Effects of calcium chelators, divalent cations and sulfhydryl reagents on calcium uptake and motility of bovine spermatozoa

Addition of 1 mM Ca/EGTA complex (1:1 ratio) to an incubation medium containing 1.5 mM Ca2+ produced a notable increase in the Ca2+ cycling in ejaculated bovine spermatozoa. Similar results were also obtained with the Ca/EDTA and Ca/EDTA complexes or with the heavy metal chelator DTPA (50 microM). Ba2+, Ni2+ or Co2+ added at 0.1 mM concentration abolished the stimulatory effect of the Ca/EGTA complex on Ca2+ cycling, whereas it did not affect the calcium movement in the absence of the calcium chelator complex. It is concluded that small amounts of these cations should be bound to the plasma membrane of bovine spermatozoa and inhibit the cellular calcium influx. 0.1 mM Cd2+ and NEM or 1 mM diamide produced a calcium efflux from the spermatozoa together with an inhibition of cellular motility and an increase in glutamate-oxaloacetate transaminase release. Conversely the impermeant sulfhydryl reagent mersalyl caused a net calcium efflux but did not alter the cellular motility nor the transaminase release. It is suggested that the permeant thiol reagents could decrease the spermatozoal mobility by impairing the mitochondrial ATP-synthesis.     

Intracellular free Ca2+ concentration in fowl spermatozoa and its relationship to motility and respiration in spermatozoa.

The ability of fowl spermatozoa to accumulate and de-esterify the intracellular fluorescent Ca2+ indicator fura-2 was established. The cytosolic Ca2+ concentrations, measured by this technique, did not change after the addition of 1 mmol EGTA l-1. Subsequently, addition of the calcium ionophore A23187 caused a reduction in cytosolic Ca2+ concentrations, presumably by efflux of Ca2+ from the spermatozoa. Intracellular free Ca2+ concentrations were then significantly increased by the addition of 1 mmol CaCl2 l-1. The motility of demembranated spermatozoa gradually decreased after the addition of EGTA alone or EGTA with A23187, but was instantly restored by the addition of CaCl2 in the presence of both EGTA and A23187. Unlike demembranated spermatozoa, intact spermatozoa maintained their motility, even after the addition of EGTA, but their motility was reduced by the addition of A23187 in the presence of EGTA. The addition of A23187 also reduced the rate of oxygen consumption, but not the ATP concentrations in intact spermatozoa. These results demonstrate that the motility and respiration of fowl spermatozoa are strongly influenced by their intracellular Ca2+ concentrations.     

Sodium-dependent calcium efflux from adrenal chromaffin cells following exocytosis. Possible role of secretory vesicle membranes.

Although cytosolic Ca2+ transients are known to influence the magnitude and duration of hormone and neurotransmitter release, the processes regulating the decay of such transients after cell stimulation are not well understood. Na(+)-dependent Ca2+ efflux across the secretory vesicle membrane, following its incorporation into the plasma membrane, may play a significant role in Ca2+ efflux after stimulation of secretion. We have measured an enhanced 45Ca2+ efflux from cultured bovine adrenal chromaffin cells following cell stimulation with depolarizing medium (75 mM K+) or nicotine (10 microM). Such stimulation also causes Ca2+ uptake via voltage-gated Ca2+ channels and secretion of catecholamines. Na+ replacement with any of several substitutes (N-methyl-glucamine, Li+, choline, or sucrose) during cell stimulation inhibited the enhanced 45Ca2+ efflux, indicating and Na(+)-dependent Ca2+ efflux process. Na+ deprivation did not inhibit 45Ca2+ uptake or catecholamine secretion evoked by elevated K+. Suppression of exocytotic incorporation of secretory vesicle membranes into the plasma membrane with hypertonic medium (620 mOsm) or by lowering temperature to 12 degrees C inhibited K(+)-stimulated 45Ca2+ efflux in Na(+)-containing medium but did not inhibit the stimulated 45Ca2+ uptake. Enhancement of exocytotic secretion with pertussis toxin resulted in an enhanced 45Ca2+ efflux without affecting calcium uptake. The combined results suggest that Na(+)-dependent Ca2+ efflux across secretory vesicle membranes, following their incorporation into the plasma membrane during exocytosis, plays a significant role in regulating calcium efflux and the decay of cytosolic Ca2+ in adrenal chromaffin cells and possibly in related secretory cells.     

