Responsiveness Of Guanylate Cyclase Research Articles

Effect of different illumination conditions and ionic environment on the guanylate cyclase activity in retina, optic nerve and optic chiasm of the rat

Guanylate cyclase is sensitive to changes of light and dark periods in incubated extracts obtained from soluble fractions of the retina, optic nerve and optic chiasm. The changes in guanylate cyclase activity found, about 100-fold between dark and light periods in those tissues, indicate a key role for this enzyme. The results showed that light inhibits strongly the retinal guanylate cyclase activity, while it increases the activity of this enzyme in the optic nerve. A generalized photo-inhibited response of guanylate cyclase was observed in all studied tissues in animals adapted to the dark. This suggests that light could act as a double stimulus gating the central circuit which promotes the hydrolysis of cGMP via cGMP phosphodiesterase-rhodopsin-transducin cascade, and by direct inhibition of the retinal guanylate cyclase activity. Finally, different responses have been observed in the guanylate cyclase activity in relation with the ion exposure depending on the studied tissue. In summary, all indicate an important role for the soluble guanylate cyclase activity in retina, and other tissues involved in the visual process such as optic nerve and optic chiasm, which have not been examined until now.

Response of guanylate cyclase to atrial natriuretic factor in epithelial cells of the frog choroid plexus

This study shows that the choroid plexus of Rana esculenta contains a guanylate cyclase particulate (GCp), similar to that identified in Mammalia, that is quite sensitive to the atrial natriuretic factor (ANF). The cytochemical tests for GCp show that ANF increases the enzymatic reaction products. Deposits are observed on the apical portion, at the basal level and along the lateral edges of the epithelial cells, with the exclusion of some intercalary epithelial cells with reaction-lacking microvilli. In particular, ANF seems to intensely stimulate the GCp activity along the lateral membranes of the epithelial cells delimiting enlarged intercellular spaces, which are probably dilated for the transport of water and solutes. These data confirm the osmoregulatory role of the hormone and its control of cephalorachidian liquid composition.

Phosphorylation of atrial natriuretic factor R1 receptor by serine/threonine protein kinases: evidences for receptor regulation.

The 130 kDa atrial natriuretic factor receptor (ANF-R1) purified from bovine adrenal zona glomerulosa is phosphorylated in vitro by serine/threonine protein kinases such as cAMP-, cGMP-dependent and protein kinase C. This phosphorylation is independent of the presence of ANF (99-126) and there is no detectable intrinsic kinase activity associated with the ANF-R1 receptor or with its activated form. In bovine adrenal zona glomerulosa cells, TPA (phorbol ester) induces a marked inhibition of the ANF-stimulated cGMP accumulation as well as of the membrane ANF-sensitive guanylate cyclase catalytic activity without any change in the binding capacity or affinity for 125I-ANF. However, we have demonstrated a significant 32P incorporation in the ANF-R1 receptor of the TPA-treated cells. The effect of TPA on the zona glomerulosa ANF-R1 receptors was abolished by calphostin C, a specific protein kinase C inhibitor. Altered ANF actions due to blunted response of guanylate cyclase to ANF could be a consequence of the ANF receptor phosphorylation by excessive activity of protein kinase C and might be involved in the pathogenesis of hypertension.

Differential effects of stimulus termination on excitation and desensitization of folic acid receptors and guanylate cyclase in Dictyostelium discoideum

