Metabolic regulation of steroidogenesis in isolated adrenal cells of rat. Relationship of adrenocorticotropin-, adenosine 3':5'-monophosphate-and guanosine 3':5'-monophosphate-stimulated steroidogenesis with the activation of protein kinase.

The data presented with the isolated adrenal cells, in the present study, show that adrenocorticotropin in the physiological concentration range stimulates the synthesis of guanosine 3':5'-monophosphate(cyclic GMP), protein kinase activity, and steroidogenesis in a concentration-dependent manner without detectable rise in the levels of adenosine 3':5'-monophosphate (cyclic AMP). Millimolar concentrations of cyclic AMP and cyclic GMP, which stimulate corticosterone synthesis, also activate kinase activity and steroidogenesis in a sigmoid concentration-response manner. The process of phosphorylation activated by corticotropin, cyclic AMP and cyclic GMP is not inhibited by cycloheximide or actinomyin D. It is therefore proposed that the hormonal responses mediated by cyclic GMP and cyclic AMP are via the protein kinase enzymatic steps, and the inhibitory effect of cycloheximide and actinomycin D in corticotropin-stimulated steroidogenesis follows this step. In conjuction with our previous observations that the biosynthetic steps from (20S)-20-hydroxycholesterol to corticosterone are neither inhibited by cycloheximide nor affected by cyclic GMP, it is inferred that the rate-limiting step of adrenal steroidogenesis is the transformation of cholesterol to (20S)-20hydroxycholesterol and this very step is regulated by cyclic GMP and cyclic AMP. Of further significance are the findings that micromolar cincentrations of cyclic AMP and cyclic GMP, which do not stimulate steroidogenesis, effectively stimulate protein kinase activity in a concentration-dependent manner. It is therefore concluded that all cyclic-nucleotide-dependent protein kinase activities of the cell are not necessarily related to steroidogenesis.     

Inhibition of aldosterone synthesis by atrial natriuretic factor

Atrial natriuretic factor (ANF) inhibits basal and stimulated aldosterone synthesis in adrenal glomerulosa cells. ANF probably acts through specific membrane receptors. Alterations in cyclic GMP and cyclic AMP levels do not account for ANF's inhibitory effect. ANF does not block angiotensin II (AngII) receptors nor does it interfere with phosphoinositide metabolism or calcium movements stimulated by adrenal agonists. ANF does not inhibit protein synthesis nor does it work by inhibiting NA+,K+-ATPase or depleting cell potassium. ANF decreases conversion of endogenous cholesterol to pregnenolone, the step stimulated by adrenocorticotropin and AngII. ANF does not affect the conversion of 20-alpha-hydroxycholesterol, which easily penetrates mitochondrial membranes to the site of the cholesterol side-chain cleavage enzyme. These results suggest that ANF inhibits the ability of endogenous cholesterol to reach or interact with the side-chain cleavage enzyme. ANF does not act like a calcium channel-blocking agent. However, ANF is less effective at high-calcium concentrations, which suggests that it may inhibit a step that calcium stimulates. Understanding ANF action will probably require identification of the specific biochemical changes (mediators) that it induces. Parallel efforts to understand how other agents stimulate steroidogenesis (particularly in the areas of protein synthesis, protein phosphorylation, and cholesterol movements) will further this understanding.     

Regional sensitivity of cyclic AMP and cyclic GMP in rat brain to central cholinergic stimulation

We determined cyclic AMP and cyclic GMP levels in 18 regions of rat brain following administration of two different centrally active cholinergic agonists. Administration of oxotremorine (2 mg/Kg IP), a muscarinic agonist, 10 minutes prior to sacrifice by exposure to high power microwave irradiation resulted in significant increases in cyclic GMP in cerebellum, brainstem, hippocampus, midbrain, thalamus and septal region. Cyclic AMP levels were significantly elevated in substantia nigra, nucleus interpeduncularis, hypothalamus, brainstem, midbrain and in the pituitary where a greater than tenfold increase was observed. Levels of plasma prolactin and corticosterone did not differ in any of the groups examined, but growth hormone was significantly lower in animals exposed to oxotremorine. Physostigmine (0.5 mg/Kg) a cholinesterase inhibitor, administered IP also produced elevations in cyclic AMP and cyclic GMP in several of the brain regions examined. These results indicate that multiple regions of the brain are responsive to central cholinergic activation of not only cyclic GMP, but also cyclic AMP system.     