Cation/proton antiport systems in Escherichia coli. Solubilization and reconstitution of delta pH-driven sodium/proton and calcium/proton antiporters

Uptake of 22Na+ and 45Ca2+ into everted membrane vesicles from Escherichia coli was measured with imposed transmembrane pH gradients, acid interior, as driving force. Vesicles loaded with 0.5 M KCl were diluted into 0.5 M choline chloride to create a potassium gradient. Addition of nigericin to produce K+/H+ exchange resulted in formation of a pH gradient. This imposed gradient was capable of driving 45Ca2+ accumulation. In another method vesicles loaded with 0.5 M NH4Cl were diluted into 0.5 M choline chloride, creating an ammonium diffusion potential. A gradient of H+ was produced by passive efflux of NH3. With an ammonium gradient as driving force, everted vesicles accumulated both 45Ca2+ and 22Na+. The data suggest that 22Na+ uptake was via the sodium/proton antiporter and 45Ca2+ via the calcium/proton antiporter. Uptake of both cations required alkaline pHout. A minimum pH gradient of 0.9 unit was needed for transport of either ion, suggesting gating of the antiporters. Octyl glucoside extracts of inner membrane were reconstituted with E. coli phospholipids in 0.5 M NH4Cl. NH4+-loaded proteoliposomes accumulated both 22Na+ and 45Ca2+, demonstrating that the sodium/proton and calcium/proton antiporters could be solubilized and reconstituted in a functional form.     

Interactions of calcium with yeast mitochondria.

The interactions of Ca2+ with mitochondria from Saccharomyces cerevisiae were explored. Mitochondria were loaded with the metallochromic dye Fluo-3 to measure the concentration of free calcium in the matrix. Addition of EGTA or Ca2+ led to fluctuations in mitochondrial free calcium between 120 and 400 nM. Ca2+ variations were slower at 4 degrees C than at 25 degrees C or in the presence of phosphate instead of acetate. The net uptake of 45Ca2+ was higher with phosphate than with acetate. The optimum pH for Ca2+ uptake was 6.8. Ruthenium red did not affect the uptake of Ca2+. Addition of antimycin-A or uncouplers led to a small and transient release of Ca2+. Addition of EGTA or the monovalent cations Na+ or K+ resulted in higher release of Ca2+. Site I but not site II dependent O2 consumption was partially inhibited by EGTA. The effect of Ca2+ on NADH oxidation is similar to results reported with enzymes from mammalian sources which use NADH, such as the pyruvate, isocitrate and oxoglutarate dehydrogenases.     

Ca2+ dependence of transverse tubule-mediated calcium release in skinned skeletal muscle fibers

Isometric force and 45Ca efflux from the sarcoplasmic reticulum were measured at 19 degrees C in frog skeletal muscle fibers skinned by microdissection. After Ca2+ loading, application of the ionophores monensin, an Na+(K+)/H+ exchanger, or gramicidin D, an H+ greater than K+ greater than Na+ channel-former, evoked rapid force development and stimulated release of approximately 30% of the accumulated 45Ca within 1 min, whereas CCCP (carbonyl cyanide pyruvate p-trichloromethoxyphenylhydrazone), a protonophore, and valinomycin, a neutral, K+-specific ionophore, did not. When monensin was present in all bathing solutions, i.e., before and during Ca2+ loading, subsequent application failed to elicit force development and to stimulate 45Ca efflux. 5 min pretreatment of the skinned fibers with 50 microM digitoxin, a permeant glycoside that specifically inhibits the Na+,K+ pump, inhibited monensin and gramicidin D stimulation of 45Ca efflux; similar pretreatment with 100 microM ouabain, an impermeant glycoside, was ineffective. Monensin stimulation of 45Ca efflux was abolished by brief pretreatment with 5 mM EGTA, which chelates myofilament-space calcium. These results suggest that: monensin and gramicidin D stimulate Ca2+ release from the sarcoplasmic reticulum that is mediated by depolarization of the transverse tubules, which seal off after sarcolemma removal and form closed compartments; a transverse tubule membrane potential (myofilament space-negative) is maintained and/or established by the operation of the Na+,K+ pump in the transverse tubule membranes and is sensitive to the permeant inhibitor digitoxin; the transverse tubule-mediated stimulation of 45Ca efflux appears to be entirely Ca2+ dependent.     

