Estrogens and progesterone increase fetal and maternal guanylate cyclase activity

Since both estrogens and cyclic guanosine 3',5'-monophosphate stimulate protein synthesis, the objective of the present investigation was to determine if estrogens and their precursors might have part of their mechanism of action through stimulation of guanylate cyclase (E.C.4.6.1.2), the enzyme that catalyzes the conversion of guanosine triphosphate to cyclic guanosine 3',5'-monophosphate. The precursors of estrogen synthesis originate from cholesterol. Cholesterol itself had no effect on guanylate cyclase activity. The precursors of estrogen synthesis generated from cholesterol, namely, progesterone, 17 alpha-OH-progesterone, androstenedione, pregnenolone, 17 alpha-OH-pregnenolone, and dehydroepinandrosterone, however, caused a 2- to 3-fold enhancement of fetal and maternal guinea pig hepatic and uterine guaynlate cyclase activity at a concentration of 1 microM. In comparative studies, similar effects were seen on immature female Sprague-Dawley rat hepatic and uterine guanylate cyclase activity. Estrone, estradiol-17 beta, estriol, and the synthetic estrogen, diethylstilbestrol, enhanced guanylate cyclase activity in the same tissues 2- to 3- fold at the 1 microM concentration. Dose-response relationships revealed that these estrogens and their precursors had their maximal effect at 0.001 microM. Estradiol-17 alpha also enhanced uterine guanylate cyclase activity, but a 1000-fold greater concentration compared to the other estrogens was necessary to show any significant effect. The data in this investigation suggest that guanylate cyclase may play a role in the mechanism of action of estrogens and their precursors.     

Plant growth-promoting hormones activate mammalian guanylate cyclase activity

In vivo injections of plant growth-promoting hormones increase the growth of animals as well as plants. Plant growth-promoting hormones and positive plant growth regulators are known to increase RNA and protein synthesis. Since cyclic GMP also increases RNA and protein synthesis, the object of the present investigation was to determine whether physiological levels of plant growth-promoting hormones and positive plant growth regulators have part of their mechanism(s) of action through stimulation of the guanylate cyclase (EC 4.6.1.2)-cyclic GMP system. Representatives of the three classes of growth-promoting hormones were investigated. Thus, auxins (indole-3-acetic acid, indole-3-butyric acid, beta-naphthoxyacetic acid, and 2,4,5-trichlorophenoxy acetic acid), gibberellins (gibberellic acid), and cytokinins [N6-benzyl adenine, kinetin (6-furfuryl aminopurine), and beta-(2-furyl) acrylic acid] all increased rat lung, small intestine, liver, and renal cortex guanylate cyclase activity 2- to 4-fold at the 1 microM concentration. Dose response curves revealed that maximal stimulation of guanylate cyclase by these plant growth regulators was at 1 microM; there was no augmented cyclase activity at 1 nM. The guanylate cyclase cationic cofactor manganese was not essential for augmentation of guanylate cyclase by these plant growth-promoting regulators. The antioxidant butylated hydroxytoluene did not block the enhancement of guanylate cyclase by these plant growth-promoting factors. These data suggest that guanylate cyclase may play a role in the mechanism of action of plant growth-promoting hormones and even of positive plant regulators at the cellular level.     

Activation of purified soluble guanylate cyclase by protoporphyrin IX.

Soluble guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] purified from bovine lung is markedly activated (30- to 40-fold) by protoporphyrin IX (Ka, 15-25 nM) and is inhibited by hematin (Ki, 3.7 microM) when MgGTP is used as substrate. Guanylate cyclase possesses specific activities (mumol of cGMP per min/mg of protein) of 0.1-0.2 (MgGTP) and 0.3-0.5 (MnGTP) and can attain values of 2-8 (MgGTP) or 1-1.4 (MnGTP) in the presence of protoporphyrin IX. Guanylate cyclase purified in this study contains heme and is activated by nitric oxide and nitrosyl-heme to the same magnitude as that by protoporphyrin IX. With the exception of hematoporphyrin IX, close structural analogs of protoporphyrin IX, including precursors and metabolites, do not activate guanylate cyclase. The insertion of iron into protoporphyrin IX to form heme or hematin renders the metalloporphyrin an inhibitor of unactivated or activated guanylate cyclase. The data suggest that protoporphyrin IX and heme could function to modulate guanylate cyclase activity.     

