Effects of guanosine 3',5'-monophosphate on glucose utilization in isolated islets of Langerhans.

Dynamic changes in total glucose utilization in isolated islets of Langerhans of the rat were determined by quantitation of the formation of 3H2O from D-[5-3H]glucose. The addition of 8-bromo-cGMP (8-Br-cGMP) or monobutyryl cGMP to the islets during a linear phase of glucose utilization resulted in concentration- and time-dependent increases in glucose utilization. Effects of the analogs of cGMP on glucose utilization were noted as early as 5 min after the onset of stimulation in the presence of 10 mM glucose. 8-Br-cGMP also increased the utilization of 1 mM glucose within 20 min. Stimulatory effects of 8-Br-cGMP were observed in the presence of cycloheximide or N-acetylglucosamine. Neither 8-bromo-cAMP (8-Br-cAMP) nor monobutyryl cAMP induced significant changes in glucose utilization at 1 or 10 mM glucose. In the presence of 3-isobutyl-1-methylxanthine (IBMX), 8-Br-cGMP, but not 8-Br-cAMP, induced a rapid change in glucose utilization. N-Methyl-N'-nitro-N-nitrosoguanidine, which activates guanylate cyclase, also stimulated glucose utilization in the presence of IBMX by 3-fold. IBMX alone did not change glucose utilization. In contrast, 8-Br-5'-GMP reduced glucose utilization, whereas 8-bromoinosine 3',5'-monophosphate and 8-bromoguanosine did not change glucose utilization. Sodium bromide did not affect glucose utilization. Glucose-stimulated insulin release was potentiated by 8-Br-cGMP, whereas insulin release from islets incubated in the absence of glucose or the presence of glyceraldehyde or 2-ketoisocaproic acid was not altered by 8-Br-cGMP. Thus, glucose utilization in pancreatic islets is modulated by cGMP, and the secretory response to 8-Br-cGMP is glucose dependent.

Autophosphorylation of cGMP-dependent protein kinase is stimulated only by occupancy of one of the two cGMP binding sites.

cGMP-Dependent protein kinase contains, per subunit, 2 binding sites for cGMP. The apparent KD values for site 1 and 2 were 12 and 55 nM. The analogues 8-benzyl-amino-cAMP and N2-monobutyryl-cGMP bind preferentially to site 1 and 2, respectively. Both analogues stimulate autophosphorylation of the enzyme at concentrations at which only half of the phosphotransferase activity of the enzyme is expressed. Complete expression of the phosphotransferase activity requires a high concentration of each analogue and is accompanied by inhibition of the autophosphorylation reactions. It is concluded that occupancy of site 1 or 2 stimulates autophosphorylation while occupancy of both sites prevents autophosphorylation.

Somatomedin and analogues of cyclic AMP increase the number of cells synthesizing DNA in cartilage from hypophysectomized rats.

The effects of somatomedin and certain nucleotides on nuclear labelling of cartilage cells with [3H]thymidine were determined by autoradiography. Segments of costal cartilage from hypophysectomized rats were incubated for 24 h in a basal medium with or without additions and then pulsed for 2 h with [3H]thymidine in the basal medium. Both somatomedin (0.1 U/ml) and Bt2cAMP (10(-4)M) increased the number of labelled nuclei, and the combined effects were more than additive. A parallelism between the effects of these agents on nuclear labelling and their effects on total thymidine incorporation into DNA was demonstrated. The 8-bromated derivative of cAMP (10(-4)M) also enhanced chondrocyte nuclear labelling, but neither 8-Br-5'-AMP (10(-4)7) nor 8-Br-cGMP (10(-4)M) exhibited actions of the cAMP analogues. It is concluded that in cartilage obtained from hypophysectomized rats and incubated under the specified conditions (1) both somatomedin and cAMP analogues increase the number of cells synthesizing DNA as well as total thymidine incorporation into DNA, (2) the effects of the hormone and cyclic nucleotide in combination are synergistic, and (3) the increased incorporation of labelled thymidine into DNA reflects increased DNA synthesis and not merely an alteration of the specific activity of the intracellular thymidine nucleotide pool.

