Effect of Ca2+ channel antagonists on the acrosome reaction of guinea pig and golden hamster spermatozoa

The effects of Ca²⁺ channel antagonists on the motility and acrosome reaction of guinea pig spermatozoa were examined by incubating the spermatozoa continuously in Ca²⁺-containing capacitating media with 10^?6 M to 10^?4 M antagonist. Antagonists tested were four voltage-gated Ca²⁺ channel antagonists (verapamil, nifedipine, nimodipine, and FR–34235) and two ligand-gated channel antagonists (NaNO₂ and Na-nitroprusside). None of these antagonists could block the acrosome reaction. Instead, three antagonists (verapamil, nimodipine, and FR-34235, each at 10^?4 M) accelerated the onset of the acrosome reaction with a subsequent decrease in sperm motility. Nifedipine and Na-nitroprusside at the same concentration caused a complete loss of sperm motility by 4 hr of incubation with no substantial effect on the rate of acrosome reaction. The detrimental effect of antagonists on the motility of spermatozoa appears to be due to a direct, Ca²⁺-independent, membrane-perturbing action of the reagents. The acrosome reaction was not inhibited when guinea pig spermatozoa were precapacitated in Ca²⁺-free medium (with a low concentration of lysolecithin) in the continuous presence of antagonists. An acceleration of the onset of the acrosome reaction by verapamil (10^?4 M) was also demonstrated in the golden hamster. These results may be interpreted as indicating that the entry of extracellular Ca²⁺ into spermatozoa, which triggers the acrosome reaction of guinea pig and hamster spermatozoa, is not mediated by Ca²⁺ channels. This is in marked contrast with the case reported in invertebrate spermatozoa. Possible mechanisms by which some of the antagonists stimulate the acrosome reaction and affect the motility of mammalian spermatozoa are discussed.     

Cationic probes: Specificity of distribution of metal-binding sites in bovine sperm

Salts of transition elements that alter the rate of sperm cell movement act at or near calcium-binding sites. After living bull sperm cells had been preincubated in VO₄^3?, Ni²⁺, Zn²⁺, Mn²⁺, and also La³⁺, they were then fixed. Crisply defined organelles and the absence of particulate deposits in the morphological controls contrasted sharply with the treated specimens; the latter contained regions of increased electron density, the nature and distribution of which depended on the test substance, reflecting the differential affinities of the specific ions. La³⁺ formed fine dense areas, mainly at the exocytic surface of the plasma membrane. VO₄^3? marks the cell surface but also left particulate densities within the cell. Ni²⁺ caused a nearly uniformly dense deposit at the surface and on the satellite fibers and axonemal microtubules. Zn²⁺ formed less uniform but coarser deposits, while in Mn²⁺ the distribution was similar to that in Zn²⁺ but much denser in the axonemal matrix and on the satellite fibers. Verapamil restricted the size and number of the opacities, while procaine permitted a similar distribution of slightly larger size reaction product. The differences in size and distribution of the enhanced densities were consistent and replicable for the individual assay substances. Vanadate, which specifically inhibits Na, K-ATPase, bound to ouabain-sensitive enzyme loci, however, completely disrupting the axonemal complex. This suggests that an important role of dynein in flagellar motion may relate to intracellular transport of Ca²⁺.     

Altered growth kinetics precede calcium-induced differentiation in mouse epidermal cells

Summary Primary cultures of newborn mouse epidermal cells proliferate rapidly and with a high growth fraction for several months when grown in medium with low calcium (0.02 to 0.1 mM). Addition of calcium to levels generally used in culture medium (1.2 mM) was followed by rapid changes in the pattern of proliferation. By using a combination of technics (a stathmokinetic method, autoradiography, [³H]thymidine incorporation into DNA, DNA flow cytometry) it was found that cell flux was blocked for 5 to 6 h, followed by a short rise in the mitotic rate at 10 h, and a gradual fall in all growth parameters until about 32 h after the calcium switch. There was no accumulation of cells in any particular cell cycle phase. The results indicate that the calcium switch is followed by a strong reduction in cell flux from G₁ whereas the majority of the cells that had left G₁ at the time of the switch completed one cell division before cessation of all proliferative activity. Both before and after the switch the primary epidermal cultures consisted of one diploid and one tetraploid G₁ DNA stemline that seemed to react in the same way to calcium. This work reported in this paper was undertaken during the tenure of an American Cancer Society-Eleanor Roosevelt-International Cancer Fellowship awarded by the International Union Against Cancer (K. E.). The project was supported by funds partly provided by the International Cancer Research Data Bank Program of the National Cancer Institute, National Institutes of Health, Bethesda, MD, under contract N01-C0-65341 (International Cancer Research Technology Transfer) and partly by the International Union Against Cancer (O.P.F.C.).     