Uptake and release of 45Ca by Myxicola axoplasm

The binding and release of 45Ca by axoplasm isolated from Myxicola giant axons were examined. Two distinct components of binding were observed, one requiring ATP and one not requiring ATP. The ATP- dependent binding was largely prevented by the addition of mitochondrial inhibitors, whereas the ATP-independent component was unaffected by these inhibitors. The ATP-independent binding accounted for roughly two-thirds of the total 45Ca uptake in solutions containing an ionized [Ca2+] = 0.54 microM and was the major focus of this investigation. This fraction of bound 45Ca was released from the axoplasm at a rate that increased with increasing concentrations of Ca2+ in the incubation fluid. The ions Cd2+ and Mn2+ were also able to increase 45Ca efflux from the sample, but Co2+, Ni2+, Mg2+, and Ba2+ had no effect. The concentration-response curves relating the 45Ca efflux rate coefficients to the concentration of Ca2+, Cd2+, and Mn2+ in the bathing solution were S-shaped. The maximum rate of efflux elicited by one of these divalent ions could not be exceeded by adding a saturating concentration of a second ion. Increasing EGTA concentration in the bath medium from 100 to 200 microM did not increase 45Ca efflux; yet increasing the concentration of the EGTA buffer in the uptake medium from 100 to 200 microM and keeping ionized Ca2+ constant caused more 45Ca to be bound by the axoplasm. These results suggest the existence of high-affinity, ATP-independent binding sites for 45Ca in Myxicola axoplasm that compete favorably with 100 microM EGTA. The 45Ca efflux results are interpreted in terms of endogenous sites that interact with Ca2+, Cd2+, or Mn2+.     

Inositol 1,4,5-trisphosphate mobilizes glucose-incorporated calcium from pancreatic islets

Mobilization of intracellular calcium from beta-cell-rich pancreatic islets of ob/ob-mice was studied by measuring unidirectional 45Ca efflux at 37 degrees and 18 degrees C during perifusion with a K+-rich medium deficient in Ca2+ and Na+. Addition of 100 microM carbachol induced a prominent peak of Ca2+ efflux from islets preexposed to glucose. After cell permeabilization with digitonin D-myo-inositol 1,4,5-trisphosphate (IP3) caused glucose-dependent mobilization of calcium. In demonstrating that not only carbachol but also IP3 can mobilize calcium incorporated in response to glucose, the present data suggests that the endoplasmic reticulum participates in glucose-induced lowering of cytoplasmic Ca2+ activity in the pancreatic beta-cells.     

Adrenergic-agonist-induced Ca2+ fluxes in rat parotid cells are not Na+-dependent.

We investigated the hypothesis that extracellular Na+ is required for the rapid mobilization of Ca2+ by rat parotid cells after adrenergic stimulation. When Na+ salts in the media were osmotically replaced with either choline chloride (+atropine) or sucrose, efflux of 45Ca2+ from preloaded cells, caused by 10 microM-(-)-adrenaline, was unchanged. Similarly adrenaline stimulated 45Ca2+ uptake into cells under nonsteady-state conditions in the presence or absence of Na+. Monensin, a Na+ ionophore, was able to elicit a modest increase in 45Ca2+ efflux, compared with controls. Studies of net 45Ca2+ flux, performed under near-steady-state conditions, showed that adrenaline caused net 45Ca2+ accumulation, whereas monensin caused net 45Ca2+ release. The effect of monensin required the presence of Na+ in the incubation medium. Both 1 mM-LaCl3 and 0.1 mM-D-600 prevented adrenaline-stimulated 45Ca2+ uptake into cells, but had no effect on monensin-induced changes. We conclude that (1) the rapid mobilization of Ca2+ by adrenergic agonists seen in rat parotid cells does not require a Na+out greater than Na+in gradient and (2) the nature of the monensin effect is quite different from the adrenergic-agonist-induced response.     

