreases the apparent Ca(2+) requirement for severing to 10(-7) m and eliminates the conformational change observed at 10(-6) m Ca(2+). The large decrease in Ca(2+) requirement for severing is not due to a change in Ca(2+) binding nor to Ca(2+)-independent activation of the C-terminal half per se. Thus, the tail helix latch is primarily responsible for transmitting micromolar Ca(2+) information from the gelsolin C-terminal half to the N-terminal half. Occupation of submicromolar Ca(2+)-binding sites primes gelsolin for severing, but gelsolin cannot sever because the tail latch is still engaged. Unlatching the tail helix by 10(-6) m Ca(2+) releases the final constraint to initiate the severing cascade.     

Effect of nitrogen and oxygen active compounds on exchange of Ca2+ and H+ through the myometrial cell membrane

While using myometrium sarcolemma vesicles the action of sodium nitroprusside, NO2-, NO3- and H+ on delta pH-dependent Ca(2+)-transport and passive permeability for H+ vesicles sarcolemma was estimated in the wide concentration range (10(-10)-10(-3) M) of the substances tested. In order of studying calcium transport 45Ca2+ was used, while for H+ translocation registrating via sarcolemma delta pH-indicator 14C-methylamine was applied. Sodium nitroprusside was displayed as weakly effective, while nitrite-anions essentially increased delta pH-dependent Ca2+ transport in the physiologically significant nanomolar concentration region, however in the micromolar region these substances effect failed to differ from the control and restored its intensity starting at 10(-4) M and more. Under the experiment sodium nitroprusside produces considerable quantities of NO2-. Effectory action of NO3- was similar as of NO2-. In the micromolar region the compounds estimated increased considerably sarcolemma passive permeability for H+. Hydrogen peroxide decreased delta pH-dependent Ca(2+)-transport by 10(-8) M and 10(-3) M while at the concentration equal to 10(-3) M increased the sarcolemma passive permeability for proton. Sodium nitroprusside and NO2(-)-effect on the vesicles passive permeability for proton failed to be prevented by dithiotriitol, while H2O2 action was completely removed. The conclusion about the complex concentration-dependent character of the active oxygen metabolities to the sarcolemma transport processes was made, and it's noticeable that the important role in vivo, probably could be played by NO (NO2-) stable nitric metabolities.     

Mode-specific inhibition of sodium-calcium exchange during protein phosphatase blockade

The effects of the protein phosphatase inhibitors calyculin A and okadaic acid on Na(+)/Ca(2+) exchange activity were examined in transfected Chinese hamster ovary cells expressing the bovine cardiac Na(+)/Ca(2+) exchanger. Incubating the cells for 5-10 min with 100 nM calyculin A reduced exchange-mediated (45)Ca(2+) uptake or Ba(2+) influx by 50-75%. Half-maximal inhibition of (45)Ca(2+) uptake was observed at 15 nM calyculin A. The nonselective protein kinase inhibitors K252a and staurosporine provided partial protection against the effects of calyculin A. Okadaic acid, another protein phosphatase inhibitor, nearly completely blocked exchange-mediated Ba(2+) influx. Chinese hamster ovary cells expressing a mutant exchanger in which 420 out of 520 amino acid residues were deleted from the central hydrophilic domain of the exchanger remained sensitive to the inhibitory effects of calyculin A and okadaic acid. Surprisingly, Na(o)(+)-dependent Ca(2+) efflux appeared to be only modestly inhibited, if at all, by calyculin A or okadaic acid. We conclude that protein hyperphosphorylation during protein phosphatase blockade selectively inhibits the Ca(2+) influx mode of Na(+)/Ca(2+) exchange, probably by an indirect mechanism that does not involve phosphorylation of the exchanger itself.     

Novel tacrine derivatives that block neuronal calcium channels

A new series of tacrine (9-amino-1,2,3,4-tetrahydroacridine) derivatives were synthesized and their effects on 45Ca(2+) entry into bovine adrenal chromaffin cells stimulated with dimethylphenylpiperazinium (DMPP) or K(+), studied. At 3 microM, compound 1 did not affect (45)Ca(2+) uptake evoked by DMPP. Compounds 14, 15 and 17 inhibited the effects of DMPP by 30%. Compounds 3, 9 and tacrine blocked the DMPP signal by about 50%. Compounds 5 and 12 were the most potent blockers of DMPP-stimulated 45Ca(2+) entry (90%); the rest of the compounds inhibited the effects of DMPP by 70-80%. Compounds 1, 3, 4, 8, 10, 11, 13, 16, 17 and tacrine inhibited 45Ca(2+) uptake induced by K(+) about 20%. Compounds 6, 14 and 15 inhibited the K(+) effects by 10% or less. Compounds 7, 9, 12 and 18 blocked the K(+) signal by 30% and, finally, compounds 2 and 5 inhibited the K(+)-induced 45Ca(2+) entry by 50%. None of the new compounds was as effective as diltiazem (IC(50)=0.03 microM) in causing relaxation of the rat aorta precontracted with 35 mM K(+); the most potent was compound 7 (IC(50)=0.3 microM). Compounds 5, 6, 8, 9, 10 and 13 had IC(50)s around 10 microM and compounds 3, 4, 11 and 12 around 20 microM. Blockade of Ca(2+) entry through neuronal voltage-dependent Ca(2+) channels, without concomitant blockade of vascular Ca(2+) channels, suggests that some of these compounds might exhibit neuroprotectant effects but not undesirable hemodynamic effects.     

