Divalent Cation Ionophore Research Articles

Sodium- and calcium-dependent steps in the mechanism of neonatal rat cardiac myocyte killing by ionophores: II. The calcium-carrying lonophore, A23187

Several lines of evidence indicate a role for elevated intracellular Ca 2+ in mechanisms of cell killing induced by a wide variety of agents. Cardiac myocytes are susceptible to killing under various conditions, including ischemia and exposure to monensin. In order to delineate the Ca 2+-dependent cell killing mechanism(s) to which cardiac myocytes are susceptible, we have investigated the mechanism by which they are killed by Ca 2+ plus the divalent cation ionophore A23187. Evidence has been obtained for two Ca 2+-mediated injury steps followed by a Na +-mediated step leading to cell death detected as membrane permeabilization to trypan blue dye. The first Ca 2+-mediated step requires the presence of A23187 and low extracellular Ca 2+ concentrations (1–100 μ m) and is inhibited by Mn 2+ and Ni 2+ ions. The second Ca 2+-dependent step requires extracellular Ca 2+ concentrations in approximately the physiological range (>1 m m), is not dependent on ionophore, and is not inhibited by Mn 2+. Arachidonic acid release occurs during both Ca 2+-mediated steps, but mostly during the second step. The second injury step is characterized by visible cell swelling and release of lactate dehydrogenase enzyme activity. The Na +-dependent step requires extracellular Na + equal to or greater than half the physiological concentration (i.e., ≥75 m m). Li +, which has a smaller ionic radius than Na +, can partially substitute for it in the Na +-dependent step, whereas K +, Cs +, Rb +, and NH 4 + (which have larger ionic radii) cannot.

Prostaglandins suppress VLDL secretion in primary rat hepatocyte cultures: relationships to hepatic calcium metabolism.

In primary cultures of rat hepatocytes, prostaglandin E2 and prostaglandin D2 (PGE2 and PGD2) inhibited the secretion of very low density lipoprotein (VLDL)-associated apoB, triacylglycerol, and cholesterol. These effects were concentration-dependent and remained apparent for at least 3 days of culture without an effect on the apoB/triacylglycerol ratio of the secreted VLDL. Prostaglandins had no effect on the overall synthesis of triacylglycerol but triacylglycerol accumulated within the cells, without intracellular accumulation of apoB. PGE2, when added to the medium together with glucagon, increased the inhibition of VLDL secretion, compared to that observed with glucagon alone. However, PGE2 did not increase the stimulatory effect of glucagon on ketogenesis. Unlike glucagon, the prostaglandins did not inhibit fatty acid synthesis nor did they stimulate ketogenesis or production of cAMP. Thus, of all the parameters of hepatic lipid metabolism studied, PGE2 and PGD2 selectively affected VLDL. Selective inhibition of VLDL secretion was also observed with the calcium antagonist verapamil. The divalent cation ionophore A23187 also inhibited VLDL release but, in contrast, also inhibited fatty acid and cholesterol synthesis. The results suggest that VLDL secretion is modulated at some optimal cell calcium concentration that may be mediated selectively by agents such as prostaglandins.

Excretion in the house cricket: role of second messengers in Malpighian tubule function

Three factors influencing diuresis, two stimulatory and one inhibitory, have been isolated from the corpora cardiaca of Acheta domesticus by means of high-performance liquid chromatography. Diuretic factor 1 provoked a fast-acting response (15 min to peak), whereas diuretic factor 2 caused a slow-acting (45 min to peak), longer lasting response. When dibutyryl-cAMP was added to the bath it increased secretion in a manner similar to diuretic factor 1. In unstimulated preparations, the divalent cation ionophore A23187 mimicked the action of antidiuretic factor, reducing the secretion rate by approximately 80%. In preparations pretreated with diuretic factor 1 or cAMP, addition of ionophore reduced the secretion rate by 60% or more, but the tubules continued to secrete at the reduced rate for at least 1 h. In contrast, when preparations were pretreated with diuretic factor 2, the addition of A23187 reduced secretion to zero within 15 min. Methanolic extracts of corpora cardiaca increased cAMP levels in tubule broken-cell preparations in a dose-dependent manner over the range of 0.03–1.0 corpus cardiacum equivalents, with a maximal increase of approximately 2 times that of the controls. Similar increases in cAMP levels were produced by homogenates of corpora cardiaca, diuretic factor 1, and sodium fluoride. In contrast, diuretic factor 2 significantly reduced cAMP concentrations. Possible mechanisms by which these three factors interact to regulate tubule secretion are discussed.