The effect of isoproterenol and its analogs upon adenosine 3′,5′-monophosphate and guanosine 3′,5′-monophosphate levels in mouse parotid gland in vivo: Relationship to the stimulation of DNA synthesis

The ability of a large number of catecholamine analogs to stimulate DNA synthesis in the mouse parotid gland in vivo was compared to their effect on the levels of adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP) in this tissue. In the normal parotid gland the level of cyclic GMP is very low (10^?9 moles/kg wet wt), being only 1/800th of the cyclic AMP concentration. Isoproterenol increases the levels of cyclic AMP and cyclic GMP 30- and 3-fold, respectively. The increase in cyclic AMP is biphasic with an apparent early maximum at 2.5 min and a main peak at 15 min while the increase in cyclic GMP is monophasic with maximum levels at 15 min. Other analogs showed a similar effect on cyclic AMP levels but the time course of increases in cyclic GMP was very variable with peak stimulation as early as 1 min in some cases. The ability of analogs to cause the accumulation of cyclic AMP was correlated with their capacity to activate adenylate cyclase in parotid extracts and to act as β-adrenergic agonists in other systems. All compounds which raised cyclic AMP levels stimulated DNA synthesis but a number of other analogs also stimulated DNA synthesis. The effects of these analogs have been correlated with their ability to raise the intracellular concentration of cyclic GMP. Cholinergic agents also cause the accumulation of cyclic GMP but the effect of the analogs does not appear to be mediated through the cholinergic system as atropine does not block their effects and cholinergic agonists do not stimulate DNA synthesis. It is suggested that cholinergic agonists and the catecholamine analogs act primarily on the duct and acinar cells, respectively.Significant with inhibitors of the rises in cyclic nucleotide levels suggest that in isoproterenol stimulation it is the rise in cyclic GMP which is the more significant event in relation to stimulation of DNA synthesis.     

Development of Adrenocortical Cyclic Nucleotide (Cyclic AMP and Cyclic GMP) and Corticosterone Orcadian Rhythms in Male and Female Rats

The daily rhythm of the adrenocortical cyclic nucleotides (cyclic AMP and cyclic GIMP) was studied in infant male and female Wistar rats before and after the establishment of an adult-like daily rhythm of plasma corticosterone. As in this strain the rhythm of corticosterone is known to be present on postnatal day 18, pups of 2 and 3 weeks of age were studied. The dams and the pups as well as the young adult animals were kept on a controlled 12L-12D photoperiod. Groups of 8-10 pups were killed at 4-hr intervals throughout the day. Plasma corticosterone levels and adrenal cyclic AMP and cyclic GMP concentrations were simultaneously measured and the daily patterns established. Pups of 2 weeks of age showed neither plasma corticosterone nor adrenal cyclic AMP rhythms whereas pups of 3 weeks of age exhibited a typical adult-like circadian rhythm for both variables. The patterns for adrenal cyclic GMP differed according to sex: In female pups no cyclic GMP circadian rhythm could be detected at either 2 or 3 wk. In male pups of 3 wk a typical mature rhythm for adrenal cyclic GMP was evident whereas in younger male pups (2 wk) a circadian rhythm was detected. This circadian rhythm, however, differed from mature circadian rhythm in that its peak was located at 1300 hr instead of 0700 hr. These results demonstrate that, unlike that of cyclic AMP the adrenal cyclic GMP circadian rhythm does not appear at the same time as the plasma corticosterone circadian rhythm. Moreover, a circadian rhythmicity for adrenal cyclic GMP can be found in the absence of any corticosterone circadian rhythm. These facts argue against the view of cyclic GMP being a mediator of ACTH-stimulated steroidogenesis.     

Development of Adrenocortical Cyclic Nucleotide (Cyclic AMP and Cyclic GMP) and Corticosterone Orcadian Rhythms in Male and Female Rats

The daily rhythm of the adrenocortical cyclic nucleotides (cyclic AMP and cyclic GIMP) was studied in infant male and female Wistar rats before and after the establishment of an adult-like daily rhythm of plasma corticosterone. As in this strain the rhythm of corticosterone is known to be present on postnatal day 18, pups of 2 and 3 weeks of age were studied. The dams and the pups as well as the young adult animals were kept on a controlled 12L-12D photoperiod. Groups of 8–10 pups were killed at 4-hr intervals throughout the day. Plasma corticosterone levels and adrenal cyclic AMP and cyclic GMP concentrations were simultaneously measured and the daily patterns established. Pups of 2 weeks of age showed neither plasma corticosterone nor adrenal cyclic AMP rhythms whereas pups of 3 weeks of age exhibited a typical adult-like circadian rhythm for both variables. The patterns for adrenal cyclic GMP differed according to sex: In female pups no cyclic GMP circadian rhythm could be detected at either 2 or 3 wk. In male pups of 3 wk a typical mature rhythm for adrenal cyclic GMP was evident whereas in younger male pups (2 wk) a circadian rhythm was detected. This circadian rhythm, however, differed from mature circadian rhythm in that its peak was located at 1300 hr instead of 0700 hr. These results demonstrate that, unlike that of cyclic AMP the adrenal cyclic GMP circadian rhythm does not appear at the same time as the plasma corticosterone circadian rhythm. Moreover, a circadian rhythmicity for adrenal cyclic GMP can be found in the absence of any corticosterone circadian rhythm. These facts argue against the view of cyclic GMP being a mediator of ACTH-stimulated steroidogenesis.     