Cimetidine partially blocks gastrin's activation of gastric mucosal guanylate cyclase

The objective of the present investigation was to determine if gastrin at physiological concentrations has part of its mechanism(s) of action through stimulation of guanylate cyclase (EC 4.6.1.2). Human gastrin (I), pentagastrin, tetragastrin, and gastrin-related tetrapeptide all increased cyclic GMP levels and guanylate cyclase activity in rat gastric mucosa, whole stomach, and duodenum. Maximal stimulation was seen at 1 microM with all of the above. There was no further enhancement of guanylate cyclase with increasing the concentration to the millimolar range. The ED50 for human gastrin and pentagastrin was 0.01 microM, whereas the ED50 was 0.1 microM for tetragastrin and the tetrapeptide. No enhancement of guanylate cyclase activity was seen with decreasing the concentration to 1 nM of the respective gastrins. Cimetidine utilized at 1 microM or 1 mM concentrations partially blocked the augmentation by gastrin suggesting that part of this enhancement was through the histamine 2 receptor which has been shown to be important in pentagastrin-stimulated gastric acid release. Since the block was only partial these data would also indicate that some part of gastrin's activation of this enzyme is not mediated through the histamine 2 receptor.     

Purification of soluble guanylate cyclase from rat liver

Soluble guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] has been purified from rat liver and exhibited a single protein band on polyacrylamide gels coincident with activity and indicative of a molecular weight of 150,000. The apparent specific activity of the purified enzyme was 276 nmol of cyclic GMP formed per mg per min with Mn²⁺ as the cation cofactor and 23.8 nmol of cyclic GMP formed per mg per min with Mg²⁺. This represented 9200-fold and 7400-fold purifications of Mn²⁺ and Mg²⁺ activities, respectively. The specific activity of soluble guanylate cyclase was not constant with protein concentration. At all stages of purification, increasing the enzyme concentration in the guanylate cyclase assay increased the apparent specific activity of the preparation. The purified enzyme could be activated by nitroprusside, nitric oxide, arachidonate, linoleate, oleate, and superoxide dismutase. However, the degree of activation was dependent upon the concentration of enzyme protein assayed.     

Nitric oxide activates guanylate cyclase and increases guanosine 3′:5′-cyclic monophosphate levels in various tissue preparations

Nitric oxide gas (NO) increased guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] activity in soluble and particulate preparations from various tissues. The effect was dose-dependent and was observed with all tissue preparations examined. The extent of activation was variable among different tissue preparations and was greatest (19- to 33-fold) with supernatant fractions of homogenates from liver, lung, tracheal smooth muscle, heart, kidney, cerebral cortex, and cerebellum. Smaller effects (5- to 14-fold) were observed with supernatant fractions from skeletal muscle, spleen, intestinal muscle, adrenal, and epididymal fat. Activation was also observed with partially purified preparations of guanylate cyclase. Activation of rat liver supernatant preparations was augmented slightly with reducing agents, decreased with some oxidizing agents, and greater in a nitrogen than in an oxygen atmosphere. After activation with NO, guanylate cyclase activity decreased with a half-life of 3-4 at 4 degrees but re-exposure to NO resulted in reactivation of preparations. Sodium azide, sodium nitrite, hydroxylamine, and sodium nitroprusside also increased guanylate cyclase activity as reported previously. NO alone and in combination with these agents produced approximately the same degree of maximal activation, suggesting that all of these agents act through a similar mechanism. NO also increased the accumulation of cyclic GMP but not cyclic AMP in incubations of minces from various rat tissues. We propose that various nitro compounds and those capable of forming NO in incubations activate guanylate cyclase through a similar but undefined mechanism. These effects may explain the high activities of guanylate cyclase in certain tissues (e.g., lung and intestinal mucosa) that are exposed to environmental nitro compounds.     

Heat-stable enterotoxin of Escherichia coli: in vitro effects on guanylate cyclase activity, cyclic GMP concentration, and ion transport in small intestine.

