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Sperm nucleus decondensing activity (NDA) of eggs has been investigated using the gametes of the sea urchin, Lytechinus variegatus. To assay for NDA, isolated sperm heads were exposed to homogenate from mature unfertilized ova and then examined with phase-contrast optics. NDA is retained by dialysis and requires Ca++ at the time of homogenization as well as at the time of assay. Other divalent cations (Mg++, Sr++, Ba++, Mn++) can substitute for Ca++ but are much less effective. NDA is stable to cold (-20 degrees C, -76 degrees C) but not to heat (56 degrees C). The pH activity optimum is in the range 7-9. NDA is present in the supernatant after centrifugation at 150,000 g for 105 minutes. It elutes shortly following the void volume on a Sephacryl S-200 column, with an approximate M.W. of 100,000 daltons.

Reintroduction of Ca ions to cat adrenal glands perfused at room temperature with Krebs solution lacking Ca and Mg, evoked a catecholamine secretory response that was directly proportional to the concentration of Ca reintroduced. This secretory response inactivated quickly, was abolished by nM concentrations of nifedipine and was potentiated dramatically by nM concentrations of BAY-K-8644. Excess Ca antagonized the inhibitory effects of nifedipine and this drug inhibited competitively the potentiating effects of BAY-K-8644. These data suggest 1st) that extracellular divalent cations deprivation activates specific Ca channels; 2nd) that the dihydropyridine BAY-K-8644 increases the release of catecholamines by Ca reintroduction by activating and/or delaying the inactivation of Ca channels; and 3rd) that the access of the dihydropyridine-type Ca agonist and antagonists to their "intra-channel" site of action requires the pre-activation of Ca channels.

Previous studies from this laboratory described myocardial depression in an arterially perfused rabbit interventricular septum following perfusion with acute septic plasma. Calcium is critical for maintenance of cardiac contractility on a beat-to-beat basis. We have investigated calcium flux in the septal tissue to determine whether altered calcium flux explains the impaired cardiac function during sepsis. Twenty-two rabbit septa were perfused with control and septic perfusate (cryo-precipitated plasma + RBCs) and calcium flux determined in seven experiments. Perfusate cations (Ca++, Na+, K+, and H+) were measured, tissue function and arterial pressure were monitored. Developed tension decreased 46%, acceleration of tension change fell 42%, and arterial pressure decreased 26%, all highly significant. All septa recovered after return to control perfusate. The septic perfusate Ca++ was significantly lower than control perfusate, while K+ and H+ were significantly elevated. Ion flux studies, however, could not demonstrate altered calcium flux associated with the depressed contractility.

Apamin is a bee venom neurotoxin of 18 amino-acids containing two disulfide bridges. Current clamp and voltage clamp experiments have shown that externally applied apamin blocks specifically at low concentration (0.1 microM) the Ca2+-dependent slow K+ conductance which mediates the long-lasting after-hyperpolarization in neuroblastoma cells and rat muscle cells in culture. The apamin-sensitive Ca2+-dependent slow K+ conductance is voltage-dependent and tetraethylammonium (TEA) insensitive. It is distinct from the high conductance Ca2+-dependent K+ channel revealed by patch clamp experiments. Biochemical characterization of the apamin receptor in rat striated muscle, neuroblastoma cells, rat synaptosomes, smooth muscles and hepatocytes was carried out with the use of a radiolabelled monoiodo-apamin derivative (125I-apamin) of high specific radioactivity (2 000 Ci/mmol). The dissociation constant of the apamin-receptor complex is between 15 and 60 pM for all tissue preparations. The density of binding sites is very low; it varied between 1 and 40 fmol/mg of protein. Radiation inactivation analysis indicates a molecular weight for the apamin receptor of 250 000 daltons whereas affinity labelling with 125I-apamin results in covalent labelling of a single polypeptide chain with a molecular weight of about 30 000 daltons. We conclude that the apamin-sensitive Ca2+-dependent K+ channel is probably a large oligomeric structure containing one subunit of 30 000 daltons.