Excretion in the house cricket ( Acheta domesticus): Relative contribution of distal and mid-tubule to diuresis

The secretion rates of tubule fluid and monovalent cations (Na +, K +) from the distal and mid-segments of single isolated Malpighian tubules of Acheta domesticus were compared. Under control conditions, the secretion rate of the distal region was four times that of the mid-tubule, and the rates remained stable for at least 3 h. Thus, although the distal segment comprised only 22% of the immersed tubule length, it contributed more than 55% of the total fluid secretion. Initial cation concentrations in the fluid secreted by the mid-tubule were more than 3-fold greater than in the distal segment, although the cation secretion rates, normalized for volume and immersed tubule length, were identical. Dibutyryl cAMP increased the rate of tubule fluid formation in both segments whereas methanolic extracts of corpora cardiaca increased fluid secretion in the mid-tubule while causing the distal segment secretory rate to decline to near zero within 90 min. In all cases, regardless of whether secretion rate was increased or decreased, both cAMP and corpora cardiaca shifted the Na +:K + ratio to 1:1, from the control value of 1:2. The divalent cation ionophore, A23187, inhibited secretion in both segments, albeit with differing time courses. The implications for these observations on the use of Acheta as a standard bioassay were discussed.

Down-regulation of cystic fibrosis transmembrane conductance regulator gene expression by agents that modulate intracellular divalent cations.

In cystic fibrosis (CF), epithelial cells are unable to normally up-regulate apical membrane Cl- secretion in response to agents which increase cyclic AMP, but they do increase Cl- secretion in response to increases in intracellular Ca2+. Since intracellular divalent cations regulate the expression of many genes, we hypothesized that mobilization of intracellular Ca2+ and/or other divalent cations might modulate not only Ca(2+)-dependent Cl- channels but also cystic fibrosis transmembrane conductance regulator (CFTR) gene expression. To evaluate this concept, HT-29 human colon carcinoma cells were cultured under various conditions designed to manipulate intracellular divalent cation concentrations and CFTR gene expression was quantified at the levels of transcription, mRNA accumulation, mRNA half-life, and protein. Exposure to the divalent cation ionophores A23187 and ionomycin (agents which increase intracellular divalent cation concentrations) caused dose- and time-dependent reductions of CFTR mRNA levels, which could be blocked by the use of Ca(2+)- and Mg(2+)-free media. Ionophore-induced CFTR gene modulation was also observed with T84 human colon carcinoma cells and freshly isolated normal human bronchial epithelial cells. Incubation of HT-29 cells with thapsigargin, an agent that releases Ca2+ from intracellular stores, or in medium containing increased extracellular concentrations of Ca2+ or Mg2+ also caused down-regulation of CFTR mRNA levels. Transcription run-on analysis showed that, parallel with the decrease in CFTR mRNA levels, A23187 reduced the rate of transcription of the CFTR gene, while CFTR mRNA transcript half-life was unaffected. Consistent with the down-regulation of CFTR gene expression, CFTR protein levels also decreased after exposure to A23187. Thus, despite the independence of Ca(2+)-dependent Cl- channels and cyclic AMP-dependent CFTR-related Cl- channels in epithelial cells, increases in intracellular divalent cation concentrations down-regulate the expression of the CFTR gene at the transcriptional level, with consequent decreases in CFTR mRNA and protein.