Ionomycin releases calcium from the sarcoplasmic reticulum and activates Na+/Ca2+ exchange in vascular smooth muscle cells

Ionomycin (1 microM) produced a large spike in cytosolic free Ca2+ [( Ca2+]i). The ionophore had no effect on [Ca2+]i if the sarcoplasmic reticulum had previously been Ca2+ depleted by stimulating neurohormone receptors. Ionomycin markedly increased 45Ca2+ efflux and decreased total cell Ca2+ by 60 to 70% in 1 min. Replacing extracellular Na+ [( Na+]o) with choline or N-methyl-D-glucamine strongly inhibited the effects of ionomycin on 45Ca2+ efflux and total Ca2+. Ionomycin caused similar peak increases in [Ca2+]i in the presence and absence of [Na+]o, but the exponential fall from the peak was faster in the presence of [Na+]o. Dimethylbenzamil, a potent blocker of Na+/Ca2+ exchange in these cells, strongly inhibited the effects of ionomycin on 45Ca2+ efflux and total cell Ca2+. We conclude that the increase in cytosolic free Ca2+ produced by ionomycin may be sufficient to activate the plasma membrane Na+/Ca2+ exchanger which removes Ca2+ from the cytosol and helps restore basal [Ca2+]i.     

Regulation of acrosomal matrix dispersion in digitonin-permeabilized guinea pig spermatozoa

Digitonin-permeabilized guinea pig spermatozoa undergo acrosomal matrix dispersion in response to 2.0 mM CaCl2. In this report, the effects of pH and metal ions on matrix dispersion in permeabilized spermatozoa are examined. Calcium-induced dispersion of the acrosomal matrix was dependent on the calcium concentration; the response was not observed at concentrations of CaCl2 less than 50 microM. Magnesium could not substitute for calcium and, in fact, had a retarding effect on the calcium-induced response. Matrix dispersion was also found to be pH-dependent. The induction of matrix dispersion was inhibited at pH 5.6 and pH 9.5 relative to the responses observed at pH 6.3 and pH 7.8. Nigericin induced acrosomal matrix dispersion in the absence of added calcium, indicating a possible role of Na+/H+ exchange across the outer acrosomal membrane in initiating the matrix modification. Sodium was required for the action of nigericin; the ionophore was ineffective in medium in which choline chloride or sucrose was substituted for NaCl. In contrast, the calcium-induced dispersion of the acrosomal matrix occurred in the absence of sodium. Furthermore, low concentrations of calcium inhibited an adenosine triphosphatase activity associated with isolated acrosomal apical segments. These data are consistent with the hypothesis that calcium induces alkalinization of the acrosome, leading to matrix dispersion. However, permeabilized spermatozoa incubated at either pH 9.5 or in the presence of 50 mM NH4Cl at pH 7.5 failed to undergo spontaneous matrix dispersion, suggesting that elevated intraacrosomal pH alone was not sufficient to initiate the reaction. The proposed alternative hypothesis is that calcium initiates matrix dispersion by a mechanism in which elevated intraacrosomal pH may be a secondary response.     

Does cyclic AMP mobilize Ca2+ for amylase secretion from rat parotid cells?

Both dibutyryl cAMP and carbachol stimulated amylase released from rat parotid cells incubated in Ca2+-free medium containing 1 mM EGTA. Cells preincubated with 10 microM carbachol in Ca2+-free, 1 mM EGTA medium for 15 min lost responsiveness to carbachol, but maintained responsiveness to dibutyryl cAMP. Dibutyryl cAMP still evoked amylase release from cells preincubated with 1 microM ionophore A23187 and 1 mM EGTA for 20 min. Although carbachol stimulated net efflux of 45Ca from cells preequilibrated with 45Ca for 30 min, dibutyryl cAMP did not elicit any apparent changes in the cellular 45Ca level. Inositol trisphosphate, but not cAMP, evoked 45Ca release from saponin-permeabilized cells. These results suggest that cAMP does not mobilize calcium for amylase release from rat parotid cells.     

