calcimycin and cobaltous-chloride

The possible mode of action of an inhibitory neuropeptide SchistoFLRFamide (PDVDHVFLRFamide) on locust oviduct visceral muscle was studied by examining its ability to inhibit calcium ionophore A23187-, caffeine- or phorbol ester-induced oviduct contractions. A23187-induced muscle contractions included two components: one which required the influx of extracellular Ca2+ through Ca2+ channels (blocked by cobalt ions), the other not blocked by cobalt ions. SchistoFLRFamide inhibited the cobalt-sensitive components, but not the latter. Caffeine induced two phases of contractions, a phasic contraction which was probably due to the release of intracellular Ca2+ and a tonic contraction which required the influx of extracellular Ca2+. SchistoFLRFamide inhibited the tonic contraction in a dose-dependent manner but did not influence the phasic contraction. The phorbol 12-myristate 13-acetate-induced muscle contraction required the presence of extracellular Ca2+, with Ca2+ probably entering through ligand-gated channels, but the contraction was not inhibited by SchistoFLRFamide. Based upon these results, it appears that SchistoFLRFamide inhibits muscle contraction by preventing the accumulation of free intracellular Ca2+ from extracellular stores. SchistoFLRFamide is incapable of inhibiting contraction induced by the release of intracellular stores of Ca2+. It is possible that SchistoFLRFamide closes or blocks voltage-gated and some ligand-gated Ca2+ channels in the plasma membrane.

Topics: Amino Acid Sequence; Animals; Caffeine; Calcimycin; Calcium; Calcium Channels; Cobalt; Depression, Chemical; Dose-Response Relationship, Drug; Drug Interactions; Female; Grasshoppers; Ion Channel Gating; Molecular Sequence Data; Muscle Contraction; Muscle, Smooth; Neuropeptides; Nifedipine; Oviducts; Tetradecanoylphorbol Acetate

Pineal arylalkylamine N-acetyltransferase (N-acetyltransferase) controls large daily changes in melatonin production. It is generally thought that the activity of this enzyme is controlled by norepinephrine acting exclusively via elevation of cyclic AMP. However, norepinephrine also elevates pineal intracellular Ca2+ concentration ([Ca2+]i), and it is not known whether Ca2+ is involved in regulating N-acetyltransferase activity other than through its established role in cyclic AMP production. In this study, the issue of whether Ca2+ enhances the effects of cyclic AMP on N-acetyltransferase activity was investigated. The effects of cyclic AMP protagonists (isobutylmethylxanthine, N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate, 8-bromoadenosine 3',5'-cyclic monophosphate, and adenosine 3',5'-cyclic monophosphothioate, Sp-diastereomer) were examined in combination with [Ca2+]i protagonists (A23187, ionomycin, and phenylephrine). All [Ca2+]i protagonists potentiated the effects of cyclic AMP protagonists. For example, ionomycin potentiated the effects of low concentrations of 8-bromoadenosine 3',5'-cyclic monophosphate, and A23187 potentiated the effects of isobutylmethylxanthine without altering cyclic AMP accumulation. These findings indicate that Ca2+ and cyclic AMP probably act physiologically in a coordinated manner to stimulate N-acetyltransferase activity; these second messengers could act directly at one or more sites or through indirect actions mediated by kinases.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Arylamine N-Acetyltransferase; Bucladesine; Calcimycin; Calcium; Cells, Cultured; Cobalt; Cyclic AMP; Drug Synergism; Female; Norepinephrine; Organ Culture Techniques; Phenylephrine; Pineal Gland; Rats; Rats, Sprague-Dawley

