Accelerated calcium ion uptake in murine thymocytes induced by concanavalin A.

The mechanism of enhancement of Ca2+ uptake by the T cell mitogen concanavalin A (Con A) was studied in murine thymocytes. Native Con A enhanced the rate of Ca2+ uptake as much as 9-fold, an increase being observed within five minutes after Con A addition. The effect of Con A was reversed completely by alpha-methyl mannopyranoside (alpha-MM). Increased Ca2+ uptake was observed with increasing concentrations of Con A, between 2 and 400 microgram/ml, indicating that the stimulation of Ca2+ uptake is not restricted to mitogenic lectin concentrations (0.5-2 microgram/ml). Succinyl Con A exhibits only a slight effect in the same concentration ranges as native Con A. Ca2+ uptake, both in the absence and presence of Con A, is strongly dependent on energy metabolism and is carrier mediated. The augmentation of Ca2+ uptake by native Con A is due to an enhanced Vmax. Uptake of the anion, CrO42-, by thymocytes, found to be a non-saturable process, was also enhanced by Con A. The effect of Con A on CrO42- permeability appears to be independent of its effect on Ca2+ uptake.     

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-.     

Biphasic stimulation of cellular calcium concentration by 3,5,3'-triiodothyronine in rat thymocytes

3,5,3'-Triiodothyronine (T3) produced a rapid and transient increase in 45Ca uptake and cytoplasmic free calcium concentration in rat thymocytes, which is the most rapid effect of T3 in this system. This effect was manifested in cells suspended in medium containing 1 mM calcium. The T3 effect on 45Ca uptake was evident at 15-30 s, reached maximum at 30-60 s, and returned to control values at 5 min. The T3 effect on cytoplasmic free calcium concentration was seen after 30 s, reached maximum at 7 min, and returned to control values after 24 min. In cells suspended in Ca2+-free medium, T3 produced a similar rapid increase in 45Ca uptake, which was sustained for at least 60 min, but T3 failed to change cytoplasmic free calcium concentration. Alprenolol (10 microM) blocked the stimulatory effects of T3 on these two functions in a similar fashion. From these results, I suggest that in rat thymocytes T3 influences cellular calcium economy through a biphasic mechanism in which T3 first increases calcium uptake which, in turn, is followed by a release of calcium from intracellular pool(s), resulting in a further increase in cytoplasmic free calcium concentration and the activation of Ca2+ -regulated systems. Moreover, the present study provides further support for the postulate that in the rat thymocyte calcium serves as the first messenger for the plasma membrane-mediated stimulatory effects of T3 on several metabolic functions.     

Immunologic and molecular characterizations of T cell-derived T cell activating factor

Culture supernatants from several subclones of a human T hybrid line (24A) stimulated with PMA showed co-stimulatory activity in the proliferation of Con A-stimulated murine thymocytes, but did not show any IL 2 activity. Some subclones did not show co-stimulatory activity even when stimulated with PMA, excluding the possibility of a carry-over effect. The factor found in the culture supernatants increased IL 2 production in normal T cells stimulated with a suboptimal concentration of PHA. The factor also induced IL 2 production in a T hybrid clone, T-394.1, when the latter was stimulated with a suboptimal concentration of mitogens, indicating a direct effect by this T cell-derived factor on mitogen-stimulated T cells inducing IL 2 production. This factor also induced the generation of other lymphokines such as BCDF and IFN-gamma. Northern blot analysis showed that the factor induced an increase in mRNA for IL 2 as well as IL 2 receptor. These results indicated that T cells could secrete a factor with IL 1-like activity. However, Northern blot analysis showed that mRNA from a T hybrid clone does not cross-react with cDNA for IL 1 (beta) derived from human monocytes.     

Calcium ion uptake in isolated pancreas cells induced by secretagogues.