The hepatic response to Ca2+ is inhibited by Mg2+ and enhanced by phenylephrine or ouabain

Net hepatic Ca2+ efflux, K+ uptake and glycogen breakdown in response to the alpha 1-adrenergic agonist phenylephrine were studied. Rat livers were perfused with CO2/bicarbonate-buffered solutions containing 10 microM Ca2+ and different amounts of Mg2+. K+-free medium and/or ouabain were used to block (Na+ + K+)-ATPase-dependent K+ uptake. In some experiments a sharp increase in extracellular Ca2+ concentrations was produced by infusing CaCl2 into the medium entering the liver. Perfusion with K+-free medium and ouabain enhanced the phenylephrine-induced Ca2+ efflux and diminished the glycogenolytic response, indicating a dissociation of Ca2+ release and glycogenolysis. Exogenous Ca2+ had practically no effect if livers were perfused with regular medium containing 1.2 mM Mg2+. In the presence of phenylephrine and if extracellular Mg2+ concentrations were lowered by omitting Mg2+ from the medium or by preperfusion with EGTA, exogenous Ca2+ was glycogenolytically effective and also produced a transient K+ uptake. Increased extracellular concentrations of Mg2+ inhibited the effects of exogenous Ca2+. In the presence of phenylephrine, higher concentrations of Mg2+ were needed than in the absence of alpha 1-adrenergic agonist to achieve a similar degree of inhibition. In one respect ouabain effects were comparable to those of phenylephrine: the glycoside also increased the metabolic response to exogenous Ca2+ and diminished the sensitivity towards Mg2+. Phenylephrine and ouabain may both enhance the permeability of plasma membranes for Ca2+.     

Regulation of Ca2+ transport by isolated organelles of a rat insulinoma. Studies with endoplasmic reticulum and secretory granules

The regulation of extramicrosomal Ca2+ concentration maintained by suspensions of rat insulinoma microsomes was studied using Ca2+-selective minielectrodes. The Ca2+-transporting activity was MgATP dependent and correlated with the endoplasmic reticulum marker NADPH-cytochrome c reductase. When incubated in a high KCl medium containing Mg2+ and phosphate, the microsomes lowered [Ca2+] within less than 10 min to around 0.2 microM. They had a high Ca2+-sequestering activity since they were able to take up and retain several small Ca2+ additions. No evidence for a Na+/Ca2+ countertransport was obtained. The accumulated Ca2+ was released by the Ca2+ ionophore A23187 or upon transforming ATP into ADP using glucose plus hexokinase. The addition of ADP, at concentrations present in cells, resulted in a dose-dependent and reversible net Ca2+ efflux from the microsomes until a higher [Ca2+] steady state was reached. This was specific for ADP since GDP, UDP, CDP, IDP, and the nonhydrolyzable analogue methylene-ADP as well as AMP and cAMP did not reproduce the effect. Insulin secretory granules were unable to lower medium [Ca2+] or to take up a pulse addition of Ca2+. However, most of the large granular calcium content was released by A23187. The addition of Na+ and lowering or increasing medium pH by 0.2 pH unit did not induce Ca2+ uptake or efflux from the secretory granules. The results indicate that insulinoma endoplasmic reticulum but not insulin secretory granules may play a critical role in the regulation of cytosolic Ca2+. A variation in cellular ADP content following secretagogue addition might modulate Ca2+ fluxes across the endoplasmic reticulum and contribute in raising cytosolic Ca2+.     

Spermine. A regulator of mitochondrial calcium cycling

Steady-state free Ca2+ concentrations have been measured with a Ca2+ electrode using suspensions of isolated rat liver mitochondria or saponin-treated hepatocytes. Mitochondria, when incubated in the presence of Mg2+ and MgATP2-, maintain a steady-state pCa2+ (-log [Ca2+]) of approximately 6.1 (0.8 microM). Addition of spermine lowered this value to a pCa2+ of 6.6 (0.25 microM). Spermine was the most effective polyamine, giving half-maximal effects at 170 microM and maximal effects at 400 microM. With saponin-permeabilized hepatocytes, spermine addition similarly showed that the mitochondria buffered the steady-state medium-free Ca2+ at a level approximating the cytosolic free Ca2+ concentration of intact hepatocytes. The initial rate of Ca2+ uptake by the mitochondrial Ca2+ uniporter was investigated using Ca2+-depleted mitochondria incubated in the presence of succinate and 0.3 mM free Mg2+. Under control conditions, Ca2+ uptake was not observed at free Ca2+ concentrations below 0.5 microM. Spermine (350 microM) increased the rate of Ca2+ uptake at all Ca2+ concentrations below 4.5 microM, but at higher Ca2+ concentrations, it was inhibitory. Spermine also affected mitochondrial Ca2+ efflux by decreasing the apparent Km from 16 to 3.8 nmol of Ca2+/mg of mitochondrial protein with no change of Vmax. Experiments with 45Ca2+ confirmed that spermine increased mitochondrial Ca2+ cycling at 0.2 microM free Ca2+. Hepatic spermine contents are reported to be about 1 mumol/g, wet weight, suggesting that this polyamine may have an important physiological role in intracellular calcium homeostasis.     

