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.

Effect of different gonadal states on the forskolin stimulated adenylate cyclase activity in rat brain.

Forskolin-stimulated adenylate cyclase activity, measured in the hypothalamus and cerebral cortex differs in male and female rats. The gonadal steroid treatment performed induced changes in the studied adenylate cyclase activity probably in relation to the sex of the animals. The stimulated-forskolin adenylate cyclase activity in the hypothalamus from orchidectomized males showed more sensitivity than ovariectomized females. Finally, in male rats, the effects of castration on the hypothalamic enzymatic activity were partially restored by the administration of testosterone dipropionate. On the other hand, estradiol decreased the forskolin-adenylate cyclase activity in the female hypothalamus and cerebral cortex. The results show that the forskolin-stimulated adenylate cyclase activity may be related with the sex and/or the gonadal state of experimental animals.

Guanylate cyclase activity in retina optic nerve and optic chiasm of rats under different illumination conditions.

In the present work we have measured the guanylate cyclase activity in soluble fractions from several tissues relevant to the visual response under different illumination conditions. Guanylate cyclase was sensitive to changes of light/dark periods in incubated extract obtained from soluble fractions of retina, optic nerve and optic chiasm. The changes in soluble 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 inhibit strongly the soluble retinal guanylate cyclase activity; while it increases the activity of this enzyme in the optic nerve. A generalized photoinhibited response of soluble guanylate cyclase was observed in all studied tissues in prolonged dark adapted animals. The effect of Na+ 1 and 10 mM, and free Ca++ 28 eta M and 2.8 microM on the guanylate cyclase activity was performed in the studied tissues. The enzymatic activity appeared to be inversely related in the retina and optic nerve with regard to the ion exposure, which may involve different ionic control mechanisms. All indicate an active role for the soluble guanylate cyclase in the phototransduction process not only in retina, also in other tissues relevant in the visual response.

Guanylate cyclase activity in the inner ear and auditory nerve of the rat.

Novel molecular mediation in the sound transductional process is implicitly suggested. We investigated the presence of the cGMP-synthesis enzyme, guanylate cyclase, in Corti's organ and auditory nerve of the rat. The soluble guanylate cyclase activity found was sensitive to changes of sound intensity in the different acoustic media used, suggesting a potential role for the system that involves this enzyme. The guanylate cyclase activity appeared to be inversely related (in the inner ear) with the sound intensity to which the animals were exposed; different behavior was observed for the auditory nerve. The enzymatic activity found in Corti's organ, a direct bio-receptor of sound, represents the first reported enzymatic activity of this type in this tissue, which apparently could be influenced by the intensity of a physical stimulus such as sound. Finally, an adequate ionic environment appears to play a potential role in the expression of the changes observed, indicating that it may function according to the requirements of the biological sensor.

Effect of ovarian steroids on hypothalamic norepinephrine-induced adenylate cyclase activity.

The adenylate cyclase activity induced by norepinephrine (10(-8)-10(-9) M) was studied in hypothalamic particulate fractions from female rats. The effect of estradiol on this activity was studied in rats that were ovariectomized in diestrus 1, injected with estradiol benzoate (50 micrograms/kg body weight), and killed 48 h later. The effect of progesterone was studied in fractions from female cycling rats injected in the morning of diestrus 2 with progesterone (2 mg/rat); these animals were killed 30 or 48 h after the steroid injection. The blockade of norepinephrine-induced adenylate cyclase activity by alpha- and beta-blocking agents (10(-8)-10(-9) M) was also evaluated. The enzymatic activity was determined by monitoring the capacity to produce cAMP from ATP at saturated levels; cAMP was assayed by radioimmunoassay. At 48 h after administration, estradiol benzoate increased the norepinephrine-induced adenylate cyclase activity in the hypothalamus. This effect was not changed by the presence of phenoxybenzamine, an alpha-adrenoceptor blocker, but was greatly reduced by propranolol, the beta-adrenoreceptor blocker. In contrast, the progesterone, at 30 and 48 h after injection, decreased the hypothalamic adenylate cyclase activity, and this effect was preferentially antagonized in the presence of phenoxybenzamine rather than propranolol. These results suggest that estrogen and progesterone act selectively on one or another type of adrenergic receptor at the hypothalamic level.