A partially purified preparation of the heat-stable enterotoxin of Escherichia coli caused a rapid and persistent increase in electric potential difference and short-circuit current when added in vitro to the luminal surface of isolated rabbit ileal mucosa. As little as 1 ng/ml produced an easily detectable response. Under short-circuit condition, the enterotoxin abolished net Cl- absorption; this change was half that produced by theophylline, which stimulated net secretion. The enterotoxin did not change cyclic AMP concentration but caused large and persistent increases in cyclic GMP concentration. The electrical and nucleotide responses exhibited similar and unusually broad concentration-dependences and maximal effects could not be demonstrated. Theophylline elevated cyclic GMP concentration 3-fold both in the presence and absense of the enterotoxin, suggesting no effect of the toxin on cyclic GMP phosphodiesterase. Guanylate cyclase [GTP pyrophosphatelyase(cyclizing); EC 4.6.1.2] activity in a crude membrane fraction from intestinal epithelial cells was stimulated 7-fold by the enterotoxin. These results suggest that guanylate cyclase stimulation is the basis for the toxin's diarrheagenic effect.     

Bromocriptine enhances guanylate cyclase activity

Bromocriptine and its parent compound alpha-ergocryptine were investigated with respect to their ability to interact with the guanylate cyclase (E.C.4.6.1.2)-cyclic GMP system in vitro in the rat pituitary and ovary. Both bromocriptine and alpha-ergocryptine enhanced guanylate cyclase two- to threefold in both of these tissues over a concentration range of 1 nM to 1 microM. Since bromocriptine is thought to be a dopamine agonist in the pituitary, dopamine's effects on guanylate cyclase were also tested. Dopamine caused a twofold enhancement of guanylate cyclase activity in the pituitary and ovary. When bromocriptine and dopamine were used in combination, bromocriptine had to be in equal or a greater concentration with respect to dopamine in vitro to enhance guanylate cyclase activity. These findings suggest that bromocriptine's effect at the level of the pituitary and ovary may be mediated through enhancement of guanylate cyclase activity.     

Activation of guanylate cyclase by superoxide dismutase and hydroxyl radical: a physiological regulator of guanosine 3'',5''-monophosphate formation.

Partially purified soluble rat liver guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] was activated by superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1). This activation was prevented with KCN or glutathione, inhibitors of superoxide dismutase. Guanylate cyclase preparations formed superoxide ion. Activation by superoxide dismutase was further enhanced by the addition of nitrate reductase. Although guanylate cyclase activity was much greater with Mn2+ than with Mg2+ as sole cation cofactor, activation with superoxide dismutase was not observed when Mn2+ was included in incubations. Catalase also decreased the activation induced with superoxide dismutase. Thus, activation required the formation of both superoxide ion and H2O2 in incubations. Activation of guanylate cyclase could not be achieved by the addition of H2O2 alone. Scavengers of hydroxyl radicals prevented the activation. It is proposed that superoxide ion and hydrogen peroxide can lead to the formation of hydroxyl radicals that activate guanylate cyclase. This mechanism of activation can explain numerous observations of altered guanylate cyclase activity and cyclic GMP accumulation in tissues with oxidizing and reducing agents. This mechanism will also permit physiological regulation of guanylate cyclase and cyclic GMP formation when there is altered redox or free radical formation in tissues in response to hormones, other agents, and processes.     

Cyclic GMP affecting the tracheal nonadrenergic noncholinergic inhibitory system

An association between guanosine 3',5'-monophosphate (cyclic GMP) and the nonadrenergic noncholinergic inhibitory system (NANCIS) has been demonstrated in the isolated bovine tissue (Bowman and Drummond, 1984). In order to investigate this association in the guinea pig trachea, we used cyclic GMP derivatives, guanylate cyclase activators (N-methylhydroxylamine (NMH) and nitroglycerin (NG)] and inhibitors [oxyhemoglobin (HbO2) and methylene blue (MB)]. Under general anesthesia paralysis, the animals were ventilated and hourly injected with atropine (0.2 mg/kg) and propranolol (1 mg/kg). Cervical segment of the trachea was converted to a closed tracheal pouch and then filled with Kreb's solution augmented with atropine (1 microM) and propranolol (3.5 microM). A decrease in the pouch pressure (Pp) reflected NANCIS nerve transmural stimulation (TS)--or drug-induced relaxation. Pharmacological agents were applied intravenously. At 2-11 min after injection, NMH and NG decreased baseline Pp and reduced TS-induced relaxation. NMH, which is more potent than NG in activating particulate guanylate cyclase activity, potentiated the TS-induced relaxation at high frequencies, but NG did not. HBO2 inhibited the TS-induced relaxation at high but not at low frequencies. In contrast, MB inhibited the relaxation at low but not high frequencies. The results suggest that activation of particulate or membrane bound guanylate cyclase potentiates NANCIS-induced decrease in Pp. Therefore, there is a possible association between cyclic GMP and the NANCIS in the guinea pig trachea.     