Down-regulation of cystic fibrosis transmembrane conductance regulator gene expression by agents that modulate intracellular divalent cations.

In cystic fibrosis (CF), epithelial cells are unable to normally up-regulate apical membrane Cl- secretion in response to agents which increase cyclic AMP, but they do increase Cl- secretion in response to increases in intracellular Ca2+. Since intracellular divalent cations regulate the expression of many genes, we hypothesized that mobilization of intracellular Ca2+ and/or other divalent cations might modulate not only Ca(2+)-dependent Cl- channels but also cystic fibrosis transmembrane conductance regulator (CFTR) gene expression. To evaluate this concept, HT-29 human colon carcinoma cells were cultured under various conditions designed to manipulate intracellular divalent cation concentrations and CFTR gene expression was quantified at the levels of transcription, mRNA accumulation, mRNA half-life, and protein. Exposure to the divalent cation ionophores A23187 and ionomycin (agents which increase intracellular divalent cation concentrations) caused dose- and time-dependent reductions of CFTR mRNA levels, which could be blocked by the use of Ca(2+)- and Mg(2+)-free media. Ionophore-induced CFTR gene modulation was also observed with T84 human colon carcinoma cells and freshly isolated normal human bronchial epithelial cells. Incubation of HT-29 cells with thapsigargin, an agent that releases Ca2+ from intracellular stores, or in medium containing increased extracellular concentrations of Ca2+ or Mg2+ also caused down-regulation of CFTR mRNA levels. Transcription run-on analysis showed that, parallel with the decrease in CFTR mRNA levels, A23187 reduced the rate of transcription of the CFTR gene, while CFTR mRNA transcript half-life was unaffected. Consistent with the down-regulation of CFTR gene expression, CFTR protein levels also decreased after exposure to A23187. Thus, despite the independence of Ca(2+)-dependent Cl- channels and cyclic AMP-dependent CFTR-related Cl- channels in epithelial cells, increases in intracellular divalent cation concentrations down-regulate the expression of the CFTR gene at the transcriptional level, with consequent decreases in CFTR mRNA and protein.

Discriminatory effects of protein kinase inhibitors and calcium ionophore on endothelial ICAM-1 induction.

Intercellular adhesion molecule 1 (ICAM-1) is a proinflammatory adhesion glycoprotein induced by cytokines such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) as well as lipopolysaccharide (LPS). Little is known, however, concerning the intracellular regulatory mechanisms that modulate ICAM-1 expression in endothelial cells. We probed the involvement of protein kinase function and intracellular calcium ion upon ICAM-1 expression of human umbilical vein endothelial cells activated alternatively by TNF-alpha, IL-1 beta, LPS, or phorbol 12-myristate 13-acetate (PMA). Methodologies for the detection of ICAM-1 included both enzyme-linked immunosorbent assay and immunoprecipitation from biosynthetically labeled cells. The protein kinase inhibitor H-7 blocked induction of ICAM-1 by all of the activators; nonlinear regression analysis revealed 50% inhibitory concentration (IC50) values of 6-10 microM. Another kinase inhibitor, HA1004, did not block expression of the adhesion molecule at concentrations up to 50 microM. In contrast, the kinase inhibitor staurosporine dose dependently inhibited ICAM-1 expression triggered by PMA (IC50 67 +/- 4 nM) but, at similar concentrations, did not inhibit ICAM-1 expression induced by the other inflammatory stimuli. The divalent cation ionophore ionomycin (0.5 microM) interacted synergistically with PMA but not with cytokines or LPS in upregulating ICAM-1. We conclude from these data that although PMA-induced ICAM-1 expression may be triggered through activation of protein kinase C, ICAM-1 induction by IL-1 beta, TNF-alpha, or LPS may involve distinct regulatory pathway(s).

Does Ca2+ release by acetylcholine enhance the synthesis of inositol 1,4,5-trisphosphate in smooth muscle of the porcine coronary artery?