Preparation, production and functional characterization of anti cAMP and cGMP antibodies.

The present work was intended to evaluate the preparation of antigens, as well as the production and characterization of anti cAMP and cGMP antibodies. Such antibodies were obtained from rabbits, and we used 2'O-succinyl cyclic nucleotide derivate, conjugated with human serum albumin, as antigen. The characterization of the antibodies was monitored by their immunoreactivity with the labelled antigen [125I]-cyclic nucleotide. This assay consists in a competition between a labelled and an unlabelled antigen for a fixed number of binding sites present in the specific antibody. The antibodies were specific for the inducing antigens. Cross-reactivity tests showed low degree competition between the immunogen and other antigens. The very high affinity, high quality and specificity of the generated antibodies indicate that they may be used not only in radioimmunoassay and immunocytochemistry methodologies, but also as bioblockers of physiological pathways.

Sexual differences in hypothalamic adenosine 3',5'-cyclic monophosphate content.

Hypothalamic adenosine 3',5'-cyclic monophosphate (cAMP) concentration was measured in the morning (10:00) and afternoon (16:00) in castrated and intact rats of both sexes to evaluate a possible sexual difference. Castration produced different effects on the hypothalamic cAMP concentration. In females, ovariectomy significantly reduced total hypothalamic cAMP concentration, whereas in males, orchidectomy elevated hypothalamic cAMP content. This observation was independent of the time of day at which the animals were killed. On the other hand, administration of the alpha-adrenoreceptor blocking agent phenoxybenzamine or the beta-adrenoreceptor blocker propranolol lowered the concentration of cAMP in the hypothalamus. However, this effect was selective and related to the time of day when the animals were killed. Phenoxybenzamine reduced the hypothalamic cAMP content in males killed in the afternoon, whereas the effect of propranolol was significant in males killed in the morning. These results indicate a response of hypothalamic cAMP to adrenoreceptor blockers in male rats opposite to that reported previously from our laboratory in female rats, suggesting that the functional behavior of the hypothalamic adrenergic response is strongly sex related.

Effect of progesterone on the hypothalamic cAMP concentration in estradiol-primed rats.

The effect of progesterone on the concentration of adenosine 3',5' cyclic monophosphate (cAMP) in the hypothalamus of ovariectomized estradiol primed rats was studied. Progesterone (2 mg/rat), injected 3 days after the priming dose of estradiol benzoate into ovariectomized rats, produced 4 h later an increase in the hypothalamic nucleotide content; whereas the lowest levels of cAMP were found 24 h after progesterone injection suggesting a biphasic response. The increase of hypothalamic cAMP observed at 4h of progesterone injection was blocked by the administration of an alpha(phenoxybenzamine) or a beta-adrenoblocker (propranolol) suggesting the participation of both, alpha and beta adrenergic receptors. Nevertheless, the administration of diethyldithiocarbamate, a blocker of norepinephrine synthesis has no effect to prevent the increase of hypothalamic cAMP induced by progesterone. Since no changes were observed in the hypothalamic cAMP concentration in the animals killed by afternoon, it may be inferred that the effects of progesterone on this parameter may depended on the time of the day at which it was administered.

Effect of progesterone on the hypothalamic cAMP concentration in estradiol-primed rats.

The effect of progesterone on the concentration of adenosine 3,5 cyclic monophosphate (cAMP) in the hypothalamus of ovariectomized estradiol primed rats was studied. Progesterone (2 mg/rat), injected 3 days after the priming dose of estradiol benzoate into ovariectomized rats, produced 4 h later an increase in the hypothalamic nucleotide content; whereas the lowest levels of cAMP were found 24 h after progesterone injection suggesting a biphasic response. The increase of hypothalamic cAMP observed at 4h of progesterone injection was blocked by the administration of an alpha(phenoxybenzamine) or a beta-adrenoblocker (propranolol) suggesting the participation of both, alpha and beta adrenergic receptors. Nevertheless, the administration of diethyldithiocarbamate, a blocker of norepinephrine synthesis has no effect to prevent the increase of hypothalamic cAMP induced by progesterone. Since no changes were observed in the hypothalamic cAMP concentration in the animals killed by afternoon, it may be inferred that the effects of progesterone on this parameter may depended on the time of the day at which it was administered.