The role of calcium in the initiation of superoxide release from alveolar macrophages

The role of calcium in the release of superoxide anion (O2-) was examined in alveolar macrophages after stimulation with the soluble stimuli: concanavalin A (Con A), N-formyl methionyl phenylalanine (FMP), and the calcium ionophore. A23187. The release of O2- by Con A was unaffected over a wide range of extracellular calcium concentrations (20 microM to 3 mM), whereas increasing the extracellular calcium above 2 mM inhibited FMP-stimulated O2- release. In contrast, A23187 did not stimulate O2- release in calcium-free medium (less than or equal to 30 microM). The addition of EGTA (50 microM) to calcium-free medium had no effect on Con A stimulation of O2- release or FMP-stimulated O2- release. These results suggest that, for the three soluble stimuli, there are different roles for Ca+2 in the activation and transmission of stimulatory signals across the cell membrane. Con A- or FMP-stimulated calcium efflux from calcium-loaded cells in either calcium-free medium or 0.5 mM calcium-containing medium. In calcium-free medium, FMP transiently retarded 45Ca+2 uptake, while in 0.5 mM calcium-containing medium, FMP transiently stimulated 45Ca+2 uptake. For either Con A or FMP, calcium efflux preceded O2- release by 30-45 sec. Quinine, an agent that blocks membrane hyperpolarization in macrophages, completely blocked O2- release by concanavalin A or FMP and inhibited 45CA+2 efflux by 50% or more for both agents. These results support the hypothesis that redistribution of cellular Ca+2 is one of the initial steps leading to the release of O2-.     

Reversible desensitization of fibroblasts to cadmium receptor stimuli: evidence that growth in high zinc represses a xenobiotic receptor.

The xenobiotic Cd2+ triggers the production of inositol trisphosphate and releases stored Ca2+ in certain cell types, apparently by binding to a zinc site in the external domain of an "orphan" receptor (no known endogenous stimulus). Cd2+ and bradykinin evoke similar spikes in cytosolic free Ca2+. Growth in high Zn2+ (100-200 microM) abolished the free Ca2+ spike evoked by Cd2+ without affecting the spike produced by bradykinin. Growth in high Zn2+ almost abolished Cd(2+)-evoked production of [3H]inositol mono-, bis-, and trisphosphate. Bradykinin-evoked [3H]inositol phosphate production was not affected by growth in high Zn2+. Growth in high Zn2+ nearly prevented the stimulation of 45Ca2+ efflux by Cd2+ without affecting the stimulation of 45Ca2+ efflux by bradykinin or histamine. Removing Zn2+ from the culture medium and incubating the cells for several hours fully restored responsiveness to Cd2+. Cycloheximide, actinomycin D, or tunicamycin prevented the restoration of Cd2+ responsiveness, indicating that resensitization requires macromolecular synthesis. Growth in high Zn2+ reversibly abolished Ca2+ mobilization evoked by two additional stimuli: a decrease in extracellular pH or Na+ concentration. These findings support the hypothesis that the three stimuli (Cd2+ or a decrease in external pH or Na+ concentration) activate the same orphan receptor. Growth in high Zn2+ apparently desensitizes the cells to the Cd2+ receptor stimuli by repressing receptor synthesis.     

Effects of monensin on metabolism of isolated rat islets of Langerhans.

