Induction of mouse epidermal ornithine decarboxylase by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate: dependence on calcium availability

The role of calcium in epidermal ornithine decarboxylase (ODC) induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) was determined in adult mouse skin pieces incubated in serum-free minimal essential medium (MEM). Addition of TPA to skin pieces incubated in serum-free MEM, which contains 1.82 mM Ca2+ and 0.83 mM Mg2+, resulted in about a 200-fold increase in epidermal ODC activity at about 8 h after TPA treatment. TPA failed to induce epidermal ODC in skin pieces incubated in calcium-free medium. Similarly, chelation of extracellular calcium by ethyleneglycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) prevented ODC induction by TPA, which could be resumed upon calcium restoration in the medium. Furthermore, calcium ionophore A23187, which facilitates efflux of Ca2+ across cellular membranes, induced ODC activity in incubated skin pieces. Epidermal ODC activity increased by TPA appears to be the result of an increase in both the amount of ODC protein and the level of hybridizable ODC messenger. Inhibition of the induction of ODC activity by EGTA was the result of the inhibition of the amount of active ODC protein and the level of ODC mRNA.     

Interaction of Penicillium notatum phospholipase B with divalent cations

The interaction between Penicillium notatum phospholipase B and divalent cations such as Ca2+ and Mg2+ was studied. When the purified enzyme, present at concentrations of submicrogram to microgram per ml, was incubated with submillimolar to millimolar concentrations of CaCl2 or MgCl2, the enzymatic activity was remarkably decreased (to no more than 30% of original activity, when the enzyme was incubated with 2 mM CaCl2 for 15 min). The inhibitory effect of divalent cations was reversible, since dialysis against a metal chelator, such as EDTA or EGTA, substantially restored the enzymatic activity. Atomic absorption analysis showed the purified enzyme molecule to be present in a complex with Ca2+ at a ratio approaching 1:1, and this Ca2+ binding was shown to be extremely tight, since repeated dialyses of the enzyme molecules against EDTA or EGTA could remove the divalent cations only in a gradual manner. During this process, the enzyme activity increased also gradually. The remnant fraction of tightly bound Ca2+ was released from the enzyme molecule after the denaturation of the enzyme by treatment with guanidine hydrochloride, and the apoenzyme recovered its substantial activity after removal of the denaturing agent by dialysis. On the other hand, the content of Mg2+ in the purified enzyme molecule was lower than that of Ca2+, and the association of Mg2+ with the enzyme was much weaker in comparison to that of Ca2+. Atomic absorption analysis of the enzyme exposed to exogenous Ca2+ showed a fast removal, by dialysis, of unbound and weakly bound divalent cation, followed by a gradual removal of endogenous Ca2+ and a concomitant increase of enzymatic activity, which are similar to data obtained for the purified enzyme. Results shown in this report suggest some regulatory roles of divalent cations, especially of Ca2+, in the enzymatic function of P. notatum phospholipse B.     

The effect of intracellular calcium ions on adrenaline-stimulated adenosine 3'':5''-cyclic monophosphate concentrations in pigeon erythrocytes, studied by using the ionophore A23187.