Inhibition of growth and guanylate cyclase activity of an undifferentiated prostate adenocarcinoma by an extract of the balsam pear (Momordica charantia abbreviata).

We have recently described the presence of a guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] inhibitor (GCI) in an aqueous extract of the balsam pear (Momordica charantia abbreviata). Because the guanylate cyclase-cyclic GMP system is though to be involved in cell growth, DNA and RNA synthesis, and possible malignant transformation, we examined the effect of the aqueous extract containing GCI on an undifferentiated adenocarcinoma of the rat prostate and concanavalin-A-stimulated [3H]thymidine incorporation into cultured splenic lymphocytes, a process thought to be mediated by cyclic GMP. The results demonstrate that the extract of the balsam pear blocks both the growth of the rat prostatic adencarcinoma in vitro and [3H]thymidine incorporation into DNA. DNA histograms from flow cytometry indicated that the extract containing GCI inhibited in the G2 + M phase of the cell cycle, a presumed locus of cyclic GMP effects. In addition, guanylate cyclase activity was significantly greater in the tumor than normal prostate tissue and was decreased by the extract containing GCI. Cyclic GMP levels in the tumor in culture wer also decreased by addition of the extract. It remains to be determined whether or not the anti-tumor agent and GCI are the same substance.     

Products of reaction catalyzed by purified rat liver guanylate cyclase determined by 31p NMR spectroscopy.

Products of the reactions catalyzed by highly purified preparations of soluble guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] from rat liver were identified and quantified with 31P NMR spectroscopy. Utilization of this technique necessitated modification of the standard assay conditions; higher concentrations of enzyme and substrate (2 mM), Mg2+ instead of Mn2+, and longer incubation times (up to 46 hr) at 30 degrees C were used. Revision of our reported procedure for purificaton of guanylate cyclase [Tsai, S-C., Manganiello, V. C. & Vaughan, M. (1978) J. Biol. Chem. 253, 8452-8457] to include chromatography on Ultrogel AcA34 and agarose-hexane-GTP provided an enzyme with specific activity higher than in our earlier preparations. 31P NMR spectra obtained during incubation of this enzyme showed that the rates of GTP disappearance and cyclic GMP (cGMP) accumulation were constant for approximately 16 hr. They indicated, however, that the preparations were contaminated with inorganic pyrophosphatase. This was removed by preparative electrophoresis, yielding enzyme with specific activities (900-1300 nmol/min per mg of protein) higher than those reported for guanylate cyclases from rat liver or lung. With this preparation, cGMP and PPi were the only products of GTP detected, consistent with the assumption that the guanylate and adenylate cyclase reactions are analogous.     

Occurrence of a hormone-sensitive inhibitory coupling component of the adenylate cyclase in S49 lymphoma cyc- variants.

The influence of somatostatin was studied on cyclic AMP levels and adenylate cyclase activity in cyc- variants of S49 lymphoma cells. These cells are deficient in the guanine nucleotide site that mediates hormone-induced adenylate cyclase stimulation, but their cyclase can be stimulated by forskolin. Somatostatin maximally decreased the 30 microM forskolin-stimulated cyclic AMP levels by 35%. Half-maximal suppression occurred at about 0.1 nM somatostatin. Somatostatin (up to 1 microM) had no effect on the 100 microM forskolin-stimulated adenylate cyclase activity in cyc- membrane preparations when guanine nucleotides were not present. In the presence of GTP, however, which by itself caused a small decrease in activity, somatostatin maximally inhibited the enzyme by 20-25%. GTP was half-maximally effective at 0.1 microM, and half-maximal inhibition by somatostatin was observed at 0.1- 1 nM. In the presence of the stable GTP analog guanosine 5'-O-(3-thiotriphosphate) (1 microM), which decreased the stimulated activity by about 40% after a short lag period, somatostatin (1 microM) did not cause a further decrease in final activity but reduced the lag period by about 50%. The data indicate that membranes of cyc- variants contain a regulatory site that mediates both guanine nucleotide and hormone-induced inhibition of the adenylate cyclase and suggest that the mechanisms of activation and inactivation of this inhibitory site are similar to those of the stimulatory component missing in cyc-membranes.     