1. Secretagogues of pancreatic enzyme secretion: pancreozymin, carbamylcholine, gastrin I, the octapeptide of pancreozymin, caerulein and the Ca2+ ionophore A 23187 stimulate 45Ca uptake into isolated rat pancreatic cells, whereas adrenaline, isoproterenol, secretin, dibutyrylic cyclic adenosine 3',5'-monophosphate and dibutyrylic cyclic guanosine 3',5'-monophosphate have no effect on 45Ca uptake. 2. A graphical analysis of the Ca2+ uptake curves reveals at least two phases: a fast phase, probably due to binding of Ca2+ to the membrane and a slow phase representing Ca2+ transport into cells. Both phases are stimulated by pancreozymin and carbamylcholine. 3. The 45Ca-exchangeable pool size is increased by both carbamylcholine and pancreozymin, whereas a significant increase of total content of cell calcium was too small to be detected. 4. Atropine blocks the stimulatory effect of carbamylcholine completely but not that of pancreozymin. The Ca2+ antagonist D600 blocks the stimulatory effects of both carbamylcholine and pancreozymin only partially. 5. The data suggest that secretagogues of pancreatic enzyme secretion act by increasing the rate of Ca2+ transfer into the cell most probably through an increase of the cell membrane permeability for Ca2+.     

Nonthermal 60 Hz sinusoidal magnetic-field exposure enhances 45Ca2+ uptake in rat thymocytes: dependence on mitogen activation

The effect of a 60 Hz sinusoidal magnetic field of nonthermal intensity on Ca2+ metabolism in rat thymic lymphocytes (thymocytes) was assessed in resting cells and in cells activated with the mitogen Concanavalin A (Con A). A 60 min exposure at 37 degrees C to an induced electric field of 1.0 mV/cm produced an average 2.7-fold increase in Con A-dependent 45Ca2(+)-uptake compared to non-exposed, isothermal control cells. In contrast, 45Ca2+ uptake remained unaltered during exposure of resting thymocytes. It was also found that thymocytes with a diminished ability to mobilize Ca2+ in response to Con A were most sensitive to the 60 Hz magnetic field. Although the precise mechanism of field interaction is at present unknown, modulation of Ca2+ metabolism during cell activation may represent a common pathway for field coupling to cellular systems.     

The role of the L3T4 molecule in mitogen and antigen-activated signal transduction

We investigated the role of the L3T4 molecule in mitogen and antigen-initiated signal transduction in the L3T4(+) murine T cell hybridoma, 3DT52.5.9 and an L3T4(-) variant, 3DT52.5.24. Both Concanavalin A (Con A) and specific antigen stimulated increases in cytosolic-free calcium ([Ca2+]i), phosphatidylinositol turnover, and interleukin-2 (IL-2) production in both cell lines. About 85% of the stimulated rise in [Ca2+]i was from an extracellular source. Anti-L3T4 monoclonal antibody (MAb) inhibited 90% of antigen- and 50% of Con A-stimulated increases in [Ca2+]i and IL-2 production but had no effect on the ability of either activation signal to stimulate phosphatidylinositol turnover in the parent L3T4(+) cells. Stimulus-response coupling in the L3T4(-) cells was unaffected by the MAb. The anti-L3T4-insensitive increase in [Ca2+]i induced by Con A was inhibited by EGTA, suggesting that this mitogen also stimulated an influx of Ca2+ via an additional transport mechanism distinct from that stimulated by antigen. The fact that anti-L3T4 antibodies inhibit antigen and Con A-stimulated Ca2+ transport and IL-2 production without affecting phosphatidylinositol turnover suggests that L3T4 may play a critical role in modulating the activation of the T cell receptor-associated Ca2+ transporter in T cell stimulus-response coupling.     

Interaction of levamisole and mercaptans during thymocyte proliferation induced by concanavalin A.

Levamisole enhances[³H]thymidine uptake of murine thymocytes stimulated by concanavalin A (Con A). The proliferative response of thymocytes to Con A can also be enhanced by addition of mercaptans. Six different mercaptans were examined for this effect; three of them, 2-mercaptoethanol, cysteamine, and l-cysteine, stimulated the Con A response. Addition of levamisole to an optimal stimulatory dose of 2-mercaptoethanol or cysteamine resulted in complete inhibition of cell proliferation. Three other mercaptans, penicillamine, d-cysteine, and glutathione, failed to enhance the Con A response and, in fact, were mildly inhibitory. Levamisole gave only slightly less than normal stimulation in the presence of these mercaptans. In the absence of Con A neither levamisole nor the mercaptans stimulated cell proliferation. Oxidized 2-mercaptoethanol reacted analogously to reduced 2-mercaptoethanol both in the presence and absence of levamisole. We have interpreted these results as suggesting that the effect of levamisole is dependent upon the state of activation of the lymphocyte.     

Delta 9-tetrahydrocannabinol suppresses concanavalin A induced increase in cytoplasmic free calcium in mouse thymocytes.