Two modes of secretion in pancreatic acinar cells: involvement of phosphatidylinositol 3-kinase and regulation by capacitative Ca(2+) entry

In pancreatic acinar cells, muscarinic agonists stimulate both the release of Ca(2+) from intracellular stores and the influx of extracellular Ca(2+). The part played by Ca(2+) released from intracellular stores in the regulation of secretion is well established; however, the role of Ca(2+) influx in exocytosis is unclear. Recently, we observed that supramaximal concentrations of acetylcholine (>or=10 microM) elicited an additional component of exocytosis despite reducing Ca(2+) influx. In the present study, we found that supramaximal exocytosis was substantially inhibited (approximately 70%) by wortmannin (100 nM), an inhibitor of phosphatidylinositol 3-kinase. In contrast, exocytosis evoked by a lower concentration of acetylcholine (1 microM) was potentiated (approximately 45%) by wortmannin. Exocytosis stimulated by 1 microM acetylcholine in the absence of extracellular Ca(2+) was, like supramaximal exocytosis, inhibited by wortmannin. The switch to a wortmannin-inhibitable form of exocytosis depended upon a reduction in Ca(2+) entry through store-operated Ca(2+) channels, as the switch in exocytotic mode could also be brought about by the selective blockade of these channels by Gd(3+) (2 microM), but not by inhibition of noncapacitative Ca(2+) entry by SB203580 (10 microM). We conclude that supramaximal doses of acetylcholine lead to a switch in the mode of zymogen granule exocytosis by inhibiting store-dependent Ca(2+) influx.     

Loss and Ca2+-Dependent Retention of Scinderin in Digitonin-Permeabilized Chromaffin Cells: Correlation with Ca2+-Evoked Catecholamine Release

Exposure of chromaffin cells to digitonin causes the loss of many cytosolic proteins. Here we report that scinderin (a Ca(2+)-dependent actin-filament-severing protein), but not gelsolin, is among the proteins that leak out from digitonin-permeabilized cells. Chromaffin cells that were exposed to increasing concentrations (15-40 microM) of digitonin for 5 min released scinderin into the medium. One-minute treatment with 20 microM digitonin was enough to detect scinderin in the medium, and scinderin leakage levelled off after 10 min of permeabilization. Elevation of free Ca2+ concentration in the permeabilizing medium produced a dose-dependent retention of scinderin. Results were confirmed by immunofluorescence microscopy of digitonin-permeabilized cells. Subcellular fractionation of permeabilized cells showed that scinderin leakage was mainly from the cytoplasm (80%); the remaining scinderin (20%) was from the microsomal fraction. Other Ca(2+)-binding proteins released by digitonin and also retained by Ca2+ were calmodulin, protein kinase C, and calcineurins A and B. Scinderin leakage was parallel to the loss of the chromaffin cell secretory response. Permeabilization in the presence of increasing free Ca2+ concentrations produced a concomitant enhancement in the subsequent Ca(2+)-dependent catecholamine release. The experiments suggest that: (1) scinderin is an intracellular target for Ca2+, (2) permeabilization of chromaffin cells with digitonin in the presence of micromolar Ca2+ concentrations retained Ca(2+)-binding proteins including scinderin, and (3) the retention of these proteins may be related to the increase in the subsequent Ca(2+)-dependent catecholamine release observed in permeabilized chromaffin cells.     

Inhibitory Ca(2+)-regulation of myosin light chain kinase in the lower eukaryote, Physarum polycephalum: role of a Ca(2+)-dependent inhibitory factor.

ATP-dependent interactions between myosin and actin in the lower eukaryote, Physarum polycephalum, are inhibited by micromolar levels of Ca2+. This inhibition is mediated by the binding of Ca2+ to myosin, the phosphorylation of which is required if Ca2+ is to inhibit the activities of myosin (Kohama, K., Trends Pharmacol. Sci. 11, 433-435 (1990)). As the first step to examine whether Ca2+ also regulates phosphorylation in the actomyosin system, we purified myosin light chain kinase (MLCK) of 55 kDa almost to homogeneity. The MLCK activity was high whether or not Ca2+ was present. However, a Ca(2+)-dependent inhibitory factor (CIF) purified from Physarum (Okagaki et al., Biochem. Biophys. Res. Commun. 176, 564-570 (1991)) was shown to reduce the MLCK activity in a Ca(2+)-dependent manner. Using crude preparations, not only MLCK but also myosin heavy chain kinase and actin kinase were shown to be inhibited by Ca2+ half-maximally at micromolar levels. Since CIF is the only Ca(2+)-binding protein in the preparations, we propose that this inhibitory Ca(2+)-regulation of the kinases for actomyosin is mediated by CIF.     