Immunohistochemical localization of guanylate cyclase within neurons of rat brain.

The immunohistochemical localization of guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] has been examined in rat neocortex, caudate-putamen, and cerebellum by using specific monoclonal antibodies. Immunofluorescence could be seen within somata and proximal dendrites of neurons in the these regions. A nuclear immunofluorescence reaction to guanylate cyclase was characteristically absent. The staining pattern for guanylate cyclase was coincident with previously described localizations of cyclic GMP immunofluorescence within medium spiny neurons of the caudate-putamen and pyramidal cells of the neocortex. Cerebellar guanylate cyclase immunoreactivity was primarily confined to Purkinje cells and their primary dendrites, similar to the pattern reported for cyclic GMP-dependent protein kinase localization. Guanylate cyclase immunofluorescence was abolished when the monoclonal antibodies were exposed to purified enzyme prior to incubation of the tissue slices or when control antibody was substituted for the primary antibody. Immunohistochemical localization of cyclic AMP in these same tissues was readily distinguished from that of guanylate cyclase or cyclic GMP, showing uniform fluorescence throughout the cell bodies of neurons and glial elements.     

Effect of in vitro organic nitrate tolerance on relaxation, cyclic GMP accumulation, and guanylate cyclase activation by glyceryl trinitrate and the enantiomers of isoidide dinitrate

Previously, it was shown that the D enantiomer of isoidide dinitrate was 10-fold more potent than the L enantiomer and 10-fold less potent than glyceryl trinitrate for stimulating cyclic GMP accumulation and relaxation of isolated rat aorta. In the present study, these organic nitrates were tested for their ability to induce tolerance to organic nitrate-induced relaxation, cyclic GMP accumulation, and guanylate cyclase activation in rat aorta in vitro. To compensate for the differences in vasodilator potency, tolerance was induced by incubating isolated rat aorta with concentrations of organic nitrates 1,000-fold greater than the EC50 for relaxation. Under these conditions, the EC50 for relaxation was increased significantly for each organic nitrate and to a similar degree on subsequent reexposure. These data suggest that the potential for inducing in vitro tolerance to relaxation was the same for the three organic nitrates tested. When activation of soluble guanylate cyclase by these compounds was assessed, the enantiomers of isoidide dinitrate were equipotent, but less potent than glyceryl trinitrate, suggesting that the site of enantioselectivity is not guanylate cyclase itself. In blood vessels made tolerant to organic nitrates by pretreatment with glyceryl trinitrate, vasodilator activity, cyclic GMP accumulation, and guanylate cyclase activation were attenuated on reexposure to each organic nitrate. In addition, differences in the potency of the three organic nitrates and the enantioselectivity of isoidide dinitrate for relaxation were abolished in tolerant tissue, whereas the potency difference between glyceryl trinitrate and isoidide dinitrate for activation of guanylate cyclase was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

A light-activated GTPase in vertebrate photoreceptors: Regulation of light-activated cyclic GMP phosphodiesterase