It has been shown that delta-9-tetrahydrocannabinol (THC) suppresses thymocyte, lymph node, and splenic lymphocyte proliferation in response to a mitogenic stimulus. It has also been reported that increases occur in the cytosolic free calcium concentration (Ca2+) in mitogen treated lymphocytes. In an attempt to understand a portion of the molecular basis of the THC induced suppression of lymphocyte proliferation, we have examined the effects of THC on the Concanavalin A (Con A) induced cytosolic free Ca2+ mobilization in mouse thymocytes measured by fluorescent Ca2+ probes and spectrofluorometry. The results show that a 10 minute pretreatment with THC suppresses the normal rise in intracellular free Ca2+ in response to Con A. A THC concentration of 4 micrograms/ml (13 microM) was suppressive and the drug vehicle, DMSO, had no effect. In addition, we found that THC pretreatment did not inhibit the binding of FITC labeled Con A to the thymocytes suggesting that the drug did not interfere with lectin binding to the cell surface. To further define the nature of the Ca2+ response affected by THC, mouse thymocytes containing fura-2 were exposed to Con A either in the presence or absence of Ca(2+)-containing medium. It was observed that THC abrogated both intracellular release (measured in Ca(2+)-free medium) as well as extracellular Ca2+ influx. These results suggest that a portion of the proliferation defect in THC treated lymphocytes may be related to a drug induced inhibition of Ca2+ mobilization that normally occurs following mitogen treatment.     

Concanavalin A-induced translocation of part of the GTP-binding activity from the membrane to the cytosol in murine thymocytes

Concanavalin A (Con A) stimulation resulted in the rapid redistribution of part of the GTP-binding activity from the membrane to the cytosol in murine thymocytes. This change in GTP-binding activity was dependent on the Con A concentration. To investigate the relationship between this redistribution and phospholipase C (PLC) activity, the effect of GTP gamma S on the cytosol PLC activity was also examined, and it was found that GTP gamma S enhanced the phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis activity in the cytosol of Con A-stimulated thymocytes more than in that of unstimulated thymocytes. This enhancement by GTP gamma S was also dependent on the Con A concentration. The results suggest that in murine thymocytes, the GTP-binding protein (G-protein) involved in the regulation of PLC activity may be translocated from the membrane to the cytosol upon Con A stimulation. Besides, the dose dependence curve for the change in the GTP gamma S-binding activity was similar to that for inositol phosphates formation in Con A-stimulated thymocytes, suggesting that the translocation of the G-protein is closely related to PLC activation. Furthermore, the effects of cytosol fractions containing the 38-43 and 23-28 kDa GTP-binding subunits of G-proteins on the PIP2 hydrolysis activity of partially purified PLC were examined. The fraction containing the 23-28 kDa subunit evidently enhanced the PLC activity but that containing the 38-43 kDa subunit enhanced the activity to a much lower extent. Moreover, the 23-28 kDa subunit fraction of Con A-stimulated thymocytes was more effective as to enhancement of the PLC activity than that of unstimulated thymocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

12-0-tetradecanoylphorbol-13-acetate and concanavalin A enhance glucose uptake in thymocytes by different mechanisms

The effects of the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) and the plant lectin concanavalin A (Con A) on glucose uptake in murine thymocytes were studied. TPA induces a rapid dose-dependent increase in the uptake of 2-deoxyglucose and in the transport of 3-0-methylglucose. Con A also elicits a time- and dose-dependent enhancement of 2-deoxyglucose uptake. The effect of Con A, however, is less pronounced. The effect of combined treatment of thymocytes with Con A and TPA is not additive. Cytochalasin B completely inhibits the basal, as well as TPA- and Con A-enhanced, 2-deoxyglucose uptake. Dexamethasone markedly inhibits basal 2-deoxyglucose uptake, but is less inhibitory to enhanced 2-deoxyglucose uptake induced by TPA and Con A. The effect of TPA on 2-deoxyglucose uptake and 3-0-methylglucose transport is refractory to inhibition by isobutyl methyl xanthine, dibutyryl cyclic AMP, and ethyleneglycol tetraacetic acid. These agents markedly inhibit the enhancement of 2-deoxyglucose (2-DOG) uptake by Con A. p-Bromophenacyl bromide, an inhibitor of phospholipase A2, also selectively inhibits Con A enhancement of 2-DOG uptake. Taken together, the results suggest that Con A and TPA exert their stimulatory effect on glucose uptake by different activating mechanisms, but they may share a final common transport pathway.     

