{"title":"Effects of hydroxyl radical scavengers KCN and CO on ultraviolet light-induced activation of crude soluble guanylate cyclase.","authors":"J O Karlsson, K L Axelsson, R G Andersson","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>The crude soluble guanylate cyclase (GC) from bovine mesenteric artery was stimulated by ultraviolet (UV) light (366 nm). Addition of free radical scavengers, dimethylsulfoxide or superoxide dismutase and/or catalase to the GC assay did not abolish the stimulatory effect of UV light. On the contrary, the UV light-induced activation was enhanced in the presence of these scavengers. KCN (1 mM) did not affect the UV light-induced activation, while 0.1 mM of CO potentiated the activation. These results may indicate that UV light is operating through a direct interaction with the ferrous form of the GC-heme.</p>","PeriodicalId":15406,"journal":{"name":"Journal of cyclic nucleotide and protein phosphorylation research","volume":"10 4","pages":"309-15"},"PeriodicalIF":0.0,"publicationDate":"1985-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"14000246","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Evidence for forskolin binding sites in rat myocardium.","authors":"K Schmidt, W R Kukovetz","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Forskolin binding sites have been identified in a preparation of rat myocardium using [3H]forskolin and [3H]14,15-dihydroforskolin ([3H]DHF) as radioligands. It could be shown that both ligands bind to the same site with only a slight difference in their affinities. Using the filtration method the binding sites were characterized by an affinity of about 250 nM for forskolin and about 650 nM for dihydroforskolin (DHF). The binding capacity was about 5 pmol/mg protein with both compounds. When using the centrifugation technique similar binding affinities were obtained; the binding capacity, however, was around 3 fold higher than with the filtration method. Under all conditions only one single group of independent binding sites was found. In contrast, stimulation of adenylate cyclase by forskolin was found to be negatively cooperative. These results indicate that stimulation of adenylate cyclase by forskolin is a very complicated mechanism and the binding procedure of forskolin to its binding sites can represent only a small part of this process.</p>","PeriodicalId":15406,"journal":{"name":"Journal of cyclic nucleotide and protein phosphorylation research","volume":"10 5","pages":"425-38"},"PeriodicalIF":0.0,"publicationDate":"1985-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"15178324","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Modulation of forskolin binding to rat brain membranes.","authors":"K B Seamon, R Vaillancourt, J W Daly","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>High affinity binding sites for [3H]forskolin have been identified in rat brain membranes. These sites have a Kd of 15 nM and a Bmax of about 200 fmol/mg protein. The binding of [3H]forskolin to those high affinity sites in rat brain membranes is increased about two-fold by addition of MgCl2 or MnCl2. Smaller increases are observed in the presence of calcium, sodium, or potassium. The binding of [3H]forskolin is also increased in the presence of NaF or GppNHp, agents that are known to activate adenylate cyclase through the stimulatory guanine nucleotide regulatory protein (Ns). The increase in [3H]forskolin binding in the presence of NaF or GppNHp is due to an increase in the number of binding sites with no change in the apparent Kd for the binding sites. The NaF- and GppNHp-stimulated binding requires the presence of magnesium or manganese. The binding of [3H]forskolin to rat brain membranes is reduced in membranes that are heated or pretreated with chymotrypsin, trypsin, or N-ethylmaleimide. NaF stabilizes the binding sites to thermal denaturation. The data demonstrate that the number of high affinity forskolin binding sites are increased under conditions that promote the activation of the catalytic protein of adenylate cyclase by the Ns protein. It is suggested that the high affinity forskolin binding sites are associated with a complex of the catalytic protein and the activated Ns protein.