Fluoride Stimulation Research Articles

Oxytocin-sensitive Adenylate Cyclase in Frog Bladder Epithelial Cells: ROLE OF CALCIUM, NUCLEOTIDES, AND OTHER FACTORS IN HORMONAL STIMULATION

Abstract The effects of Ca2+, Mg2+, pH, temperature, and nucleotides (ATP or GTP) on the oxytocin-sensitive adenylate cyclase of frog bladder epithelial cells were studied. The site of the action of these parameters on the binding step, receptor-cyclase coupling, and the adenylate cyclase system as a whole was deduced from a comparison of their effects on basal, and oxytocin- and fluoride-sensitive activities and on the apparent Km of the system for oxytocin. Minute amounts of Ca2+ were found necessary for the stimulation of adenylate cyclase by oxytocin. Elimination of Ca2+ by treatment with ethylene glycol bis(β-aminoethyl ether)-N,N'-tetraacetic acid led to inhibition of the response, which was completely restored by the addition of 10-6 m free Ca2+. Calcium concentrations higher than 5 x 10-5 m were inhibitory, with no change in the apparent Km for oxytocin. Basal activity remained unchanged throughout the range of Ca2+ concentrations tested. The measurement of oxytocin-sensitive adenylate cyclase activity as a function of the ATP concentration in the medium showed that the hormone increases both the maximum velocity of the adenylate cyclase and its affinity for ATP. When low ATP concentrations were used (between 10-6 and 2.5 x 10-4 m), the activation ratio (stimulated to basal activities) showed an optimum for 10-5 m ATP. When low GTP concentrations (10-8 to 10-6 m) were used, the magnitude of this optimum increased and moved toward the lowest concentrations. These results were taken as evidence for the existence of regulatory sites binding either GTP or ATP; the occupancy of these sites enhances the velocity of the adenylate cyclase reaction to a greater extent in the presence of oxytocin than under basal conditions. Oxytocin increases the affinity of the regulatory site for ATP or GTP. Lowering the pH of the incubation medium from 8.0 to 7.0 resulted in a loss of affinity for oxytocin, with no change either in maximum stimulation or fluoride-sensitive activity, suggesting an effect of pH primarily located at the hormone-binding step. Thermal denaturation at 43° for ½ to 30 min did not make it possible to dissociate the hormonal and fluoride stimulation processes. Magnesium experiments confirmed the existence of two actions of Mg2+ ions acting as cosubstrate with ATP and as a regulatory agent for the cyclase system. Effects of varying Mg2+ concentration on fluoride-sensitive activity must be interpreted with caution because of possible formation of poorly dissociated complexes between (F-, Mg2+, and HPO42-).

Requirement for Guanosine Triphosphate in the Prostaglandin Activation of Adenylate Cyclase of Platelet Membranes

Abstract Adenylate cyclase in purified platelet membrane was activated by prostaglandin E1 (PGE1) when 2 mm ATP was used as substrate. When the substrate concentration was lowered to 0.1 mm (using an analogue of ATP, 5'-adenylyl imidodiphosphate (AMP-PNP) which has been shown to be a substrate for adenylate cyclase but is not degraded by ATPase), the PGE1 activation of adenylate cyclase was markedly reduced or abolished, without affecting the fluoride stimulation. The PGE1 activation of the enzyme was restored by the addition of GTP to the incubation medium. The effect was evident even at 1 µm GTP, and the optimal effect was obtained at 0.1 mm GTP. The GTP effect was seen at all concentrations of PGE1 used in these experiments. Moreover, GTP effect also was evident at lower substrate concentrations. GTP did not appear to change the apparent affinity of the substrate to the enzyme, but markedly increased the PGE1-stimulated enzyme activity.

Adenyl cyclase in the toad bladder

Preparation of hormone sensitive adenyl cyclase and some properties of this enzyme were investigated in mucosal cells of toad bladder. This tissue responds to antidiuretic hormone by an increase in the active transport of Na +, enhancement of permeability to water, urea and certain other small molecules, and by an increase in osmotic water flow across the tissue. These actions of the hormone are all thought to be mediated by adenyl cyclase. Antidiuretic hormones, NaF and Mg 2+ were all found to stimulate the enzyme although the stimulation by Mg 2+ of basal enzyme activity was slight. The hormonal activation was usually greater that of fluoride under optimal conditions. The apparent K m for ATP as derived by measurement of half maximal activity was approx. 1·10 −4 M. Optimal pH was 7·5. At high concentrations both theophylline and, to a lesser extent, cyclic AMP inhibited adenyl cyclase activity. The adenyl cyclase activity was highest in the whole homogenate and the sediment from a 1000 × g centrifugation, results that are in accord with a localization of the enzyme in the cell membrane. Barely detectable activity occurred in the 10 000 × g supernatant. When high concentrations of protein were present in the enzyme assay mixture, inhibition of enzyme activity was observed. The enzyme was labile and deterioration set in when homogenization was too vigorous or prolonged, or when the cells were disrupted by sonication. Ageing of the preparation, even when frozen at −80°, resulted in loss of activity, the loss being more marked for hormonal stimulation than for fluoride stimulation. Divalent cations had marked effects on the enzyme activity. Concentrations of Mg 2+ from 1 to 25 mM had a stimulatory effect upon the enzyme and upon stimulation by hormone and fluoride. Greater concentrations had an inhibitory effect. Ca 2+ was inhibitory at all concentrations above 1·10 −5 M. Thus optimal conditions for the activity of adenyl cyclase have been defined and the preparation should allow further elucidation of the action of hormones on active Na + transport and osmotic water flow in this tissue.

Histochemical and Quantitative Studies of the In Vitro Dehydrogenase Activity of Spleen in Hypersplenism

Abstract Tetrazolium chloride was used to visualize the in vitro dehydrogenase activity of splenic tissues from cases with and without hypersplenism. In addition, quantitative measurements of such activity were made in the presence and absence of fluoride, malonate and azide. The data indicate that spleen slices from cases of hypersplenism differed from the controls and were characterized by (a) increased endogenous dehydrogenase activity of the lymphocytes, particularly around the follicles, (b) a decreased azide stimulation in those cases having an initial thrombocytopenia which responded to splenectomy, and (c) a decreased fluoride stimulation in those cases having an initial leukopenia which responded to splenectomy. Routine histological studies confirmed other reports that a constant structural feature of the spleen in hypersplenism is a perifollicular lymphocytic rimming.