A possible role was investigated of the Ca2+ released by acetylcholine (ACh) in the ACh-induced synthesis of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) in smooth muscle of the porcine coronary artery. In Ca(2+)-free solution, 10 microM ACh transiently increased the cellular concentration of Ca2+ ([Ca2+]i) and Ins(1,4,5)P3. Divalent cation ionophores abolished the increase in [Ca2+]i but not the synthesis of Ins(1,4,5)P3 induced by subsequent application of 10 microM ACh in Ca(2+)-free solution, suggesting that the Ca2+ released by Ins(1,4,5)P3 following application of ACh does not act to accelerate the ACh-induced synthesis of Ins(1,4,5)P3 in smooth muscle of the porcine coronary artery.

Defibrotide inhibits Ca2+ dependent neutrophil activation: implications for its pharmacological activity in vascular disorders.

Defibrotide (DEF) is a polydeoxyribonucleotide agent provided with profibrinolytic and antithrombotic properties. Moreover, an antiischemic, cardioprotective effect of the drug has recently been demonstrated in experimental animals. Increasing evidence exists of the important role played by neutrophils in the development of tissue damage during chronic and acute ischemia and in the early phases of reperfusion. In order to evaluate whether the overall cytoprotective effect of DEF could be based, at least in part, on a neutrophil-involving mechanism, the authors studied the in vitro effects of the drug on human neutrophil activation triggered by several specific stimuli. The drug dose-dependently (10-100 microM) inhibited enzyme release, superoxide anion generation, and chemiluminescence induced in neutrophils by the chemoattractant oligopeptide fMLP and by the divalent cation ionophores A23187 and ionomycin. The increase of extracellular calcium concentration from 0.5 to 2.0 mM antagonized the inhibitory effect of DEF. The use of the fluorescent probe Fura 2/AM led them to show that DEF is able to reduce the cytosolic free calcium increase following specific stimulation by affecting extracellular calcium entrance. Such a behavior resembles that of calcium-antagonistic drugs, thus suggesting that DEF works, at least in part, similarly to calcium entry blockers. Such an activity on cell calcium translocation could represent the underlying molecular mechanism of cytoprotection. Finally, the inhibitory action on neutrophil functions may play a role in tissue protection during ischemic injury.

Effect of ionophore A23187 on the membrane permeability in mouse fibroblasts

Addition of the divalent cation ionophore A23187 to transformed mouse fibroblasts (3T6) resulted in an increase in the cell membrane permeability to normally impermeant solutes (e.g., nucleotides). The membrane permeability was assessed by following the efflux of prelabeled adenine nucleotides, the influx of p-nitrophenyl phosphate in cells attached to plastic dishes and reconstitution of intracellular protein synthesis in the presence of exogenously added normally impermeant factors required for macromolecular synthesis. The permeability change of 3T6 cells was found to be dependent on the specific presence of external calcium ion. The permeabilization was found to occur preferably in alkaline pH and specific to certain transformed cells. It is preceded by rapid efflux of K +, influx of Na + and partial hydrolysis of cellular nucleotides in 3T6 cells. Similar ion fluxes were previously found tc precede cell permeabilization by electrogenic ionophores for monovalent ions and by exogenous ATP. Our data suggest that a calcium dependent process caused the K + release and excess Na + entry, causing dissipation of the membrane potential and subsequent formation of aqueous channels.

Content of significant amounts of a cytotoxic end-product of lipid peroxidation in human semen

(E)-4-Hydroxy-2-nonenal (HNE), a cytotoxic end-product of lipid peroxidation, is present in significant amounts in human semen (0.902 +/- 0.190 microM; mean +/- s.e.; n = 18). The addition of the divalent cation ionophore A23187 to suspensions of human spermatozoa resulted in increased production of HNE. Exogenous HNE was powerfully spermicidal and as little as 50 microM caused an irreversible loss of motility of human spermatozoa within minutes. The addition of human seminal plasma protected spermatozoa from the toxic effects of HNE.