1. The bivalent cation ionophore A23187 was used to increase the intracellular concentration of Ca2+ in pigeon erythrocytes to investigate whether the increase in cyclic AMP content caused by adrenaline might be influenced by a change in intracellular Ca2+ in intact cells. 2. Incubation of cells with adrenaline, in the concentration range 0.55--55 muM, resulted in an increase in the concentration of cyclic AMP over a period of 60 min. The effect of adrenaline was inhibited by more than 90% with ionophore A23187 (1.9 muM) in the presence of 1 mM-Ca2+. This inhibition could be decreased by decreasing either the concentration of the ionophore or the concentration of extracellular Ca2+, and was independent of the concentration of adrenaline. 3. The effect of ionophore A23187 depended on the time of incubation. Time-course studies showed that maximum inhibition by ionophore A23187 was only observed when the cells were incubated with the ionophore for at least 15 min before the addition of adrenaline. 4. The inhibition by ionophore A23187 depended on the concentration of extracellular Ca2+. In the absence of Mg2+, ionophore A23187 (1.9 muM) inhibited the effect of adrenaline by approx. 30% without added Ca2+, by approx. 66% with 10 muM-Ca2+ and by more than 90% with concentrations of added Ca2+ greater than 30 muM. However, even in the presence of EGTA [ethanedioxybis(ethylamine)tetra-acetate](0.1--10 mM), ionophore A23187 caused an inhibition of the cyclic AMP response of at least 30%, which may have been due to a decrease in cell Mg2+ concentration. 5. The addition of EGTA after incubation of cells with ionophore A23187 resulted in a partial reversal of the inhibition of the effect of adrenaline. 6. Inclusion of Mg2+ (2 mM) in the incubation medium antagonized the inhibitory action of ionophore A23187. This effect was most marked when the ionophore A23187 was added to medium containing Mg2+ before the addition of the cells. 7. The cellular content of Mg2+ was decreased by approx. 50% after 20 min incubation with ionophore A23187 (1.9 muM) in the presence of Ca2+ (1 mM) but no Mg2+. When Mg2+ (2 mM) was also present in the medium, ionophore A23187 caused an increase of approx. 80% in cell Mg2+ content. Ionophore A23187 had no significant effect on cell K+ content. 8. Ionophore A23187 caused a decrease in cell ATP content under some conditions. Since effects on cyclic AMP content could also be shown when ATP was not significanlty lowered, it appeared that a decrease in ATP in the cells could not explain the effect of ionophore A23187 on cyclic AMP. 9. Ionophore A23187 (1.9 muM), with 1 mM-Ca2+, did not enhance cyclic AMP degradation in intact cells, suggesting that the effect of ionophore A23187 on cyclic AMP content was mediated through an inhibition of adenylate cyclase rather than a stimulation of cyclic AMP phosphodiesterase. 10. It was concluded that in intact pigeon erythrocytes adenylate cyclase may be inhibited by intracellular concentrations of Ca2+ in the range 1-10 muM.     

The role of extra- and intracellular calcium in pepsinogen extrusion by isolated gastric glands]

It was found that carbacholine stimulated pepsinogen extrusion by isolated guinea pig stomach glands which were incubated in Ca(2+)-free medium, containing EGTA (0.25 mM). This effect could be imitated by caffeine (10 mM), a specific activator of Ca2+ release from intracellular pools. Extracellular Ca2+ in the concentrations over 0.125 mM increased pepsinogen extrusion which was stimulated by carbacholine. The interdependence between the level of pepsinogen extrusion and Ca2+ concentration in the medium had S-shaped character. La3+ ions (10(-4) mM) inhibited pepsinogen extrusion already in the first minutes after its activation by carbacholine. When testing other cations (Sr2+, Mg2+, Ba2+) it was found that only Sr2+ had some influence on pepsinogen extrusion. Thus, it can be concluded that both intra- and extracellular Ca2+ take part in the activation of pepsinogen extrusion. Obviously the role of extracellular Ca2+ consists in the support of reactivity of stomach glands to the action of stimulators of secretion.     

Modulation of endothelial cell shape by SPARC does not involve chelation of extracellular Ca2+ and Mg2+.