Stimulation-Evoked Ca2+ Fluxes in Cultured Bovine Adrenal Chromaffin Cells Are Enhanced by Forskolin

Forskolin, 1 microM, increased acetylcholine (ACh)-stimulated 45Ca uptake by chromaffin cells. The stimulatory effects of forskolin decreased with increasing concentration of ACh. The attenuation of the effect of forskolin on 45Ca uptake as a function of ACh concentration correlated well with changes in the forskolin effect on ACh-evoked catecholamine (CA) release. Forskolin increased excess KCl- and veratrine-evoked CA release and 45Ca uptake. Forskolin by itself stimulated 45Ca efflux and enhanced ACh-, excess KCl-, and veratrine-stimulated 45Ca efflux. High doses of forskolin inhibited both ACh-evoked 45Ca uptake and CA release. The inhibitory action of forskolin was specific to receptor-mediated response because excess KCl- and veratrine-stimulated 45Ca uptake and CA release were not inhibited. Forskolin, 0.3-30 microM, dose-dependently increased caffeine-stimulated CA release and 45Ca efflux in the absence of Ca2+ in the medium, and the effects were mimicked by dibutyryl cyclic AMP. These results suggest that cyclic AMP increases stimulation-induced CA release by enhancing calcium uptake across the plasma membrane and/or altering calcium flux in an intracellular calcium store.     

Blockade of K+ contractures in skeletal muscle by opioid drugs: a nonstereospecific effect

The effect of several opioid drugs was tested on the K+ contractures in frog's skeletal muscle. These contractures are produced by the entrance of extracellular Ca2+ ions via the voltage-dependent, slow Ca2+ channels located in the T tubules. Morphine and other opioid agonists in concentrations ranging from 10(-10) to 10(-5) M inhibited K+ contractures. The stereoisomers, dextrorphan and levorphanol, were found to have identical potency in inhibiting high K+ contractures, suggesting that this was a nonstereospecific blockade of voltage-dependent calcium channels by the opioid drugs despite the low effective drug concentrations. In agreement with this conclusion it was found that the inhibition of K+ contractures by the opioids was not antagonized by naloxone. It also was observed using a sucrose gap apparatus that these opioid drugs in concentrations used to block the high K+ contractures did not reduce the K+-induced membrane depolarization. Raising the bathing solution Ca2+ concentration from 1.08 to 5 mM produced a reversal of the opioid-induced block of K+ contractures. Finally it was shown that while opioids completely blocked K+ contractures, they did not produce any effect on caffeine contractures showing that opioids do not deplete intracellular Ca2+ stores or inhibit the release of Ca2+ from intracellular sarcoplasmic reticulum stores. It was concluded that several opioid drugs in very low concentrations block K+ contractures in frog's skeletal muscle by a nonstereospecific block of voltage-dependent slow calcium channels.     

Guanine Nucleotide Regulation of [125I]β-Endorphin Binding to Rat Brain Membranes: Monovalent Cation Requirement

The binding of [125I]beta h-endorphin to rat brain membranes was investigated in the presence of GTP and guanylyl-5'-imidodiphosphate. In contrast to the binding of the mu-selective opioid agonist, [3H][D-Ala2,MePhe4,Glyol5]enkephalin, and the delta-selective opioid agonist, [3H][D-penicillamine2, D-penicillamine5]enkephalin, [125I]beta h-endorphin binding was not affected by GTP or guanylyl-5'-imidodiphosphate in a concentration-dependent manner in the absence of cations. However, in the presence of NaCl, the inclusion of either GTP or guanylyl-5'-imidodiphosphate resulted in a concentration-dependent inhibition of [125I]beta h-endorphin binding. This inhibition was significantly greater than the decrease in [125I]beta h-endorphin binding observed in the presence of sodium alone. Although GTP most potently inhibited [125I]beta h-endorphin binding in the presence of sodium, inhibition of [125I]beta h-endorphin binding by GTP was also observed in the presence of the monovalent cations lithium and potassium, but not the divalent cations magnesium, calcium, or manganese. The effect produced by GTP in the presence of NaCl was mimicked by GDP, but not by GMP or other nucleotides. Unlike [125I]beta h-endorphin, the binding of the putative sigma receptor agonist, (+)-[3H]SKF 10,047, was not significantly altered by GTP or guanylyl-5'-imidodiphosphate in the absence or presence of sodium.     