LaATP is shown to be an effective inhibitor of the calcium ATPase of sarcoplasmic reticulum because the binding of LaATP to cE.Ca2 results in the formation of lanthanum phosphoenzyme, which decays slowly. Steady-state activity of the calcium ATPase in leaky sarcoplasmic reticulum vesicles is inhibited 50% by 0.16 microM LaCl3 (15 nM free La3+, 21 nM LaATP) in the presence of 25 microM Ca2+ and 49 microM MgATP (5 mM MgSO4, 100 mM KCl, 40 mM 4-morpholinepropanesulfonic acid, pH 7.0, 25 degrees C). However, 50% inhibition of the uptake of 45Ca and phosphorylation by [gamma-32P]ATP in a single turnover experiment requires 100 microM LaCl3 (28 microM free La3+) in the presence of 25 microM Ca2+; this inhibition is reversed by calcium but inhibition of steady-state turnover is not. Therefore, binding of La3+ to the cytoplasmic calcium transport site is not responsible for the inhibition of steady-state ATPase activity. The addition of 6.7 microM LaCl3 (1.1 microM free La3+) has no effect on the rate of dephosphorylation of phosphoenzyme formed from MgATP and enzyme in leaky vesicles, while 6.7 mM CaCl2 slows the rate of phosphoenzyme hydrolysis as expected; 6.7 microM LaCl3 and 6.7 mM CaCl2 cause 95 and 98% inhibition of steady-state ATPase activity, respectively. This shows that inhibition of ATPase activity in the steady state is not caused by binding of La3+ to the intravesicular calcium transport site of the phosphoenzyme. Inhibition of ATPase activity by 2 microM LaCl3 (0.16 microM free La3+, 0.31 microM LaATP) requires greater than 5 s, which corresponds to approximately 50 turnovers, to reach a steady-state level of greater than or equal to 80% inhibition. Inhibition by La3+ is fully reversed by the addition of 0.55 mM CaCl2 and 0.50 mM EGTA; this reactivation is slow with t1/2 approximately 9 s. Two forms of phosphoenzyme are present in reactions that are partially inhibited by La3+: phosphoenzyme with Mg2+ at the catalytic site and phosphoenzyme with La3+ at the catalytic site, which undergo hydrolysis with observed rate constants of greater than 4 and 0.05 s-1, respectively. We conclude, therefore, that La3+ inhibits steady-state ATPase activity under these conditions by replacing Mg2+ as the catalytic ion for phosphoryl transfer. The slow development of inhibition corresponds to the accumulation of lanthanum phosphoenzyme. Initially, most of the enzyme catalyzes MgATP hydrolysis, but the fraction of enzyme with La3+ bound to the catalytic site gradua

Topics: Adenosine Triphosphate; Animals; Calcimycin; Calcium Chloride; Calcium-Transporting ATPases; Cobalt; Egtazic Acid; Kinetics; Lanthanum; Magnesium; Muscles; Phosphorylation; Protein Binding; Rabbits; Sarcoplasmic Reticulum

This report describes a series of studies on the regulation of teleocalcin secretion by primary cultures of rainbow trout corpuscles of Stannius, endocrine glands believed to be unique to bony fishes. Teleocalcin release by these cultured cells was stimulated specifically by calcium in a dose-related fashion. Magnesium did not mimic the effects of added calcium and varying the osmotic pressure had no effect on hormone release. The addition of either ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or cobalt chloride blocked the stimulatory effects of added calcium, whereas the calcium ionophore A23187 mimicked the effects of calcium on hormone release. Mammalian and piscine pituitary hormones (prolactin, growth hormone and gonadotrophic hormone) had no effect on teleocalcin secretion. Inconclusive results were obtained with the calcium channel blockers, verapamil and nifedipine. The results are discussed in relation to calcium-regulated secretion of calcitonin and parathyroid hormone, as well as the known physiological effects of teleocalcin in fish.