SPARC (secreted protein, acidic and rich in cysteine) is an extracellular, Ca(2+)-binding protein that inhibits the spreading of newly plated cells and elicits a rounded morphology in spread cells. In this study, I investigated whether the rounding effect of SPARC depends on the ability of the protein to chelate Ca2+ at the cell surface. Bovine aortic endothelial cells were plated in the presence of different concentrations of SPARC and Ca2+; control experiments were performed with 1 mM EGTA and with Mg2+. Quantitative estimates of cell rounding were calculated according to a rounding index. SPARC, at concentrations between 0.15 and 0.58 microM, elicited rounding (or prevented spreading) of cells cultured for 16-38 h in 0.5-2.0 mM Ca2+. Addition of 0.5-2.0 mM Mg2+ to cells previously rounded in the presence of SPARC did not abrogate the effect of SPARC. When the levels of extracellular Ca2+ were adjusted with 1 mM EGTA to maximum values ranging from 7.1 to 320 microM, cells displayed a rounded morphology in the presence of exogenous SPARC. Although the rounding induced by 1 mM EGTA was essentially reversed by the inclusion of 2 mM Ca2+, cultures containing these reagents together with SPARC maintained the rounded phenotype. These results do not support a mechanism that involves the abstraction of Ca2+ from proteins at the cell surface or the provision of Ca2+ from native extracellular SPARC to cells. Therefore, SPARC does not appear to act as a local chelator of extracellular Ca2+ and Mg2+ and presumably exerts its function as a modulator of cell shape via a different pathway.     

Participation of calcium in flagellar shortening and regeneration in Chlamydomonas reinhardii

Cation chelators cause flagellar shortening in Chlamydomonas reinhardii. Most effective are EDTA and EGTA (1 mM) but pyrophosphate (10 mM) also is effective. Addition of 5 mM Ca2+ after shortening caused by 4 mM EGTA results in flagellar regeneration. Other divalent cations can replace Ca2+ with the following relative activities: Ca2+ greater than Sr2+ = Mn2+ much greater than Ba2+ = Mg2+. Although the specific ion requirement to reverse shortening is not clear, it is possible that all of the ions act by displacing one bound cation, presumably Ca2+. A specific requirement for Ca2+ in flagellar regeneration could be demonstrated, however, because as little as 50 microM EGTA in the presence of 500 microM Mg2+ delayed regeneration and prevented full regeneration. Ca2+ at 100 microM overcame this inhibition.     

Regulation of Cyclic AMP Levels by Calcium in Bovine Adrenal Medullary Cells

Both nicotine and histamine have been reported to increase cyclic AMP levels in chromaffin cells by Ca(2+)-dependent mechanisms. The present study investigated whether Ca2+ was an adequate and sufficient signal for increasing cyclic AMP in cultured bovine adrenal medullary cells. Depolarization with 50 mM K+ caused a two- to three-fold increase in cellular cyclic AMP levels over 5 min, with no change in extracellular cyclic AMP. This response was abolished by omission of extracellular Ca2+ and by 100 microM methoxyverapamil, and was unaffected by 1 microM tetrodotoxin and by 1 mM isobutylmethylxanthine. Veratridine (40 microM) also increased cellular cyclic AMP levels by two- to fourfold. This response was abolished by either methoxyverapamil or tetrodotoxin. The Ca2+ ionophore A23187 (10-50 microM) had little or no effect on cellular cyclic AMP levels. When the concentration of K+ used to depolarize the cells was reduced to 12-15 mM, the catecholamine release was similar to that induced by 50 microM A23187, and the cyclic AMP response was almost abolished. The results suggest that Ca2+ entry into chromaffin cells is a sufficient stimulus for increasing cellular cyclic AMP production. The possible involvement of a Ca2+/calmodulin-dependent isozyme of adenylate cyclase is discussed.     

Activation of potassium channels in erythrocytes of marine teleost Scorpaena porcus

To assess the possibility of stimulating Ca2+-activated K+ channels, marine fish erythrocytes were incubated at 20-22 degrees C in saline containing a Ca2+-ATPase inhibitor (orthovanadate), a Ca2+ ionophore (A23187), propranolol or Pb2+. Incubation of the cells for up to 2 h under control conditions or in the presence of 5 mM NH4VO3 and 1 mM Ca2+ did not affect the intracellular K+ and Na+ concentrations. About 50% cellular K+ was lost from erythrocytes incubated in the presence of 0.01 mM A23187, 1 mM EGTA and 0.4-1.0 mM Ca2+. There was a significant loss of cellular K+ after the addition of 0.05-0.2 mM propranolol to the incubation medium. The stimulatory effect of propranolol on the K+ efflux was independent of external Ca2+. Blockers of Ca2+ transport, verapamil and Co2+, caused only a small decrease in the K+ loss induced by propranolol. The treatment of erythrocytes with 1-2 microM Pb2+ led to a minor K+ loss, but at a Pb2+ concentration of 20-50 microM, about 70% cellular K+ was lost. The K+ efflux induced by propranolol or Pb2+ was completely blocked by 1 mM quinine. The induced K+ loss from the erythrocytes was accompanied by a slight increase in the intracellular Na+ concentration. These data indicate the possibility of inducing Ca2+- and Pb2+-activated potassium channels in erythrocytes of S. porcus. A distinctive feature of the cells is a high sensitivity to propranolol, which activates K+ channels in the absence of external Ca2+.     