Norepinephrine--stimulated activity of hypothalamic adenylate cyclase varies throughout the estrous cycle of the female rat.

The activity of hypothalamic adenylate cyclase was studied throughout the estrous cycle of the female rat. The activity of the enzyme was determined in particulate fractions obtained from hypothalami of rats killed at 10.00 h and 16.00 h of the 4-day estrous cycle. The activity was assayed in the presence of norepinephrine (10(-8) to 10(-3) M) by the capacity to produce adenosine 3',5' cyclic monophosphate. The basal activity of adenylate cyclase was higher in the morning of estrus than at any other time during the cycle. Norepinephrine-stimulated adenylate cyclase activity, as assessed by the apparent affinity (Kd) and apparent maximum effect, varied during the cycle, showing highest affinity, lowest Kd, in the afternoon of proestrus. The highest level of apparent maximum effect was also found in the afternoon of proestrus declining on diestrous day 2, diestrous day 1 and estrus. The norepinephrine stimulated activity was significantly inhibited by phenoxybenzamine, an alpha-blocker, in the morning of diestrus day 1, whereas on the day of diestrus day 2 and proestrus it was inhibited by the beta-adrenoblocker, propranolol. A similar degree of inhibition by alpha- and beta-blockers was observed in the morning of estrus. These results indicate that the hypothalamic adenylate cyclase coupled to adrenergic receptors shows dynamic changes throughout the estrous cycle.

Norepinephrine: stimulated activity of hypothalamic adenylate cyclase varies throughout the estrous cycle of the female rat

The activity of hypothalamic adenylate cyclase was studied throughout the estrous cycle of the female rat. The activity of the enzyme was determined in particulate fractions obtained from hypothalami of rats killed at 10.00 h and 16.00 h of the 4-day estrous cycle. The activity was assayed in the presence of norepinephrine (10(-8) to 10(-3) M) by the capacity to produce adenosine 3',5' cyclic monophosphate. The basal activity of adenylate cyclase was higher in the morning of estrus than at any other time during the cycle. Norepinephrine-stimulated adenylate cyclase activity, as assessed by the apparent affinity (Kd) and apparent maximum effect, varied during the cycle, showing highest affinity, lowest Kd, in the afternoon of proestrus. The highest level of apparent maximum effect was also found in the afternoon of proestrus declining on diestrous day 2, diestrous day 1 and estrus. The norepinephrine stimulated activity was significantly inhibited by phenoxybenzamine, an alpha-blocker, in the morning of diestrus day 1, whereas on the day of diestrus day 2 and proestrus it was inhibited by the beta-adrenoblocker, propranolol. A similar degree of inhibition by alpha- and beta-blockers was observed in the morning of estrus. These results indicate that the hypothalamic adenylate cyclase coupled to adrenergic receptors shows dynamic changes throughout the estrous cycle

Norepinephrine--stimulated activity of hypothalamic adenylate cyclase varies throughout the estrous cycle of the female rat.