Mechanism of inhibition of net ion transport across frog corneal epithelium by calcium channel antagonists

Summary In the isolated bullfrog cornea, three calcium channel antagonists had dose-dependent inhibitory effects on the Cl-originated short-circuit current (SCC). Their order of decreasing potency was bepridil, verapamil and diltiazem. One millimolar diltiazem inhibited the SCC by 98% and subsequent incubation with the calcium ionophore A23187 had no restorative effect. Increasing the bathing solution Ca concentration from 0.05 to 15mm, however, decreased diltiazem's inhibitory efficacy. This antagonist depolarized the intracellular potential differenceV _m from –54 to –18 mV (tear: reference) and the voltage divider ratioFR ₀ decreased from 0.58 to 0.30, suggesting an increase in basolateral membrane electrical resistance. Additional indication of a basolateral membrane effect by the drug was that preincubation with 10⁵ m amphotericin B in Cl-free Ringer's did not eliminate the inhibitory effect of the drug on the Na- and K-elicited SCC. In the absence of amphotericin B in Cl-free Ringer's (SCC=0), 1 ×10³ m diltiazem depolarized theV _m from –78 to –9 mV suggesting that the increase in basolateral membrane resistance was due to K channel blockade. Diltiazem (1×10³ m) significantly decreased cyclic AMP content; however, isoproterenol in the presence of the drug increased cyclic AMP fourfold without having any restorative effect on the inhibited SCC. Therefore, the inhibition of the Cl-originated SCC resulting from an increase in basolateral membrane K resistance is not caused by a decline in cyclic AMP content. In plasma membrane-enriched fractions prepared from broken cell preparations of bovine corneal epithelium, 1×10³ m diltiazem had no inhibitory effects on either Na,K-ATPase or Ca,Mg-ATPase activities. These latter effects further point to the selectivity of diltiazem as an inhibitor of K-channel activity, but do not preclude a Ca-channel blocker effect by the drug in the micromolar range.     

Effects of saponin extracts from Solanum elaeagnifotium fruits on ion uptake by clover seedlings

Solanum elaeagnifolium Cav. fruits contain high concentrations of steroidal saponins. Treatment of 3-day-old clover seedlings with aqueous fruit extracts modified Ca²⁺ uptake without significantly altering K⁺ and H₂PO₄⁻ uptake. The extracts increased Ca²⁺ uptake in the concentration range of 0.2 to 20 m M Ca²⁺. Uptake curves could be represented by two phases. In the lower phase (0.2-1.0 m M Ca²⁺), this change could be related to an increase in V_max. Pretreatment of seedlings with saponin extracts significantly reduced ATP-dependent Ca²⁺ uptake and Ca²⁺-dependent ATPase activity in a fraction isolated from root homogenates by centrifugation at 1500 g for 15 min. Saponins purified from S. eleagnifolium extracts by thin-layer chromatography modified in vitro the Ca²⁺-ATPase activity of this fraction, indicating that the steroid may act directly on Ca²⁺ transport across membranes.     

Central actions of calcitonin on body temperature and intestinal motility in rats: evidence for different mediations

The effects of intracerebroventricular (i.c.v.) administration of calcitonin and PGE2 on intestinal motility and body temperature were examined in conscious rats chronically fitted with intraparietal electrodes in the small intestine, a cannula in a cerebral lateral ventricle and a subcutaneous thermistor probe. Both calcitonin and PGE2 restored the fasted pattern of intestinal motility in fed rats and induced an increase in body temperature. Indomethacin, an inhibitor of the cyclooxygenase with calcium antagonistic properties, and TMB-8, an intracellular calcium antagonist, blocked the effects of calcitonin on intestinal motility and body temperature. Piroxicam, an inhibitor of the cyclooxygenase which does not affect calcium uptake blocked the thermic but not the intestinal effects of calcitonin. TMB-8 but not indomethacin or piroxicam partially blocked the effects of PGE2 on both intestinal motility and body temperature. It is concluded that the central hyperthermic effect of calcitonin is mediated through the formation and the release of prostaglandins whereas the central action of calcitonin on digestive motility results from intracerebral effects on calcium fluxes.     

Stimulatory effects of human pancreatic polypeptide on rat pancreatic acini

The effect of human pancreatic polypeptide (HPP) on rat pancreatic acini has been studied. It was found that HPP stimulated amylase and lipase release from the acini. The secretory response of acini to HPP was dose-dependent in a sigmoidal fashion. Between 10(-9) M and 10(-8) M concentration of HPP there was a slow increase of enzyme release to about 40-60% over basal release. At concentrations of HPP above 10(-8) M there was a rapid increase of enzyme release, amounting to 4-6 times over basal release at 10(-6) M concentration of HPP. The potency of HPP compared to other secretagogues at 10(-7) M concentration was 45% of CCK, 60% of carbachol and 75% of secretin. HPP did not inhibit the effect of CCK, secretin and carbachol on amylase release. The amylase release stimulated by HPP was accompanied by an increase in 45Ca2+ efflux. Atropine or dibutyryl cyclic GMP did not influence the effect of HPP. It is concluded that HPP stimulates the release of enzymes from rat pancreatic acini and that Ca2+ may be a mediator for this secretion.     

Computer search of calcium binding sites in a gene data bank: use of learning techniques to build an expert system

Using a learning set of 28 sequences able to bind calcium (each sequence is 12 residues long), we have built two filters by learning on this set. The first filter uses a pattern-matching technique and the second one takes into account the environment of amino-acids. These two filters have been used to find new calcium-binding proteins in a data bank. The results are discussed.