Calcium influx is not required for thyrotropin-releasing hormone stimulation of prolactin release from GH3 cells

TRH stimulation of prolactin release from GH3 cells is dependent on Ca2+; however, whether TRH-induced influx of extracellular Ca2+ is required for stimulated secretion remains controversial. We studied prolactin release from cells incubated in medium containing 110 mM K+ and 2 mM EGTA which abolished the electrical and Ca2+ concentration gradients that usually promote Ca2+ influx. TRH caused prolactin release and 45Ca2+ efflux from cells incubated under these conditions. In static incubations, TRH stimulated prolactin secretion from 11.4 +/- 1.2 to 19 +/- 1.8 ng/ml in control incubations and from 3.2 +/- 0.6 to 6.2 +/- 0.8 ng/ml from cells incubated in medium with 120 mM K+ and 2 mM EGTA. We conclude that Ca2+ influx is not required for TRH stimulation of prolactin release from GH3 cells.     

Cellular structure and function of mouse adrenocortical tumor cells Y-1 in the post-treatment state of low Ca2+

Under lowered Ca2+ content and in the post-treatment state of low Ca2+, we studied the cellular structure and functioning in mouse adrenocortical tumor cells, Y-1. These cells had been maintained in Ham F12 medium containing 10% fetal calf serum. The Ca2+ present in this complete medium was 0.39 mM. Under a slightly lowered Ca2+ (0.29 mM) produced by EGTA, the cells had many blebs on their surfaces and specific functional activity decreased in steroidogenesis as did ACTH reactivity. In the post-treatment state of a low Ca2+, the cellular surface was covered with many short microvilli and there was greater cellular activity than in the control cells. When the Ca2+ concentration was below 0.17 mM, the cellular structure and functioning were disturbed, and there was no recovery even at the physiological Ca2+ after the removal of EGTA.     

Cyclic nucleotide-gated channels of rat olfactory receptor cells: divalent cations control the sensitivity to cAMP

cAMP-gated channels were studied in inside-out membrane patches excised from the apical cellular pole of isolated olfactory receptor cells of the rat. In the absence of divalent cations the dose-response curve of activation of patch current by cAMP had a KM of 4.0 microM at -50 mV and of 2.5 microM at +50 mV. However, addition of 0.2 or 0.5 mM Ca2+ shifted the KM of cAMP reversibly to the higher cAMP concentrations of 33 or 90 microM, respectively, at -50 mV. Among divalent cations, the relative potency for inducing cAMP affinity shifts was: Ca2+ > Sr2+ > Mn2+ > Ba2+ > Mg2+, of which Mg2+ (up to 3 mM) did not shift the KM at all. This potency sequence corresponds closely to that required for the activation of calmodulin. However, the Ca(2+)-sensitivity is lower than expected for a calmodulin-mediated action. Brief (60 s) transient exposure to 3 mM Mg2+, in the absence of other divalent cations, had a protective effect in that following washout of Mg2+, subsequent exposure to 0.2 mM Ca2+ no longer caused affinity shifts. This protection effect did not occur in intact cells and was probably a consequence of patch excision, possibly representing ablation of a regulatory protein from the channel cyclic nucleotide binding site. Thus, the binding of divalent cations, probably via a regulatory protein, controls the sensitivity of the cAMP-gated channels to cAMP. The influx of Ca2+ through these channels during the odorant response may rise to a sufficiently high concentration at the intracellular membrane surface to contribute to the desensitization of the odorant- induced response. The results also indicate that divalent cation effects on cyclic nucleotide-gated channels may depend on the sequence of pre-exposure to other divalent cations.     