The activity of hypothalamic adenylate cyclase was studied throughout the estrous cycle of the female rat. The activity of the enzyme was determined in particulate fractions obtained from hypothalami of rats killed at 10.00 h and 16.00 h of the 4-day estrous cycle. The activity was assayed in the presence of norepinephrine (10(-8) to 10(-3) M) by the capacity to produce adenosine 3,5 cyclic monophosphate. The basal activity of adenylate cyclase was higher in the morning of estrus than at any other time during the cycle. Norepinephrine-stimulated adenylate cyclase activity, as assessed by the apparent affinity (Kd) and apparent maximum effect, varied during the cycle, showing highest affinity, lowest Kd, in the afternoon of proestrus. The highest level of apparent maximum effect was also found in the afternoon of proestrus declining on diestrous day 2, diestrous day 1 and estrus. The norepinephrine stimulated activity was significantly inhibited by phenoxybenzamine, an alpha-blocker, in the morning of diestrus day 1, whereas on the day of diestrus day 2 and proestrus it was inhibited by the beta-adrenoblocker, propranolol. A similar degree of inhibition by alpha- and beta-blockers was observed in the morning of estrus. These results indicate that the hypothalamic adenylate cyclase coupled to adrenergic receptors shows dynamic changes throughout the estrous cycle.

Norepinephrine--stimulated activity of hypothalamic adenylate cyclase varies throughout the estrous cycle of the female rat.

The activity of hypothalamic adenylate cyclase was studied throughout the estrous cycle of the female rat. The activity of the enzyme was determined in particulate fractions obtained from hypothalami of rats killed at 10.00 h and 16.00 h of the 4-day estrous cycle. The activity was assayed in the presence of norepinephrine (10(-8) to 10(-3) M) by the capacity to produce adenosine 3,5 cyclic monophosphate. The basal activity of adenylate cyclase was higher in the morning of estrus than at any other time during the cycle. Norepinephrine-stimulated adenylate cyclase activity, as assessed by the apparent affinity (Kd) and apparent maximum effect, varied during the cycle, showing highest affinity, lowest Kd, in the afternoon of proestrus. The highest level of apparent maximum effect was also found in the afternoon of proestrus declining on diestrous day 2, diestrous day 1 and estrus. The norepinephrine stimulated activity was significantly inhibited by phenoxybenzamine, an alpha-blocker, in the morning of diestrus day 1, whereas on the day of diestrus day 2 and proestrus it was inhibited by the beta-adrenoblocker, propranolol. A similar degree of inhibition by alpha- and beta-blockers was observed in the morning of estrus. These results indicate that the hypothalamic adenylate cyclase coupled to adrenergic receptors shows dynamic changes throughout the estrous cycle.

Norepinephrine: stimulated activity of hypothalamic adenylate cyclase varies throughout the estrous cycle of the female rat

The activity of hypothalamic adenylate cyclase was studied throughout the estrous cycle of the female rat. The activity of the enzyme was determined in particulate fractions obtained from hypothalami of rats killed at 10.00 h and 16.00 h of the 4-day estrous cycle. The activity was assayed in the presence of norepinephrine (10(-8) to 10(-3) M) by the capacity to produce adenosine 3,5 cyclic monophosphate. The basal activity of adenylate cyclase was higher in the morning of estrus than at any other time during the cycle. Norepinephrine-stimulated adenylate cyclase activity, as assessed by the apparent affinity (Kd) and apparent maximum effect, varied during the cycle, showing highest affinity, lowest Kd, in the afternoon of proestrus. The highest level of apparent maximum effect was also found in the afternoon of proestrus declining on diestrous day 2, diestrous day 1 and estrus. The norepinephrine stimulated activity was significantly inhibited by phenoxybenzamine, an alpha-blocker, in the morning of diestrus day 1, whereas on the day of diestrus day 2 and proestrus it was inhibited by the beta-adrenoblocker, propranolol. A similar degree of inhibition by alpha- and beta-blockers was observed in the morning of estrus. These results indicate that the hypothalamic adenylate cyclase coupled to adrenergic receptors shows dynamic changes throughout the estrous cycle (Au)

Hypothalamic cyclic AMP after the injection of ovarian steroids into ovariectomized rats.