The role of Na+ and Ca2+ as well as the influence of other metal ions on the lipase action were studied with the aid of immobilisation techniques. By this method it is possible to distinguish between the action of these ions on the lipase molecule itself and their action on the substrate or product. It could be shown that both alkali and alkali earth metal ions, especially Na+, Ca2+ and Mg2+ stabilize active states of the enzyme which were detected by immobilization to controlled pore glass beads. Though Na+ as well as Ca2+ and Mg2+ stabilize the enzyme significantly, they differ in their efficacy. Reasons for this are discussed and the data compared to the findings of other authors who performed their studies with the native enzyme.

The electrophysiological properties of the cell membrane of rat spermatogenic cells were studied using the whole-cell variation of the patch-electrode voltage-clamp technique. In late primary spermatocytes and early spermatids isolated from adult testis, a transient inward current followed by a slowly developing outward current was produced when the membrane potential was made more positive than -60 mV. Early spermatogenic cells which consist of spermatogonia and early spermatocytes were isolated either from new-born rats (12-14 days old) of from adult cryptorchid rats 15-21 days after the operation. In early spermatogenic cells, some showed a slowly developing outward current with negligible initial inward current, while others showed a recognizable inward current followed by the slowly developing outward current. The inward currents are identified as Ca2+-carried current, since replacement of external Ca2+ with Mn2+ reversibly diminished the current whereas Ba2+ or Sr2+ substituted for Ca2+. The reversal potential of the outward current changed from -65 to -12 mV when [K+]o was raised from 5 to 100 mM. The outward current was independent of [Ca2+]o and was blocked by tetraethylammonium chloride. Thus the current was identified as membrane-potential-dependent K+ current. During spermatogenesis from spermatogonia to early spermatids, the density of Ca2+ current increased while the K+ current density decreased significantly.

Membrane potentials of mouse parathyroid cells were measured by means of the intracellular microelectrode method. The membrane potential in external Krebs solution containing 2.5 mM of Ca++ was -23.6 +/- 0.4 mV (mean +/- standard error of mean). The low concentration of Ca++ (1.0 mM) caused hyperpolarization of the membrane potential to -61.7 +/- 0.8 mV. The membrane potential was proportional to the logarithm of the concentration of K ion in the solution of low Ca ion. The concentration of external Na+, C1- and HPO4-- had no effect on the membrane potential. The sigmoidal transition of membrane potentials was induced by the change of Ca ion concentration in the range from 2.5 to 1.0 mM. The change of the membrane potentials in low Ca ion is originated from increase in potassium permeability of the cell membrane. The similar sigmoidal changes of the membrane potentials were observed in the solution containing 4 to 3 mM of Sr ion. The Mg and Ba ion showed smaller effect on the membrane potential. The Goldman equation was extended to divalent ions. Appling the extended membrane potential equation, ratios of the permeability coefficients were obtained as follows: PK/PCa = 0.067 for 2.5 mM Ca++, 0.33 for 1.0 mM Ca++; PK/PSr = 0.08 for 4 mM Sr++ and 0.4 for 3 mM Sr++; PK/PMg = 0.5; PK/PBa = 0.67 for all range of concentration. The Hill constants of Sr ion and Ca ion were 20; the relationship between Sr ion and Ca ion was competitive. The Hill constants of Mg and Ba ion were 1 each. The Hill constant of Ca ion was depend of the temperature; nmax = 20 at 36 degrees C, n = 9 at 27 degrees C, n = 2 at 22 degrees C. The enthalpy of Ca-binding reaction was obtained from the Van't Hoff plot as 0.58 kcal. The activation energies of the K+ permeability increase were obtained from the Arrhenius plots as 3.3 kcal and 4 kcal. The difference, 0.7 kcal, corresponds to the enthalpy change of this reaction, of which value is close to that of the Ca-binding reaction.