</p>","PeriodicalId":15406,"journal":{"name":"Journal of cyclic nucleotide and protein phosphorylation research","volume":"10 6","pages":"535-49"},"PeriodicalIF":0.0,"publicationDate":"1985-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"15198658","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Forskolin does not activate cyclic AMP synthesis in Dictyostelium discoideum in vivo or in vitro.","authors":"M Brenner, H Padh","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Cyclic AMP (cAMP) has been postulated to regulate many of the events in the development of the eukaryotic social ameba Dictyostelium discoideum. The various suggested roles of cAMP could be tested were it possible to reversibly elevate cAMP levels in the organism. To this end we examined the effect of forskolin, which activates the adenylate cyclase of many eukaryotic organisms. We found, however, that the drug does not elevate cAMP levels in intact D. discoideum. In addition, it does not stimulate the adenylate cyclase in either its basal state or activated state.</p>","PeriodicalId":15406,"journal":{"name":"Journal of cyclic nucleotide and protein phosphorylation research","volume":"9 4-5","pages":"297-303"},"PeriodicalIF":0.0,"publicationDate":"1983-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"17155395","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Separation of the catalytic and stimulatory regulatory subunits of rat brain adenylate cyclase.","authors":"L S Bradham, M G Hegazy","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>The catalytic subunit of rat brain adenylate cyclase was separated from its stimulatory subunit with loss of activation by 5'-guanylylimidodiphosphate. With respect to other properties, the resolved catalytic subunit was similar to the native enzyme. Nucleotide activation was restored after reconstitution with the isolated regulatory subunit. A key step in resolution was preliminary exposure of isolated membranes to increased ionic strength. After solubilization with nonionic detergent solution, the subunits were separated by gel filtration. The apparent molecular size of the catalytic subunit was unchanged by salt treatment, whereas the Stokes' radius of the regulatory subunit decreased from 72 A to 48 A.</p>","PeriodicalId":15406,"journal":{"name":"Journal of cyclic nucleotide and protein phosphorylation research","volume":"9 4-5","pages":"331-40"},"PeriodicalIF":0.0,"publicationDate":"1983-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"17155396","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"George Ira Drummond 1925-1984.","authors":"","doi":"","DOIUrl":"","url":null,"abstract":"","PeriodicalId":15406,"journal":{"name":"Journal of cyclic nucleotide and protein phosphorylation research","volume":"9 6","pages":"397-9"},"PeriodicalIF":0.0,"publicationDate":"1983-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"17165500","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Mepacrine-induced elevation of cyclic GMP levels and acceleration of reversal of ADP-induced aggregation in washed rabbit platelets.","authors":"I Matsuoka, T Suzuki","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Mepacrine, a phospholipase A2 inhibitor, caused concentration-dependent elevations of cyclic GMP levels without changing cyclic AMP levels in washed rabbit platelets. Mepacrine (100 microM) increased cyclic GMP levels to a peak (25-fold of basal level) within 4 min. Mepacrine had no effect on platelet guanylate cyclase and cyclic AMP phosphodiesterase but selectively inhibited cyclic GMP phosphodiesterase, indicating that mepacrine may elevate platelet cyclic GMP levels as a result of inhibiting cyclic GMP breakdown. In addition, mepacrine accelerated the disaggregation of platelets which had been aggregated maximally by ADP. This effect was associated with elevated cyclic GMP levels. Likewise, sodium nitroprusside and sodium ascorbate, which also elevate platelet cyclic GMP levels, caused marked disaggregation. The increases in cyclic GMP levels with these agents were well correlated with the extent of disaggregation, suggesting that cyclic GMP may mediate a process opposing platelet aggregation and that the mepacrine-induced acceleration of disaggregation may be mediated by cyclic GMP.</p>","PeriodicalId":15406,"journal":{"name":"Journal of cyclic nucleotide and protein phosphorylation research","volume":"9 4-5","pages":"341-53"},"PeriodicalIF":0.0,"publicationDate":"1983-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"17213424","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Homologous desensitization of rat heart cells to beta-adrenergic stimulation and the absence of alpha-adrenergic or prostaglandin E1 effects.","authors":"R A Fisher, I Harary, J A Thomas","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Epinephrine is shown to stimulate heart cell beating by both alpha- and beta-adrenergic mechanisms. The beta-adrenergic effect undergoes desensitization in one to two minutes after beta-agonist addition. A beta-antagonist did not cause desensitization. The desensitization response is quantitatively related to the concentration of cAMP produced in the cells. The