Tentacle contraction in Heliophrya erhardi (Suctoria): the role of inositol phospholipid metabolites and cyclic nucleotides in stimulus-response coupling.

Extracellular stimulation of Heliophrya erhardi with 15 V, 100 ms induces tentacle contraction. The effects of a number of pharmacological agents on response latency (time from stimulus to start of contraction) and contraction time (time for tentacles to reach 20% of their original length) were recorded. Contraction was enhanced in the presence of phorbol ester TPA and R59022, both of which increase activation of diacylglycerol-dependent protein kinase C. Lithium ions, which are known to inhibit dephosphorylation of inositol 1-phosphate, also affected the response, causing a decrease in latency and increase in contraction time. These results strongly implicate products of inositol phospholipid metabolism in stimulus-contraction coupling. The latency of contraction was reduced in the presence of IBMX, a nonspecific inhibitor of cyclic nucleotide phosphodiesterases. Application of membrane-permeable cyclic nucleotide analogs led to an increase in latency and contraction time for cAMP analog, but a decrease in both parameters for cGMP. This suggests that the two cyclic nucleotides have opposing roles in modulating the response. The possible roles and interactions of membrane phospholipid metabolites and cyclic nucleotides in the control of tentacle contraction of H. erhardi are discussed.

Role of nitric oxide in control of prolactin release by the adenohypophysis.

Nitric oxide synthase-containing cells were visualized in the anterior pituitary gland by immunocytochemistry. Consequently, we began an evaluation of the possible role of NO in the control of anterior pituitary function. Prolactin is normally under inhibitory hypothalamic control, and in vitro the gland secretes large quantities of the hormone. When hemipituitaries were incubated for 30 min in the presence of sodium nitroprusside, a releaser of NO, prolactin release was inhibited. This suppression was completely blocked by the scavenger of NO, hemoglobin. Analogs of arginine, such as NG-monomethyl-L-arginine (NMMA, where NG is the terminal guanidino nitrogen) and nitroarginine methyl ester, inhibit NO synthase. Incubation of hemipituitaries with either of these compounds significantly increased prolactin release. Since in other tissues most of the actions of NO are mediated by activation of soluble guanylate cyclase with the formation of cyclic GMP, we evaluated the effects of cyclic GMP on prolactin release. Cyclic GMP (10 mM) produced an approximately 40% reduction in prolactin release. Prolactin release in vivo and in vitro can be stimulated by several peptides, which include vasoactive intestinal polypeptide and substance P. Consequently, we evaluated the possible role of NO in these stimulations by incubating the glands in the presence of either of these peptides alone or in combination with NMMA. In the case of vasoactive intestinal polypeptide, the significant stimulation of prolactin release was augmented by NMMA to give an additive effect. In the case of substance P, there was a smaller but significant release of prolactin that was not significantly augmented by NMMA. We conclude that NO has little effect on the stimulatory action of these two peptides on prolactin release. Dopamine (0.1 microM), an inhibitor of prolactin release, reduced prolactin release, and this inhibitory action was significantly blocked by either hemoglobin (20 micrograms/ml) or NMMA and was completely blocked by 1 mM nitroarginine methyl ester. Atrial natriuretic factor at 1 microM also reduced prolactin release, and its action was completely blocked by NMMA. In contrast to these results with prolactin, luteinizing hormone (LH) was measured in the same medium in which the effect of nitroprusside was tested on prolactin release, there was no effect of nitroprusside, hemoglobin, or the combination of nitroprusside and hemoglobin on luteinizing hormone release. Therefore, in contrast to its inhibitory action on prolactin release NO had no effect on luteinizing hormone release. Immunocytochemical studies by others have shown that NO synthase is present in the folliculostellate cells and also the gonadotrophs of the pituitary gland. We conclude that NO produced by either of these cell types may diffuse to the lactotropes, where it can inhibit prolactin release. NO appears to play little role in the prolactin-releasing action of vasoactive intestinal polypeptide and substance P, but mediates the prolactin-inhibiting activity of dopamine and atrial natriuretic factor.