The response of guanylate cyclase to addition of extracellular stimuli is well documented. Here we report for the first time the response of guanylate cyclase to removal of stimuli. Three methods were employed to terminate rapidly a stimulus of folic acid. (1) Addition of a highly active folate deaminase preparation, or (2) 12-fold dilution of the stimulated cell suspension, or (3) addition of an excess concentration of a non-agonistic derivative of folic acid, i.e., 2-deaminofolic acid, which chases the folate agonist from its cell-surface receptors. Accumulation of cGMP terminated instantaneously upon addition of deaminase, but degradation of the synthesized cGMP was not observed until 10–12 s after stimulation. Also in a cGMP phosphodiesterase-lacking ‘streamer’ mutant an instantaneous termination of further cGMP accumulation was observed upon stimulus removal. This suggests that the termination of cGMP accumulation is due to inactivation of guanylate cyclase instead of a steady state of cGMP synthesis and degradation. Further accumulation of cGMP was approx. 75% reduced upon dilution of a cell suspension after stimulation with both agonists. Stimulation by 300 nM folic acid or by 30 nM N 10-methylfolic acid (a more potent agonist) yielded identical results. However, upon addition of deaminofolic acid the accumulation of cGMP continued normally if the cells had been stimulated with N 10-methylfolic acid, but only slightly in the case of a folic acid stimulus. The effect of stimulus duration on desensitization was monitored; it was observed that 50% desensitization was induced by stimulation for 1 s, while 4 s was sufficient for maximal desensitization. Short stimuli were observed to elicit high levels of desensitization without much excitation of guanylate cyclase. A desensitization-like process was observed at the level of the folate-binding chemotactic receptors as well. Relationships between the cGMP response data and folic acid receptor kinetics are discussed.

21. EFFECT OF AGE AND SITE ON BASAL AND E.COLI ST-STIMULATED INTESTINAL GUANYLATE CYCLASE ACTIVITY IN CHILDREN

E.Coli heat-stable enterotoxin (ST) induces diarrhea through the stimulation of the guanylate cyclase (GC)-cGMP system. The effect of ST on the human intestine has not been investigated nor is any information available on the activity, distribution, or development of GC activity in the human intestine. We have characterized these aspects of GC activity by measuring its basal and ST-stimulated activity in intestinal specimens, obtained operatively from 35 infants and children of 1 day to 16 years of age. GC activity was linear with protein concentration and time. Basal activity was similar in small intestine and in colon. In the small intestine, however, basal GC activity varied with age. It was maximal in children 1 day of age, then decreased linearly with age up to 8 months and became quite variable thereafter. In colon, an age-related pattern was not found. E.Coli ST stimulated GC activity in a dose-related manner. The effect of ST was linear with protein concentration and with time. In the small intestine, ST-stimulation of GC was twice that found in colon. Age affected the response of small intestinal GC to ST. Maximal response to ST was observed in children of 1 day of age and ST stimulation was significantly greater in children less than 1 year of age than in older children. In the colon, the response of GC to ST did not rhange with age. The greater responsiveness of small intestinal GC to ST in younger children may explain the severity of diarrhea induced by ST at that age.

Small and large intestinal guanylate cyclase activity in children: effect of age and stimulation by Escherichia coli heat-stable enterotoxin.

Heat-stable enterotoxin (ST) producing Escherichia coli are a common cause of diarrhea in infants. ST acts through the stimulation of the guanylate cyclase-cGMP system. The effect of ST on the human intestine has not been investigated nor is any information available on the activity, distribution, or development of guanylate cyclase activity in the human intestine. The purpose of this study, therefore, was to characterize, these aspects of guanylate cyclase activity and to study the effect of ST on the activity and responsiveness of guanylate cyclase in the intestine of infants and children of various ages. We measured guanylate cyclase activity in 35 intestinal specimens, obtained operatively, from children aged 1 day to 16 yr. Guanylate cyclase activity was linear with protein concentration and time. Basal activity was similar in small intestine and in colon. In the small intestine, however, basal guanylate cyclase activity varied with age. It was maximal in children 1 day of age, and although somewhat variable, decreased with age thereafter. In colon, an age-related pattern was not found. E. coli ST stimulated guanylate cyclase activity in all specimens in a dose-related manner. In the small intestine ST-stimulation of guanylate cyclase was twice that found in colon. Furthermore, age affected the response of small intestinal guanylate cyclase to ST. Maximal response to ST was observed in children 1 day of age and ST stimulation was significantly greater in children less than 1 yr of age than in older children. In the colon, the response of guanylate cyclase to ST did not change with age.(ABSTRACT TRUNCATED AT 250 WORDS)