alpha-adrenergic component of epinephrine action does not undergo desensitization nor does it have effects on the beta-adrenergic stimulation of cell beating or cAMP production. Other alpha-agonists and antagonists also have no effect on cAMP production. Prostaglandin E1 increased cellular cAMP by a mechanism that was additive with the beta-adrenergic mechanism. Both prostaglandin E1 and beta-agonists caused phosphorylation of both glycogen phosphorylase and glycogen synthase. Prostaglandin E1 did not desensitize the cells to beta-adrenergic stimulation of cAMP. The data show that heart cells undergo a rapid homologous desensitization to beta-agonists when treated with epinephrine or isoproterenol. The desensitization is quantitatively related to the cellular cAMP concentration, but cAMP produced by another mechanism does not desensitize the cells. No alpha-adrenergic effects on the beta-induced desensitization were observed.</p>","PeriodicalId":15406,"journal":{"name":"Journal of cyclic nucleotide and protein phosphorylation research","volume":"9 6","pages":"449-60"},"PeriodicalIF":0.0,"publicationDate":"1983-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"17217846","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Immunocytochemical evidence for 3',5'-cGMP and 3',5'-cGMP-dependent protein kinase involvement in lymphocyte proliferation.","authors":"M T Largen, B Votta","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>In preliminary experiments cyclic nucleotides and cyclic nucleotide-dependent protein kinase subunits were localized in murine splenocytes using immunofluorescence and immunoperoxidase techniques. Cyclic nucleotides, presumably protein bound, and protein kinases (PK) were found in both cytoplasm and nucleus. Following mitogen stimulation the localizations did not change. In experiments reported here using the peroxidase-antiperoxidase technique not all cells in the population were stained with antisera against the various antigens. At early times (5-60 min) following stimulation with lipopolysaccharide (LPS) or concanavalin A (ConA) the fraction of cells staining positively for cGMP and cGMP PK increased relative to non-stimulated cells. Radioimmunoassay measurements showed elevated intracellular concentrations of cGMP beginning at 15 min following mitogenic stimulation. The data presented is consistent with a role for cGMP and cGMP PK in lymphocyte activation.</p>","PeriodicalId":15406,"journal":{"name":"Journal of cyclic nucleotide and protein phosphorylation research","volume":"9 3","pages":"231-44"},"PeriodicalIF":0.0,"publicationDate":"1983-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"17382591","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Forskolin-stimulated cyclic AMP accumulation mediates protein synthesis-dependent refractoriness in C6-2B rat glioma cells.","authors":"K Barovsky, C Pedone, G Brooker","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>We have examined the roles that cyclic AMP and protein synthesis play in the development of refractoriness in C6-2B rat glioma cells using the diterpene, forskolin, a general activator of cyclic AMP-generating systems. Forskolin-stimulated cyclic AMP accumulation peaked at 30 min and declined thereafter to 10% of peak levels by 3 hr despite the continued presence of sufficient forskolin to produce 98% of the control response when the incubation medium was transferred to naive cells. C6-2B cells treated for 3 hr with forskolin were refractory to a subsequent challenge with forskolin or isoproterenol. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) increased the degree of refractoriness developed after forskolin treatment. In the presence of IBMX, the induction of refractoriness by forskolin and forskolin-stimulated cyclic AMP accumulation were similarly dependent on forskolin concentration. Pre-treatment with isoproterenol or the cyclic AMP analogue, dibutyryl cyclic AMP, induced refractoriness to forskolin. When C6-2B cells were pre-treated with forskolin plus the protein synthesis inhibitor, cycloheximide, the development of refractoriness to forskolin or isoproterenol was attenuated. Cycloheximide prevented isoproterenol- or dibutyryl cyclic AMP-induced refractoriness to forskolin. These data provide further evidence that the onset of the refractory state in C6-2B cells is mediated by cyclic AMP and is a protein synthesis-requiring process.</p>","PeriodicalId":15406,"journal":{"name":"Journal of cyclic nucleotide and protein phosphorylation research","volume":"9 3","pages":"181-9"},"PeriodicalIF":0.0,"publicationDate":"1983-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"17263034","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}