Role of ions in activation of human lymphocytes

Phytohemagglutinin (PHA)-stimulated lymphocytes were cultured in media containing varying levels of K+, Mg2+, Ca2+. Cell activation was monitored by measuring nuclear diameter and by evaluating the area of nucleolus which reacted with silver nitrate. Decreasing extracellular K+ from normal levels (5.0 mM) to 14% (0.7 mM) and decreasing extracellular Mg2+ from normal levels (1.0 mM) to 14% (0.14 mM) did not affect nuclear diameter or silver nitrate reactivity of PHA-stimulated lymphocytes. Chelation of extracellular Ca2+ with EGTA during the first 24 h after PHA stimulation completely inhibited the increases in silver reactivity and nuclear diameter associated with stimulation. Chelation of extracellular Ca2+ 48 h after PHA stimulation did not inhibit lymphocyte stimulation. Inhibitory effects of EGTA were completely reversed if CaCl2 was added to the medium within 24 h of PHA stimulation. By 48 h the effects were irreversible.     

Characterization of rat kidney proximal tubule brush border membrane-associated phosphatidylinositol phosphodiesterase

Isolated rat kidney proximal tubule brush border membrane vesicles exhibit an increase in diacylglycerol levels (20- to 30-fold) and a concomitant decrease in phosphatidylinositol when incubated with [3H]arachidonate-labeled lipids, Ca2+, and deoxycholate. Levels of free arachidonate, triglyceride, and noninositol phospholipids are not altered. These results suggest phosphatidylinositol phosphodiesterase activity is associated with rat proximal tubule brush border membrane. Presence of both deoxycholate and certain divalent cations was necessary to demonstrate enzyme activity. Optimum pH ranged from 7.0 to 8.5. Ca2+, Mg2+, and Mn2+ stimulated diglyceride production while Ba2+, Zn2+, Hg2+, and K+ were ineffective. HgCl2 inhibited Ca2+-stimulated phosphatidylinositol phosphodiesterase. Mg2+ and deoxycholate-dependent enzyme activity was shown to be phosphatidylinositol specific. Sodium lauryl sulfate, tetradecyltrimethylammonium bromide, and Triton X-100 did not activate phosphatidylinositol phosphodiesterase in the presence of Ca2+. In combination with deoxycholate, diglyceride formation was not affected by sodium lauryl sulfate, partially inhibited by Triton X-100, and completely abolished by tetradecyltrimethylammonium bromide. Diglyceride kinase activity was not found associated with brush border membrane phosphatidylinositol phosphodiesterase. ATP (1-5 mM) inhibited Ca2+- or Mg2+-stimulated, deoxycholate-dependent phosphatidylinositol hydrolysis by chelating the required divalent cation.     

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.     

Involvement of cellular calcium incorporated from the medium in production of inositol trisphosphate in A-431 cells

The property of intensive 45Ca2+ uptake by A-431 human epidermoidal carcinoma cells was indicated to be an influx, not binding to the cell surface, since the two apparent dissociation constants (Kd) between 45Ca2+ and cells were almost the same when measured in either the presence or absence of 1 mM [ethylenebis (oxyethylenenitrilo)]tetraacetic acid (EGTA); these constants were approximately 5-10 x 10(-6) and 1 x 10(-4) M, respectively, which are much higher than the chelating constant of EGTA for Ca2+ (approximately 10(-11) M). Furthermore, addition of A23187, a calcium ionophore, rapidly released the 45Ca2+ incorporated into cells at both 37 degrees C and 0 degrees C. The 45Ca2+ associated with the cells was slowly released or exchanged when cells were incubated in medium depleted of Ca2+, or in that containing 1 mM non-radioactive Ca2+. The ability of A-431 cells to respond to extracellular ATP by elevating their level of intracellular calcium ions, as well as by producing inositol trisphosphate (InsP3), was suppressed in cells depleted of cellular calcium. These data suggest that calcium ions are extensively incorporated or exchanged with those outside the cells, maintained as stored calcium, and involved in production of InsP3, when A-431 cells are stimulated by ATP to trigger the signal transduction system.     