Monensin, a univalent ionophore, is a carboxylic acid produced by Streptomyces cinnamonensis. It will complex various alkali-metal ions, but most readily binds Na+. Because of interest in the possible role of Na+ in the regulation of insulin secretion, we examined its effects on several aspects of the metabolism of isolated rat islets of Langerhans. The ionophore inhibited glucose-stimulated insulin release in a concentration-dependent manner, completely inhibiting secretion evoked by 20 mM-glucose at concentrations as low as 0.1 microM in static incubations. In perifusion experiments, both phases of insulin release were equally affected. Monensin (0.1 microM) had no significant effect on glucose oxidation as measured by the generation of 14CO2 from [14C]glucose. Monensin increased the rate of 22Na+ efflux from preloaded islets and net 22Na+ uptake over 30 min, in the absence of changes in islet volume or extracellular space. The ionophore increased the Rb+/K+ permeability of islet cells, as shown by its inhibition of 86Rb+ retention and stimulation of 86Rb+ efflux. At 0.1 microM, monensin abolished glucose-stimulated 45Ca2+ uptake by islets during 5 min incubations, and stimulated 45Ca2+ efflux from preloaded islets perifused with Ca2+-free medium, even in the complete absence of extracellular Na+. Studies of the uptake of 14C-labelled 5,5-dimethyloxazolidine-2,4-dione showed that 0.1 microM-monensin increased net intracellular pH from 7.05 to 7.13. 7 Monensin has widespread, complex, effects on the secretory responses and ion handling by the B cells, which are difficult to interpret in terms solely of actions as a Na+ ionophore.     

Ca2+ dependence of stimulated 45Ca efflux in skinned muscle fibers

Stimulation of sarcoplasmic reticulum Ca release by Mg reduction of caffeine was studied in situ, to characterize further the Ca2+ dependence observed previously with stimulation by Cl ion. 45Ca efflux and isometric force were measured simultaneously at 19 degrees C in frog skeletal muscle fibers skinned by microdissection; EGTA was added to chelate myofilament space Ca either before or after the stimulus. Both Mg2+ reduction (20 or 110 microM to 4 microM) and caffeine (5 mM) induced large force responses and 45Ca release, which were inhibited by pretreatment with 5 mM EGTA. In the case of Mg reduction, residual efflux stimulation was undetectable, and 45Ca efflux in EGTA at 4 microM Mg2+ was not significantly increased. Residual caffeine stimulation at 20 microM Mg2+ was substantial and was reduced further in increased EGTA (10 mM); at 600 microM Mg2+, residual stimulation in 5 mM EGTA was undetectable. Caffeine appears to initiate a small Ca2+-insensitive efflux that produces a large Ca2+-dependent efflux. Additional experiments suggested that caffeine also inhibited influx. The results suggest that stimulated efflux is mediated mainly or entirely by a channel controlled by an intrinsic Ca2+ receptor, which responds to local [Ca2+] in or near the channel. Receptor affinity for Ca2+ probably is influenced by Mg2+, but inhibition is weak unless local [Ca2+] is very low.     

Thyrotropin-releasing hormone-induced spike and plateau in cytosolic free Ca2+ concentrations in pituitary cells. Relation to prolactin release

Using the acetoxymethyl ester of "Quin 2," a fluorescent Ca2+-indicator, we have loaded prolactin (PRL)-producing rat pituitary cells with non-toxic concentrations of Quin 2 and quantitated changes in cytosolic free calcium concentration ( [Ca2+]i) during stimulation of PRL release by thyrotropin-releasing hormone (TRH) and 40 mM K+. TRH induced a biphasic response, with an immediate (less than 1 s) spike in [Ca2+]i from basal levels (350 +/- 80 nM) to a peak of 1-3 microM, which decayed rapidly (t 1/2 = 8 s) to a near basal nadir, then rising to a plateau in [Ca2+]i of 500-800 nM. The TRH-induced spike phase was attenuated but not abolished by prior addition of EGTA, while the plateau phase was eliminated by EGTA. Addition of 40 mM K+ caused an immediate spike in [Ca2+]i to 1-3 microM which equilibrated slowly (t 1/2 = 1 min) directly to a plateau of 600-800 nM. The K+-induced spike and plateau phases were both abolished by prior addition of EGTA. The biphasic nature of TRH action on [Ca2+]i parallels the biphasic actions of TRH on 45Ca2+ fluxes and the biphasic release of PRL by GH cells in suspension. These findings provide evidence that Ca2+-dependent agonist-mediated increases in [Ca2+]i and hormone release are linked, and may generally have two modes: an acute "spike" mode, dependent primarily on redistribution of intracellular Ca2+ stores; and a sustained "plateau" mode, dependent on influx of extracellular Ca2+.