The effect of ovarian steroids on the concentration of adenosine 3',5'-cyclic monophosphate (cAMP) in the hypothalamus was studied in ovariectomized rats. Ovariectomized rats exhibited a lower cAMP concentration than intact rats. The administration of a single dose of estradiol benzoate (50 micrograms/kg body weight) resulted 3 days later in a rise of cAMP values, but levels did not reach those observed in estrous rats. Progesterone (2 mg/rat) injected 3 days after the priming dose of estradiol benzoate produced 4 h later no further changes in hypothalamic cAMP. The changes in hypothalamic cAMP concentration induced by estrogen treatment depend, at least in part, on noradrenergic inputs, since they were prevented by the injection of the norepinephrine synthesis inhibitor, diethyldithiocarbamate. In addition, administration of the beta-blocking agent, propranolol, to estradiol- and estradiol-progesterone-treated rats lowered the concentration of cAMP in the hypothalamus in a dose-dependent manner. In contrast, the administration of an alpha-blocking agent, phenoxybenzamine, had no effect at the tested concentration. The results of this study indicate that estrogen increases cAMP concentration in the hypothalamus by a noradrenergic mechanism involving beta-receptors. Moreover, the findings suggest that estrogen induces an increase in the number of beta-receptor sites, whereas progesterone increases the apparent propranolol sensitivity for these receptor sites.

Bradykinin stimulates GTP hydrolysis in NG108-15 membranes by a high-affinity, pertussis toxin-insensitive GTPase.

In membranes of neuroblastoma x glioma hybrid (NG108-15) cells, bradykinin (EC50 approximately equal to 5 nM) stimulates GTP hydrolysis by a high-affinity GTPase (Km approximately equal to 0.2 microM). The octapeptide, des-Arg9-bradykinin, was inactive. Stimulation of GTP hydrolysis by bradykinin and an opioid agonist was partially additive. Treatment of NG108-15 cells with pertussis toxin, which inactivates Ni, eliminated GTPase stimulation by the opioid agonist but not by bradykinin. The data suggest that bradykinin activates in NG108-15 membranes a guanine nucleotide-binding protein which is not sensitive to pertussis toxin and which may be involved in bradykinin-induced stimulation of phosphoinositide metabolism in these cells.

Dual regulation of adenylate cyclase. A signal transduction mechanism of membrane receptors.

The hormone-sensitive adenylate cyclase is a multi-component system embedded in the lipid bilayer of the plasma membrane and serves as a signal transduction system for various membrane receptors. The complete system consists of various receptor molecules, which sensitize the external ligands, the effector enzyme adenylate cyclase, which catalyzes the formation of cyclic AMP from ATP, and two guanine nucleotide-binding regulatory proteins (N or G proteins), which transduce the signals from the receptors to the adenylate cyclase. Depending on the receptor type activated by a ligand, stimulatory or inhibitory, either the stimulatory or the inhibitory N protein is activated and induces stimulation or inhibition of adenylate cyclase with subsequent increase or decrease in cellular cyclic AMP levels. In this paper, the mechanisms of this hormonal signal transduction system and its regulation will briefly reviewed, with some emphasis on the cardiac system.

Hypothalamic cyclic-AMP throughout the 4-day estrous cycle of the female rat.

The concentration of adenosine 3',5'-cyclic monophosphate (cAMP) in the hypothalamus was measured in the morning (10.00 h) and in the afternoon (16.00 h) during the 4-day estrous cycle of the female rat. The values were found to rise from the lowest levels on the afternoon of diestrus 1 to the highest levels on the morning of proestrus to decline thereafter. A second less marked peak was seen on the morning of diestrus 1. In rats treated with diethyldithiocarbamate to block norepinephrine (NE) synthesis, the concentration of the nucleotide decreased and remained at steady levels indicating that cyclic variations of cAMP in the hypothalamus are related to the endogenous release of NE. Blocking alpha-adrenergic receptors with phenoxybenzamine or beta-adrenergic receptors with propranolol also resulted in a drop of the concentration of cAMP in the hypothalamus. The effect of phenoxybenzamine was almost constant during the different days of the cycle whereas that of propranolol varied showing maximal effects on diestrus 2 and proestrus and minimal on estrus and diestrus 1. These results suggests that the response of endogenously released NE in the hypothalamus mediated through beta-adrenergic receptors undergo variations during the estrous cycle, whereas those mediated through alpha-adrenergic receptors remain at constant levels.