Differences in the association of calmodulin with cyclic nucleotide phosphodiesterase in relaxed and contracted arterial strips

Changes in the concentration of cytosolic Ca2+ are assumed to alter the activity of Ca2+-calmodulin-sensitive cyclic nucleotide phosphodiesterase in intact cells. However, this assumption is based on indirect evidence and by analogy from studies of enzyme activities in broken cell systems. We have developed a procedure for estimating the fraction of Ca2+-calmodulin-sensitive phosphodiesterase that is in an activated, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) sensitive state in intact porcine coronary artery strips. The experimental approach involves homogenization of the strips and assay of cyclic guanosine monophosphate (cyclic GMP) phosphodiesterase activity under conditions that retard changes in the amount of the complex Ca2+-calmodulin-phosphodiesterase. Our findings indicate that cyclic GMP phosphodiesterase in intact coronary artery strips does associate with Ca2+-calmodulin and that interventions that change the concentration of Ca2+ in the cytosol of the intact strip change the extent of this functional association. Exposure to histamine (10 or 100 microM) or 50 mM KCl caused contraction and an increase in EGTA-sensitive cyclic GMP phosphodiesterase activity. Isoproterenol-induced relaxation of tissues that had been caused to contract with 10 microM histamine was accompanied by a reduction in EGTA-sensitive cyclic GMP phosphodiesterase activity to the same level as that present before contraction was initiated.     

The effect of divalent cations on bovine spermatozoal adenylate cyclase activity.

The effect of divalent cations on bovine sperm adenylate cyclase activity was studied. Mn2+, Co2+, Cd2+, Zn2+, Mg2+ and Ca2+ were found to satisfy the divalent cation requirement for catalysis of the bovine sperm adenylate cyclase. These divalent cations in excess of the amount necessary for the formation of the metal-ATP substrate complex were found to stimulate the enzyme activity to various degrees. The magnitude of stimulation at saturating concentrations of the divalent cations was strikingly greater with M2+ than with either Ca2+, Mg2+, Zn2+, Cd2+ or Co2+. The apparent Km was lowest for Zm2+ (0.1 - 0.2 mM) than for any of the other divalent cations tested (1.2 - 2.3 mM). The enzyme stimulation by Mn2+ was decreased by the simultaneous addition of Co2+, Cd2+, Ni2+ and particularly Zn2+ and Cu2+. The antagonism between Mn2+ and Cu2+ or Zn2+ appeared to have both competitive and non-competitive features. The inhibitory effect of Cu2+ on Mn2+-stimulated adenylate cyclase activity was prevented by 2,3-dimercaptopropanol, but not by dithiothreitol, L-ergothioneine, EDTA, EGTA or D-penicillamine. Ca2+ at concentrations of 1-5 mM was found to act synergistically with Mg2+, Zn2+, Co2+ and Mn2+ in stimulating sperm adenylate cyclase activity. The Ca2+ augmentation of the stimulatory effect of Zn2+, Co2+, Mg2+ and Mn2+ appeared to be specific.     