We have been studying the mechanism by which light and nucleoside triphosphates activate the discmembrane phosphodiesterase (oligonucleate 5'-nucleotidohydrolase; EC 3.1.4.1) in frog rod outer segments. GTP is orders of magnitude more effective than ATP as a cofactor in the light-dependent activation step. GTP and the analogue guanylyl-imidodiphosphate function equally as allosteric activators of photoreceptor phosphodiesterase rather than participating in the formation of a phosphorylated activator. Moreover, we have found a light-activated (5-fold) GTPase which participates in the modulation of photoreceptor phosphodiesterase. This GTPase activity appears necessary for the reversal of phosphodiesterase activation in vitro and may play a critical role in the in vivo regulation of light-sensitive phosphodiesterase. The K(m) for GTP in the light-activated GTPase reaction is <1 muM. The light sensitivity of this GTPase (number of photons required for half-maximal activation) is identical to that of light-activated phosphodiesterase. The GTPase action spectrum corresponds to the absorption spectrum of rhodopsin. There is, in addition, a light-insensitive GTPase activity with a K(m) for GTP of 90 muM. At GTP concentrations above 5 muM, there is no appreciable activation of GTPase activity by light. The substrate K(m) values for guanylate cyclase, light-activated GTPase, and light-activated phosphodiesterase order an enzyme array that might permit light to simultaneously cause the hydrolysis of both the substrate and product of guanylate cyclase. These findings reveal yet another facet of light regulation of photoreceptor/cyclic GMP levels and also provide a striking analogy to the GTP regulation of nonphotoreceptor, hormone-sensitive adenylate cyclase.     

Atrial natriuretic peptide, oxytocin, and vasopressin increase guanosine 3',5'-monophosphate in LLC-PK1 kidney epithelial cells

The effect of atrial natriuretic peptide (ANP), arginine vasopressin (AVP), and oxytocin (OT) on cAMP and cGMP accumulation was investigated in LLC-PK1 kidney epithelial cells. The addition of ANP, AVP, and OT to intact cells produced a time- and concentration-dependent increase in cGMP accumulation. ANP produced a 1.7-fold increase in cGMP at 10 pM and a maximal 28-fold increase in cGMP at 1 microM. ANP had no effect on basal or AVP-induced stimulation of cAMP accumulation. OT was 10-fold more potent than AVP at increasing cGMP levels, producing a 2.1-fold increase in cGMP at 0.1 nM, whereas AVP was 100-fold more potent at increasing cAMP levels. At a concentration of 1 microM, AVP and OT produced a maximal 12 to 14-fold increase in cGMP, while OT and AVP produced 50- and 90-fold increase in cAMP, respectively. The selective OT agonist [Thr4, Gly7]oxytocin was very effective at increasing cGMP, but not at increasing cAMP levels. The V2-vasopressin agonist [deamino-Pen1,Val4, D-Arg8]vasopressin did not increase cGMP levels, but produced a 20-fold increase in cAMP levels. The addition of ANP together with either AVP or OT produced an additive increase in cGMP content. Simultaneous addition of AVP and OT did not lead to a greater increase in cAMP or cGMP levels. These results suggest that the AVP- and OT-induced increase in cGMP is mediated by OT receptors, whereas the increase in cAMP is probably mediated by vasopressin receptors. ANP increased the activity of particulate guanylate cyclase by 6-fold, while AVP and OT has no effect on particulate guanylate cyclase activity. The relatively selective inhibitor of soluble guanylate cyclase, methylene blue, had no effect on the ANP-induced increase in cGMP content in intact cells, but produced a 50% inhibition of the increase in cGMP by AVP and OT. Methylene blue did not alter the stimulation of cAMP by AVP or OT. These results demonstrate that ANP, AVP, and OT increase cGMP in LLC-PK1 kidney epithelial cells. The increase in cGMP by ANP is mediated by particulate guanylate cyclase, whereas AVP and OT probably increase cGMP by interacting with OT receptors coupled to soluble guanylate cyclase.     

Rod light adaptation may be mediated by acceleration of the phosphodiesterase-guanylate cyclase cycle.

We compare the retinal rod photocurrent before and after introduction of an hydrolysis-resistant analog of GTP into the outer segment by the whole-cell patch technique. Others have shown that GTP bound to transducin leads to the hydrolysis of cyclic GMP, causing the response to light--a decrease in dark current. The hydrolysis-resistant GTP analog prolongs the response to a bright flash, which leads us to suggest that prolonged transducin activation by bright light desensitizes the rod by a prolonged decrease in dark current. Recovery from the response to a bright flash does occur after introduction of the analog; that recovery requires acceleration of cyclase activity rather than inhibition of phosphodiesterase. The analog mimics light adaptation by desensitizing the rod and speeding the recovery from a dim flash. The analog plus light or light adaptation prolongs the activities of transducin and phosphodiesterase (oligonucleate 5'-nucleotidohydrolase, EC 3.1.4.1) to mediate desensitization by reducing the dark current. Hence, this faster recovery from a dim flash would be by increased activity of guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] rather than by inhibited phosphodiesterase. Accelerated activity of guanylate cyclase may speed recovery by response truncation. We conclude that transducin, activated by photolyzed rhodopsin, may lead to increased activity of both phosphodiesterase and guanylate cyclase to mediate the desensitization and the faster recovery of the light-adapted response.     