Soluble guanylate cyclase activation by nitric oxide and its reversal: Involvement of sulfhydryl group oxidation and reduction

Pre-incubation of either crude or purified nitric oxide-stimulated soluble lung guanylate cyclase resulted in a temperature-dependent decay of enzyme activity. The decay of nitric oxide-stimulated activity during pre-incubation was associated with a reduced responsiveness of the enzyme to reactivation by a second exposure to nitric oxide. This loss of enzyme responsiveness to reactivation by nitric oxide was greater with purified guanylate cyclase than with the crude enzyme and was highly dependent upon the nitric oxide dose. The addition of dithiothreitol or other thiols to nitric oxide-stimulated enzyme markedly accelerated the decay of activity in a dose-dependent manner. In addition, thiols prevented the loss of responsiveness of guanylate cyclase to reactivation by nitric oxide. Nitric oxide-stimulated enzyme activity was, therefore, reversed by the addition of thiol reducing agents. The addition of the thiol oxidizing agents, diamide or oxidized glutathione, to nitric oxide-stimulated guanylate cyclase caused a rapid and irreversible loss of activity. The effects of diamide or oxidized glutathione on the crude enzyme were prevented by excess dithiothreitol. Dithiothreitol did not prevent the destruction of purified nitric oxide-stimulated guanylate cyclase activity by diamide or oxidized glutathione, however. The results suggest that nitric oxide activation and its reversal are linked to the reversible oxidation and reduction, respectively, of sulfhydryl groups on guanylate cyclase which are involved in enzyme activation. The results further suggest the existence of a second class of sulfhydryl groups involved in the maintenance of enzyme activity.

Activation of guanylate cyclase by nitric oxide in mouse liver: Comparison of MNNG- with protoporphyrin IX-stimulated guanylate cyclase

It is known that guanylate cyclase(GC) from various tissues is stimulated by nitric oxide, nitroso compounds and protoporphyrin IX(PTP IX). In this report, we investigated the responsiveness of mouse liver GC to MNNG or porphyrins.

Selective alterations in responsiveness of guanylate cyclase to activation by nitroso compounds during enzyme purification

Partially purified, heme-free, hepatic soluble guanylate cyclase was activated by NO, S-nitrosocysteine and NO-heme complexes to the extent of 19–34-fold in the presence of 3 mM Mg 2+, but only up to 2-fold in the presence of 3mM Mn 2+, when the GTP concentration was 1 mM. Even with Mg 2+, however, nitroprusside and nitrosoguanidine failed to activate guanylate cyclase. Dithiothreitol or cysteine and, to a much lesser extent, hematin or hemoglobin restored the capacity of nitroprusside and nitrosoguanidine to activate guanylate cyclase, and enhanced enzyme activation by NO. Restoration or enhancement of enzyme activation with thiols was unrelated to their reducing potential because nonthiol reductants such as dithionite and ascorbate failed to influence guanylate cyclase activation. Instead, thiols likely reacted with the nitroso compounds to form S-nitrosothiols, which were potent activators of guanylate cyclase. Similarly, heme-containing substances reacted with nitroso compounds to form NO-heme complexes, which are known to activate guanylate cyclase. Enzyme activation by nitroprusside and nitrosoguanidine was restored, and that by NO and NO-hemoglobin was enhanced, by addition of heated hepatic soluble fraction (devoid of guanylate cyclase activity). The heated soluble fraction appears to contain a partially heat-stable, thiol-containing component(s) of molecular weight greater than 5000, which may be in part responsible for the observed effects on enzyme activation. These data suggest that partial enzyme purification results in the removal of thiol components that are required for the full expression of guanylate cyclase activation.