Studies on cyclic nucleotide metabolism in Tetrahymena pyriformis: partial characterization of cyclic AMP- and cyclic GMP-dependent phosphodiesterases

Cyclic nucleotide phosphodiesterase [EC 3.1.4.17] was examined in tetrahymena pyriformis strain NT-1. Enzymic activity was associated with the soluble and the particulate fractions, whereas most of the cyclic GMP phosphodiesterase activity was localized in the soluble fraction; the activities were optimal at pH 8.0-9.0. Although very low activities were detected in the absence of divalent cations, they were significantly increased by the addition of either Mg2+ or Mn2+. A kinetic analysis of the properties of the enzymes yielded 2 apparent K(m) values ranging in concentration from 0.5 to 50 micron and from 0.1 to 62 micron for cyclic AMP and GMP, respectively. A Ca2+ -dependent activating factor for cyclic nucleotide phosphodiesterase was extracted from Tetrahymena cells, but this factor did not stimulate guanylate cyclase [EC 4.6.1.2] activity in this organism. On the other hand, tetrahymena also contained a protein activator which stimulated guanylate cyclase in the presence of Ca2+, although this activator did not stimulate the phosphodiesterase. The results suggested that Tetrahymena might contain 2 types of Ca2+ -dependent activators, one specific for phosphodiesterase and the other for guanylate cyclase.     

Alterations of extracellular calcium elicit selective modes of cell death and protease activation in SH-SY5Y human neuroblastoma cells

The role of intracellular Ca2+ homeostasis in mechanisms of neuronal cell death and cysteine protease activation was investigated in SH-SY5Y human neuroblastoma cells. Cells were incubated in 2 mM EGTA to lower intracellular Ca2+ or 5 mM CaCl2 to raise it. Cell death and activation of calpain and caspase-3 were measured. Both EGTA and excess CaCl2 elicited cell death. EGTA induced DNA laddering and an increase in caspase-3-like, but not calpain, activity. Pan-caspase inhibitors protected against EGTA-, but not CaCl2-, induced cell death. Conversely, excess Ca2+ elicited necrosis and activated calpain but not caspase-3. Calpain inhibitors did not preserve cell viability. Ca2+ was the death-mediating factor, because restoration of extracellular Ca2+ protected against cell death induced by EGTA and blockade of Ca2+ channels by Ni2+ protected against that induced by high Ca2+. We conclude that the EGTA treatment lowered intracellular Ca2+ and elicited caspase-3-like protease activity, which led to apoptosis. Conversely, excess extracellular Ca2+ entered Ca2+ channels and increased intracellular Ca2+ leading to calpain activation and necrosis. The mode of cell death and protease activation in response to changing Ca2+ were selective and mutually exclusive, demonstrating that these are useful models to individually investigate apoptosis and necrosis.     

Characterization of basal and methylxanthine-stimulated Ca2+ transport in abalone spermatozoa

Methylxanthines, such as 1-methyl-3-isobutylxanthine (MIX) and theophylline, stimulate abalone sperm 45Ca2+ uptake in a time- and concentration-dependent manner. MIX is the most potent compound tested, and the ability of these compounds to alter 45Ca2+ uptake resides with methyl or isobutyl substitution of the xanthine nucleus at multiple sites. Methylxanthine-stimulated 45Ca2+ uptake does not occur as a secondary consequence of cyclic nucleotide phosphodiesterase inhibition, and added cyclic nucleotides are also without effect. The dramatic elevation of intracellular cAMP concentrations and induction of the acrosome reaction of sperm incubated with methylxanthines in the presence of Ca2+ is mediated by a primary effect of methylxanthines on Ca2+ transport. Basal 45Ca2+ uptake occurs through a verapamil-insensitive site that obeys the properties of a simple diffusion-mediated process. MIX-stimulated 45Ca2+ uptake occurs through a carrier-mediated transport site that has low affinity for Ca2+ (Km = 19.9 mM) and is verapamil sensitive. 45Ca2+ uptake through both basal and MIX-stimulated sites is enhanced by low extracellular Na+ concentrations (less than or equal to 15 mM) and is not affected by either extracellular Mg2+ or K+ X 45Ca2+ uptake through both sites is pH sensitive, but this sensitivity is different for each site. These data suggest that methylxanthines can affect sperm function via primary effects on Ca2+ transport, which occur through a specific carrier-mediated site(s). It is possible that many of the previously described effects of methylxanthines on sperm function are mediated via such changes in Ca2+ conductance.