Exogenous ATP enhances calcium influx in intact thymocytes

Recent observations have indicated that exogenous adenosine triphosphate (ATP) may influence lymphocyte functions such as proliferation and cytoxicity. Here we report a novel activity of extracellular ATP--it specifically increases Ca2+ uptake in murine lymphocytes. ATP added to thymocytes increases the rate of [45Ca2+] uptake by up to 20-fold. The increased rate is seen with ATP concentrations as low as 500 microM and is half-maximal at approximately 2 mM ATP. The magnitude of stimulation by ATP is dependent on Mg2+ concentration, and ATP-Mg2+ complex is probably the true activator. Of the high-energy phosphate-containing compounds tested, including deoxy-ATP, only GTP showed a modest stimulation of calcium uptake. ADP, AMP, cyclic AMP, and adenosine did not significantly increase calcium uptake. Cellular integrity as indicated by trypan blue exclusion and ethidium bromide/acridine orange staining was unaffected by ATP. Ca2+ influx is the major mode of action of ATP in raising intrathymocyte Ca2+ levels, because neither the Ca2+ efflux nor the [45Ca2+]-Ca2+ exchange was significantly altered in the presence of ATP. Verapamil, a Ca2+ channel blocking agent, could not prevent the ATP effect, suggesting that ATP may be acting by a mechanism other than the voltage-dependent Ca2+ channel. An analysis of intracellular and extracellular ATP levels by chemiluminescence assay indicated no significant ATP entry into intact lymphocytes. Also, ATP added to the medium containing thymocytes was destroyed (approximately 50% by 20 min). The nonhydrolyzable ATP analogs, AMPPCP and AMPPNP, were unable to stimulate a significant amount of Ca2+ uptake, suggesting the involvement of a cell surface phosphotransferase activity. This was supported by the demonstration of a threefold to fivefold increase in the labeling of protein and phospholipid fractions obtained from intact thymocytes exposed to [gamma 32P]ATP for 30 min. Ca2+ is believed to play an important role in a variety of lymphocyte functions, including mitogenesis and natural killer cell activity. The data herein thus provide a potential mechanism for the action of exogenous ATP on these lymphocyte functions.     

Lectin-Induced Enhancement of Voltage-Dependent Calcium Flux and Calcium Channel Antagonist Binding

Concanavalin A (Con A), a tetravalent lectin with preferential affinity for mannosyl and glucosyl residues of membrane glycoconjugates, increased K+ depolarization-evoked uptake of 45Ca2+ in the PC12 neural cell line. Enhancement of uptake by Con A was concentration dependent, with maximal (24%) stimulation at 100 micrograms/ml of Con A, and was preferentially inhibited by mannoside and glucoside. Succinyl-Con A, a divalent analog with reduced biological potency, increased uptake by only 7%. The effect of Con A on 45Ca2+ uptake was dependent on membrane depolarization, was abolished by ionic Ca2+ channel blockers and organic Ca2+ channel antagonists, and was accompanied by an equivalent increase in Ca2+ channel 3H-labeled antagonist binding, observations suggesting that the voltage-dependent Ca2+ channel was the site of Ca2+ entry. The mechanism for enhancement of 45Ca2+ uptake by Con A appeared to be separate from that used by the Ca2+ channel agonist BAY K 8644 and independent of that involved in Ca2+ channel regulation by phorbol esters. These findings suggest that voltage-dependent Ca2+ channels may link cell surface carbohydrate interactions with intracellular effector processes.     

Age- and sex-dependent stimulation of calcium uptake in duodenal cells by prolactin in vitamin D-deficient rats

Administration of ovine-prolactin (O-PRL) stimulated Ca2+ uptake in isolated duodenal cells prepared from vitamin D-deficient rats. The time course of this effect was biphasic: uptake activity reached a peak in 2.5 hrs followed by a decrease at 5 hrs to original levels. This stimulatory effect of O-PRL was observed in vitamin D-deficient male, but not in female rats. This stimulatory effect was observed in 16- and 26-week old, but not 9 week old, animals. Increase in Ca2+ uptake in duodenal cells was not due to a decrease in intracellular Ca2+ efflux. We measured serum Ca concentration in vitamin D-deficient female rats and found that serum Ca increased in D-deficient female rats between 16 and 52 weeks whereas a minimal increase was observed in D-deficient male rats. Although prolactin was shown to stimulate duodenal Ca2+ uptake, it appears that the source of the increase in levels of serum Ca in D-deficient female rats was not derived from an increase in Ca2+ uptake by prolactin in duodenum. The increase in serum calcium with time may explain why female D-deficient rats survive longer then male.     