Role of lipid metabolism in cell killing by calcium plus ionophore A23187

Cultured fibroblasts treated with divalent cation ionophore A23187 in the presence of extracellular calcium provide a useful model system for studying mechanisms of cell death associated with elevated intracellular calcium concentrations. Cell death induced by A23187 plus calcium can be conveniently monitored as membrane permeabilization to Trypan blue dye. Because lipids are a major component of cell membranes and play an important role in determining membrane permeability, the present study was initiated to identify changes in cell lipid composition that occur during membrane permeabilization induced by calcium plus A23187. The percent label in each of the major structural lipids in biosynthetically labeled NIH3T3 fibroblasts changed less than 10% during the time course of membrane permeabilization. During the course of membrane permeabilization there was significantly increased label in lysophosphatidylinositol and lysophosphatidylcholine and reduced label in phosphatidylinositol 4,5-bisphosphate. The time course of these changes corresponded to that of the arachidonic acid release response stimulated by calcium plus A23187, not to the time course of membrane permeabilization, which occurs later. These observations are consistent with lipid metabolism induced by A23187 plus calcium playing only a possible regulatory or intermediatory role in membrane permeabilization, rather than causing direct permeabilization of the lipid phase of the membrane.

Characterization of lipoxins by combined gas chromatography and electron-capture negative ion chemical ionization mass spectrometry: Formation of lipoxin A4 by stimulated human whole blood

The lipoxins are a recent addition to the family of bioactive products derived from arachidonic acid. Here, we have prepared pentafluorobenzyl ester, trimethylsilyl ether derivatives of lipoxin A4, lipoxin B4 and pentadeuterolipoxin A4 and have characterized these products by electron-capture negative ion chemical ionization gas chromatography/mass spectrometry (NICI GC/MS). Lipoxin A4 (5S,6R,15S-trihydroxy-7,9,13-trans-11-cis-eicosa-tetraenoic acid; LXA4) was quantified following extraction from whole blood by stable isotopic dilution utilizing deuterium-labeled LXA4 as internal standard and selected ion monitoring of the [M--pentafluorobenzyl] anions. Studies with a second tritiated internal standard (e.g. [11,12-3H]LXA4) also showed that the recovery of LXA4 was greater than 80% following solid-phase extraction from whole blood, and greater than 90% from isolated cells. In addition, neither isolated neutrophils nor platelets oxidatively metabolized [11,12-3H]LXA4 when incubated in the presence or absence of stimuli. Whole blood incubated with either the ionophore of divalent cations (A23187), thrombin, or thrombin plus the chemotactic peptide formylmethionyl-leucine-phenylalanine generated both LXA4 and thromboxane, which were quantified by stable isotope dilution. The ratio of thromboxane to LXA4 formed by stimulated whole blood ranged from approximately 2:1 to 20:1. These results indicate that the lipoxins display suitable characteristics as their respective pentafluorobenzyl ester, trimethylsilyl ether derivatives for quantification by electron-capture NICI GC/MS. Moreover, they provide evidence that LXA4 can be generated from endogenous sources in whole blood following exposure to physiologically relevant stimuli.

Activation of internucleosomal DNA cleavage in human CEM lymphocytes by glucocorticoid and novobiocin. Evidence for a non-Ca2(+)-requiring mechanism(s).