Comparison of the luteinizing hormone-sensitive adenylyl cyclase of the pig ovarian follicle and corpus luteum and its susceptibility to in vitro hormone-dependent desensitization

The hormone responsiveness of the adenylyl cyclase of pig ovarian follicles or corpora lutea was examined. Adenylyl cyclase activity was assayed in 10,000 x g membrane fractions that had been prepared with or without (control) a urea extraction. In control luteal membranes there was little stimulation (less than 2-fold) or adenylyl cyclase by saturating ovine (o) LH, hCG, or (-)isoproterenol in the absence or presence of 10 microM GTP. However, in urea-treated luteal membranes, a 2- to 3-fold stimulation of adenylyl cyclase was caused by saturating oLH or hCG, and a 4- to 5-fold stimulation by (-)isoproterenol; the marked stimulation by the gonadotropins was only observed if 10 microM GTP was added. In follicular membranes, a 3- to 4-fold stimulation of adenylyl cyclase by gonadotropins was observed regardless of whether GTP was added or the membranes had been urea extracted. Stimulation of adenylyl cyclase by (-)isoproterenol was always less than 2-fold in follicular membranes. The binding affinity for [125I]hCG was similar in control follicular and luteal membranes, but there were approximately 10-fold more [125I]hCG-binding sites in follicular compared with luteal membranes. The binding affinities and number of receptor sites were not significantly changed by urea treatment. The ED50 values for hCG or (-)isoproterenol were the same in follicular and luteal membranes and were uneffected by the addition of 10 microM GTP, but the ED50 for oLH was 3-fold lower in follicular than in luteal membranes. GTP caused a dose-dependent increase in adenylyl cyclase activity in luteal and follicular membranes, and both tissues had the same ED50. A saturating hormone concentration resulted in an approximately 2-fold decrease in the ED50 for GTP. In vitro hCG-induced desensitization of the hCG-responsive adenylyl cyclase was 31% in follicular membranes, but only 11-15% in luteal membranes. Hormone-induced desensitization was not increased in incubations of luteal homogenate or membranes plus cytosol. These results establish the existence of a LH/hCG-sensitive adenylyl cyclase in the pig corpus luteum and indicate that the G-protein and catalytic moieties of the follicular and luteal adenylyl cyclase complex are functionally the same, but some difference exists in the way the LH/hCG-receptor in the two tissues interacts with the G-protein/catalytic complex.     

Epidermal growth factor enhances guanylate cyclase activity in vivo and in vitro

Epidermal growth factor (EGF) increases DNA synthesis and cell division both in vivo and in vitro. The mechanism by which EGF increases growth and DNA synthesis is unknown. Since the intracellular messenger cGMP stimulates DNA synthesis, the present investigation was designed to determine if EGF might have part of its mechanism of action through activating guanylate cyclase [EC 4.6.1.2], the enzyme that catalyzes the formation of cGMP. EGF enhanced soluble and particulate guanylate cyclase activities as well as cGMP levels 2- to 3-fold in hypophysectomized and nonhypophysectomized tissues both in vivo and in vitro. EGF increased guanylate cyclase activity 0.5 h after ip injection in mice, and this increased activity was still present 12 h later. Guanylate cyclase activity was increased to a greater extent secondary to EGF in hypophysectomized cecum compared to nonhypophysectomized cecum. Dose-response curves revealed that maximal stimulation of guanylate cyclase by EGF occurred at 1 nM. There was no augmented guanylate cyclase activity when the concentration of EGF was decreased to 0.01 nM. The data in this investigation suggest that guanylate cyclase may play a role in the mechanism of action of EGF.