Calcium ion influx during mitogenic stimulation of lymphocytes

The uptake of free calcium ion (Ca2+) in PHA- or A23187-stimulated lymphocytes was measured using 45CaCl2 and 3H-water. Augmentation of Ca2+ uptake by both mitogens was observed, but the enhanced uptake occurred transiently, sometime within 30 min of the stimulation. The total amount of calcium in quiescent lymphocytes as determined by atomic absorption spectroscopy was about 2.9 X 10(-15) g/cell. When stimulated with PHA, more calcium gradually accumulated in the cells. The maximum amount of accumulation occurred at around 40 h, and was about 2-fold higher than that of control cells. In A23187-stimulated cells, the calcium content increased within 1 h by about 4-fold, reached a maximum at about 6 h (6-fold) and thereafter, surplus calcium was pumped out. The cytosolic free calcium ion concentration (the [Ca2+]i) within single cells was measured using quin 2 or fura-2. The [Ca2+]i was about 1 X 10(-7) M, and a transient increase in the [Ca2+]i was observed in some cells within 1 min after Con A-stimulation. Another rise in the [Ca2+]i was observed around the 40th h, and the maximum expression of the IL-2 receptor was observed at about this time. Therefore the results may indicate that the IL-2-mediated lymphocyte transformation is dependent on the rise in the [Ca2+]i.     

Characterization of Ca2+ uptake by guinea pig epididymal spermatozoa

Comparative studies of Ca2+-uptake by guinea pig spermatozoa were performed with fresh epididymal sperm and with cells preincubated in a chemically defined, Ca2+-free medium for capacitation. Calcium uptake was negligible in fresh spermatozoa, but increased dramatically after 20 min of incubation at 37 degrees C in the presence of pyruvate and lactate. Spermatozoa incubated in the absence of these substrates accumulated only 34% as much 45Ca2+ as was taken up by cells in complete medium. The monosaccharides glucose, fructose, and mannose and the nonmetabolizable sugars 2-deoxyglucose and sucrose inhibited the enhancement of Ca2+-permeability. In the presence of 6 mM sucrose 45Ca2+ uptake was not influenced by external sodium chloride concentration between 0 mM and 145 mM. The respiratory activity of the capacitated spermatozoa not only was higher than that of uncapacitated cells, but it was stimulated by Ca2+. No effect of Ca2+ on respiration of fresh spermatozoa was detected. An increase in calcium uptake was associated with increasing pH of the medium. It is possible that a regulatory mechanism through the calcium permeability of the plasma membrane of guinea pig spermatozoa exists and controls the development of physiological events related with the fertilization process. The sugar composition, the availability of the energy substrates lactate and pyruvate, and the pH of the reproductive tract fluids could play an important role in the accessibility of Ca2+ into the cells in vivo, as has been demonstrated in vitro. The enhancement of calcium permeability during the preincubation could be a useful indicator to verify if capacitation has occurred.     

Comparison of the effects of phorbol 12-myristate 13-acetate and prostaglandin E1 on calcium regulation in human platelets.

We compared the effects of phorbol 12-myristate 13-acetate (PMA) with those of prostaglandin E1 (PGE1) on the calcium transient in intact platelets and on 45Ca2+ uptake in saponin-treated platelets and microsomal fractions to determine the roles of protein kinase C and cyclic AMP in calcium sequestration. In intact platelets, PMA, like PGE1, stimulated the return of the calcium transient to resting values after a thrombin stimulus, but only the PGE1 effect was reversed by adrenaline. Both PMA and PGE1, when added before saponin, stimulated ATP-dependent 45Ca2+ uptake into the permeabilized platelets. Thrombin also stimulated 45Ca2+ uptake into saponin-treated platelets. Uptake of 45Ca2+ was increased in microsomal preparations from platelets pretreated with PMA or PGE1. PMA did not increase the cyclic AMP content of control or thrombin-treated platelets, and it induced a pattern of protein phosphorylation in 32P-labelled platelets different from that with PGE1. In correlation with the increased uptake of calcium in the saponin-treated preparation, we measured a rapid translocation of protein kinase C from supernatant to cell fraction after the addition of PMA. Our results suggest that activation of protein kinase C enhances calcium sequestration independently of an effect on cyclic AMP content in platelets. This activation could play a physiological role in the regulation of the calcium transient.