Internucleosomal DNA cleavage is the key molecular event of the cytolytic phase of glucocorticoid-induced lymphocytolysis. We find that novobiocin, the topoisomerase II inhibitor, is a potent inducer of in vivo internucleosomal DNA cleavage in human CEM lymphocytes. This in vivo effect is very rapid, time- and dose-dependent, requires cellular integrity, and does not require de novo protein synthesis. Recently our data (Alnemri, E. S., and Litwack, G. (1989) J. Biol. Chem. 264, 4104-4111) suggested that activation of DNA cleavage in CEM-C7 lymphocytes by glucocorticoids is independent of calcium uptake. Similarly, the novobiocin effect is also independent of calcium uptake and does not occur in isolated CEM nuclei or in CEM cells treated previously with the divalent cation ionophore A23187. Internucleosomal DNA cleavage induced by novobiocin or glucocorticoid generates blunt-ended double-stranded DNA fragments possessing 3'-hydroxyls and 5'-phosphates. As demonstrated by gel retardation analysis and DNase I footprinting, novobiocin causes the disruption and unfolding of an in vitro reconstituted mononucleosome so that it becomes more susceptible to DNase I cleavage. Our data suggest that 1) novobiocin rapid activation of internucleosomal DNA cleavage and chromatin changes in CEM lymphocytes are molecular features of apoptosis or programmed cell death. 2) CEM lymphocytes apparently do not express a Ca2(+)-dependent endonuclease. 3) The mechanism(s) of glucocorticoid or novobiocin-induced DNA cleavage in CEM lymphocytes involves activation of a constitutive non Ca2(+)-dependent endonuclease. We propose that the majority of nuclear chromatin is maintained in a highly compact and charge-neutralized state and that disruption of this highly ordered structure, directly by novobiocin or indirectly by glucocorticoid, may lead to the exposure and unmasking of internucleosomal linker DNA regions which are substrates for a constitutive non-Ca2(+)-dependent endonuclease.

Divalent cation activation of galactosyltransferase in native mammary Golgi vesicles.

Millimolar concentrations of manganese are required for maximal activation of purified galactosyltransferase (lactose synthase, EC 2.4.1.22), the enzyme that catalyzes addition of galactosyl groups to proteins and, in lactose synthesis, to glucose. To examine manganese activation of this enzyme under in vivo conditions, we studied intact, partially purified Golgi membranes from mouse mammary glands. In intact vesicles treated with the divalent cation ionophore, A23187, activation followed Michaelis-Menton kinetics with a Km of 3 microM; maximal activation was achieved below 10 microM manganese. In both detergent-solubilized and leaky vesicles the kinetics of manganese activation were consistent with the presence of two manganese-binding sites with dissociation constants about 40 microM and 20 mM. The difference is consistent with the presence in intact vesicles of an endogenous activator too large to traverse the membrane via A23187; this activator could bind to the low affinity manganese site allowing manganese or another divalent cation such as zinc to activate the enzyme at micromolar concentrations. The Km for UDP-galactose was found to be similar in the vesicular and solubilized preparations at micromolar and millimolar manganese concentrations, respectively, providing additional evidence for this hypothesis.

Rat oocyte maturation: Role of calcium in hormone action

We studied the role of extracellular calcium (Ca 0) in oocyte maturation and oocyte-cumulus cells interaction in rat follicles in vitro. Luteinizing hormone (LH) or a gonadotropin-releasing hormone analog (GnRHa) induced full maturation at [Ca 0]= 1.3 mM. At [Ca 0]= 0.6 mM, maturation induced by LH or GnRHa was inhibited by 65%. Chelatin of [Ca 0] resulted in 45% maturation and neither hormone caused a further increase of maturation. [Ca 0]= 20 mM enhanced the response to suboptimal concentrations of GnRHa but inhibited that to LH. Divalent cation ionophores caused [Ca 0]-dependent maturation, which was fully inhibited by dibutyryl cAMP. Changes in [Ca 0] also affected oocyte-cumulus interaction. At [Ca 0]= 1.3 mM, either LH or GnRHa caused partial dispersion of the cumulus. Chelation of [Ca 0] also resulted in an almost complete dispersion of the cumulus. The ionophores, however, caused maturation with the oocyte-cumulus complex preserved intact. Our data suggest that GnRHa may induce maturation via cAMP-sensitive calcium mobilization into the oocyte-cumulus-granulosa complex.