Topics: Animals; Calcimycin; Calcium; Cells, Cultured; Cobalt; Culture Media; Egtazic Acid; Endocrine Glands; Glycoproteins; Hormones; Nifedipine; Osmotic Pressure; Pituitary Hormones; Salmonidae; Trout; Verapamil

Some of the mechanisms underlying intestinal glucagon-like immunoreactive (GLI) peptide secretion from cultured fetal rat intestinal cells were investigated using modulators of the adenylate cyclase pathway [(Bu)2cAMP, theophylline, isobutylmethylxanthine], calcium fluxes (ionomycin, A23187), and protein kinase-C (phorbol ester). All of these agents were found to stimulate GLI peptide release, to 120-230% of paired control values (P less than 0.05-0.001). (Bu)2cAMP, but not the phorbol ester, also increased the total cell content of GLI peptides over the 2-h incubation period (P less than 0.05). No synergism between any of the three pathways was detected. When the mol wt distribution of the stored and secreted GLI peptides was determined in control and (Bu)2 cAMP-stimulated samples, 68 +/- 2% of the peptide corresponded to glicentin, while the remainder eluted with the same distribution coefficient as oxyntomodulin. No 3.5K glucagon was detected in any of the extracts. GLI peptide secretion by the cells was not altered by several pancreatic glucagon secretagogues (cortisol, bombesin, and prostaglandins E1 and D2), but was stimulated by the opioid peptide beta-endorphin (1 microM; P less than 0.02). These studies have indicated that the control of secretion of fetal rat intestinal GLI peptides is complex, involving activation of any one or a combination of the three major second messenger systems. A role for the adenylate cyclase pathway in regulating GLI peptide biosynthesis is also suggested.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Bucladesine; Calcimycin; Cells, Cultured; Cobalt; Ethers; Fetus; Gastrointestinal Hormones; Glucagon-Like Peptides; Intestinal Mucosa; Intestines; Ionomycin; Kinetics; Magnesium; Magnesium Chloride; Peptides; Phorbol Esters; Rats; Rats, Inbred Strains

The kinetic parameters of the Ca2+ pump were assessed in red blood cells of essential hypertensive subjects as compared to their respective controls. Uphill Ca2+ efflux was investigated in Ca2+ -saturated intact red blood cells using a new method recently developed for human red cells (Dagher,G. and Lew, V. J. Physiol. (London), in the press). 45Ca-equilibrated cells were obtained using ionophore A23187 and Ca2+ efflux was assessed after addition of excess CoCl2 which totally inhibits Ca2+ influx and thus exposes uphill Ca2+ extrusion by the pump. The results comprise methodological aspects of the use of this technique in rat red blood cells. The determination of the maximal velocity and the Ca2+ concentration for half-maximal stimulation (KCa 0.5) did not reveal any alteration in essential hypertensives and spontaneously hypertensive rats as compared to their controls.

Topics: Adult; Aged; Animals; Calcimycin; Calcium; Calcium Radioisotopes; Cobalt; Erythrocytes; Humans; Hypertension; Ion Channels; Kinetics; Male; Membrane Potentials; Middle Aged; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Vanadates

The net Ca2+ influx was increased in human red cells in suspension by adding moderate concentrations of the Ca2+ ionophore A23187, and due to the increased cellular Ca2+ concentration [( Ca]i) the K+ channels opened (the 'Gardos effect'). At low K+ concentration and with the protonophore CCCP in the buffer-free medium the cells hyperpolarized and the extracellular pH (pH0) increased, enhancing the A23187-mediated net Ca2+ influx. This elicited a prolonged response, viz. a primary transient increase of pH0 and [Ca]i followed by one or more spontaneous pH0 and [Ca]i transients. We explored the pump-mediated Ca2+ efflux by blocking the A23187-mediated Ca2+ flux with CoCl2 at appropriate times during the prolonged response. The Ca2+ pumping was higher during the descendent than during the ascendent phase of the primary transient at equal values of [Ca]i. The data were analyzed using a mathematical model that accounts for the prolonged oscillatory response, including pH0 and [Ca]i. In conclusion, the activation of the Ca2+ pump is delayed due to slow binding of cellular calmodulin, which is a hysteretic response to a rapid increase of the cellular Ca2+ concentration. This mechanism may be important for generation and execution of transient signals in other types of cell.

Topics: Biological Transport; Calcimycin; Calcium; Cobalt; Erythrocytes; Humans; Hydrogen-Ion Concentration; Ion Channels; Potassium