Degradation of the proto-oncogene product p39mos is not necessary for cyclin proteolysis and exit from meiotic metaphase: requirement for a Ca(2+)-calmodulin dependent event.

Exit from M phase, which requires cyclin degradation, is prevented from occurring in unfertilized eggs of vertebrates arrested at second meiotic metaphase due to a cytostatic factor recently identified as p39mos, the product of the proto-oncogene c-mos. Calpain can destroy both p39mos and cyclin in vitro in extracts prepared from metaphase-arrested Xenopus eggs, but only when free Ca2+ concentration is raised to the millimolar range. When free Ca2+ concentration is raised for only 30 s to the micromolar range, as occurs in physiological conditions after fertilization, cyclin degradation is induced, byt p39mos is not degraded. Cyclin proteolysis at micromolar free Ca2+, is not inhibited by calpastatin, and therefore does not involve calpain. A cyclin mutant modified in the destruction box is found to be resistant at micromolar, but not millimolar free Ca2+, suggesting that the ubiquitin pathway mediates cyclin degradation at micromolar Ca2+ concentration whereas calpain is involved at the millimolar level. A synthetic peptide which binds Ca(2+)-calmodulin with high affinity suppresses cyclin degradation at micromolar but not millimolar free Ca2+, and this only when it is present in the extract during the first 30 s after raising free Ca2+ concentration. The inhibition of the cyclin degradation pathway by the Ca(2+)-calmodulin binding peptide can be overcome by adding calmodulin. These results strongly suggest that a Ca(2+)-calmodulin process is required as an early event following fertilization to release the cyclin degradation pathway from inhibition in metaphase-arrested eggs. In contrast, p39mos degradation is not required.     

Ca2+ microdomains in smooth muscle

In smooth muscle, Ca(2+) controls diverse activities including cell division, contraction and cell death. Of particular significance in enabling Ca(2+) to perform these multiple functions is the cell's ability to localize Ca(2+) signals to certain regions by creating high local concentrations of Ca(2+) (microdomains), which differ from the cytoplasmic average. Microdomains arise from Ca(2+) influx across the plasma membrane or release from the sarcoplasmic reticulum (SR) Ca(2+) store. A single Ca(2+) channel can create a microdomain of several micromolar near (approximately 200 nm) the channel. This concentration declines quickly with peak rates of several thousand micromolar per second when influx ends. The high [Ca(2+)] and the rapid rates of decline target Ca(2+) signals to effectors in the microdomain with rapid kinetics and enable the selective activation of cellular processes. Several elements within the cell combine to enable microdomains to develop. These include the brief open time of ion channels, localization of Ca(2+) by buffering, the clustering of ion channels to certain regions of the cell and the presence of membrane barriers, which restrict the free diffusion of Ca(2+). In this review, the generation of microdomains arising from Ca(2+) influx across the plasma membrane and the release of the ion from the SR Ca(2+) store will be discussed and the contribution of mitochondria and the Golgi apparatus as well as endogenous modulators (e.g. cADPR and channel binding proteins) will be considered.     

Effects of isoflurane on voltage-dependent calcium fluxes in rabbit T-tubule membranes: comparison with alcohols

The effects of racemic (+/-) and (+)- and (-)-stereoisomers of isoflurane on depolarization-induced (45)Ca(2+) fluxes mediated by voltage-dependent Ca(2+) channels were investigated in transverse tubule membrane vesicles from rabbit skeletal muscle. In the concentration range 0.5 to 2 mM, (+/-)-isoflurane inhibited (45)Ca(2+) fluxes and functionally modulated the effects of the Ca(2+) channel antagonist nifedipine (1-10 microM). Isoflurane-induced inhibition of (45)Ca(2+) fluxes was not significantly affected by pretreatment with either pertussis toxin (5 microg/ml) or phorbol 12-myristate 13-acetate (50 nM). Further experiments indicated that there were no significant differences between (+)- and (-)-stereoisomers of isoflurane with respect to the extent of inhibition of (45)Ca(2+) fluxes. Radioligand binding studies indicated that racemic and (+)- and (-)-isoflurane were equally effective in displacing the specific binding of [(3)H]PN 200-110 to transverse tubule membranes. There were no apparent differences between the effects of (+)- and (-)-isoflurane on the characteristics of [(3)H]PN 200-110 binding. Although the concentrations of isoflurane for the inhibitions of (45)Ca(2+) fluxes and radioligand bindings were similar, the concentrations of n-alcohols required for the inhibition of (45)Ca(2+) fluxes were lower than those for the displacement of radioligand. Comparison of the data for the displacement of [(3)H]PN 200-110 binding and the inhibition of (45)Ca(2+) fluxes by isoflurane and by n-alcohols suggested that both isoflurane and n-alcohols may have more than a single binding site. In conclusion, results indicate that isoflurane, independent of intracellular Ca(2+) levels, nonstereospecifically inhibits the function of voltage-dependent Ca(2+) channels and this effect is mediated through multiple binding sites.