Effects of A23187 in cultures containing only rat Schwann cells and sensory neurons

Serum from approximately 40% of patients with the acute Guillain-Barrésyndrome (GBS) selectively destroys myelin and myelin-related Schwann cells in cultures containing only rat dorsal root ganglion neurons and Schwann cells. To determine if the effects of GBS serum on myelin and myelin-related Schwann cells could be mediated through elevations in the intracellular concentration of calcium ions, we compared the effects of cytotoxic serum to A23187, a divalent cation ionophore. Both myelin- and nonmyelin-related Schwann cells were killed along with neurons in the presence of A23187 and extracellular calcium ions. Myelin sheaths also underwent vesicular disruption. The ultrastructural appearance of myelin and myelin-related Schwann cell lysis caused by A23187 were essentially identical to damage produced by GBS serum. These observations suggest that GBS serum factors might damage myelin and myelin-related Schwann cells in culture by an increase in myelin-related Schwann cell permeability to extracellular calcium ions. In contrast, A23187 causes necrosis of Schwann cells and neurons as well as myelin lysis by a nonselective increase in membrane permeability to extracellular calcium ions.

Effects of the divalent cation ionophore ionomycin on the performance of isolated guinea-pig atria

In isolated electrically driven left and in spontaneously beating right guinea-pig atria, the calcium ionophore ionomycin produced a concentration-dependent positive inotropic and chronotropic effect with a threshold near 10(-7) mol/l and a pD2 of 6.31 +/- 0.09 and 5.94 +/- 0.07, respectively. At low [Ca2+]o (0.5 mmol/l), the positive inotropic effect of ionomycin (3 X 10(-6) mol/l) was strongly attenuated by ryanodine and nifedipine, and slightly attenuated by pindolol and mepyramine; atropine had no effect. The positive chronotropic effect of ionomycin was slightly reduced by cimetidine or pindolol, whereas atropine, nifedipine, and ryanodine showed no inhibitory activity. The oxygen consumption of resting left atria was significantly enhanced by addition of ionomycin. It is concluded that the action of ionomycin involves at least the following mechanisms: I) release of Ca2+ from sarcoplasmic reticulum, II) influx of Ca2+ from the extracellular space, and, having little significance, III) release of catecholamines and histamine from sympathetic nerve endings and tissue mast cells. However, additional mechanisms of action of ionomycin cannot be excluded.

Ca2+ uptake during capacitation of mouse spermatozoa and the effect of an anion transport inhibitor on Ca2+ uptake

With a specially constructed chamber, Ca2+ uptake by mouse spermatozoa was monitored continuously during capacitation and the acrosome reaction. It was shown, using calcium ion-selective microelectrodes, that there was an initial uptake of Ca2+ by spermatozoa undergoing capacitation. Such net transport was also promoted by the divalent cation ionophores A23187 or ionomycin. An anion inhibitor, SITS, produced dose-dependent inhibition of Ca2+ uptake. This inhibitor reduced the incidence of capacitation as revealed by a reduction in the B pattern by chlortetracycline (CTC) assay and thus inhibited fertilization, suggesting that anions are involved in calcium uptake in mouse spermatozoa.

Phorbol 12-myristate 13-acetate (PMA) increases the expression of the nerve growth factor (NGF) gene in mouse L-929 fibroblasts

The rise of the NGF mRNA pool which takes place following exposure of L-929 fibroblasts to serum was prevented in the presence of 5 μM K-252a, a compound which inhibits several species of protein kinase activities. To characterize further this phenomenon, L-929 cells growing in a serum-free medium were exposed to cyclic nucleotide analogs, to a divalent cation ionophore or to the phorbol ester PMA. Only this latter compound induced an enhancement of the NGF mRNA pool, suggesting an involvement of protein kinase C in the upregulation of the NGF transcripts. The effects of PMA or serum also require a synthesis of protein since the level of NGF transcripts remained stable in the presence of cycloheximide.