Cyclic nucleotides and their relationship to complement-component-C2 synthesis by human monocytes.

The time courses of changes in cyclic nucleotide levels in monocytes have been studied. Histamine and prostaglandin E2 (PGE2) produced a rapid rise in cyclic AMP (peak 15 min) levels, which returned to normal within 4h, whereas cholera toxin, NaF and phosphodiesterase inhibitors produced slow sustained rises lasting over 24h. With the exception of isobutylmethylxanthine (10 mumol X 1(-1), none of these reagents altered cyclic GMP levels. alpha 1-Adrenergic and nicotinic cholinergic receptor-ligand interactions and imidazole produced rapid and relatively short-lived falls in cyclic AMP, and rises in cyclic GMP. In contrast, prostaglandin synthetase inhibitors produced delayed but more sustained falls in cyclic AMP but no rises in cyclic GMP. Agents that increased cyclic AMP decreased complement-component-C2 production, and those that decreased cyclic AMP increased C2 production. Agents that increased cyclic GMP alone (ascorbate, nitroprusside and prostaglandin F2 alpha) did not affect C2 production. Antigen-antibody complexes that stimulate C2 synthesis produced falls in cyclic AMP and rises in cyclic GMP similar to those produced by adrenergic and cholinergic ligands. Serum-treated complexes and anaphylatoxins, which inhibited C2 production, were associated with changes in cyclic AMP similar to those produced by histamine and PGE2. These data suggest that there are two transmembrane signals involved in the regulation of C2 production by monocytes. The inhibitory signal is adenylyl cyclase activation. The stimulatory signal is not so obvious, but may be Ca2+ influx, since the time courses of changes in cyclic nucleotides produced by agents that stimulate C2 synthesis are identical, and alpha 1-adrenergic agonists cause the formation of Ca2+ channels.     

Atrial natriuretic factor and sodium nitroprusside increase cyclic GMP in cultured rat lung fibroblasts by activating different forms of guanylate cyclase.

We used cultured rat lung fibroblasts to evaluate the role of particulate and soluble guanylate cyclase in the atrial natriuretic factor (ANF)-induced stimulation of cyclic GMP. ANF receptors were identified by binding of 125I-ANF to confluent cells at 37 degrees C. Specific ANF binding was rapid and saturable with increasing concentrations of ANF. The equilibrium dissociation constant (KD) was 0.66 +/- 0.077 nM and the Bmax. was 216 +/- 33 fmol bound/10(6) cells, which corresponds to 130,000 +/- 20,000 sites/cell. The molecular characteristics of ANF binding sites were examined by affinity cross-linking of 125I-ANF to intact cells with disuccinimidyl suberate. ANF specifically labelled two sites with molecular sizes of 66 and 130 kDa, which we have identified in other cultured cells. ANF and sodium nitroprusside produced a time- and concentration-dependent increase in intracellular cyclic GMP. An increase in cyclic GMP by ANF was detected at 1 nM, and at 100 nM an approx. 100-fold increase in cyclic GMP was observed. Nitroprusside stimulated cyclic GMP at 10 nM and at 1 mM a 500-600-fold increase in cyclic GMP occurred. The simultaneous addition of 100 nM-ANF and 10 microM-nitroprusside to cells resulted in cyclic GMP levels that were additive. ANF increased the activity of particulate guanylate cyclase by about 10-fold, but had no effect on soluble guanylate cyclase. In contrast, nitroprusside did not alter the activity of particulate guanylate cyclase, but increased the activity of soluble guanylate cyclase by 17-fold. These results demonstrate that rat lung fibroblasts contain ANF receptors and suggest that the ANF-induced stimulation of cyclic GMP is mediated entirely by particulate guanylate cyclase.     

Glucocorticoid modulation of adrenocorticotropin-induced melanogenesis in the Cloudman S-91 melanoma in vitro.

The acute in vitro action of adrenocorticotropin (ACTH) and corticosterone alone and in combination were determined in the Cloudman S-91 melanoma grown in vivo. Hormone-treated melanoma dice (5-240 min) were analyzed for tyrosinase activity (EC 1.14.18.1), cyclic AMP (cAMP) and cyclic GMP (cGMP). ACTH elevated cAMP levels in the S-91 melanoma. However, these increases in cAMP were not accompanied by increased tyrosinase activity. Corticosterone depressed cAMP levels while stimulating tyrosinase activity. ACTH plus corticosterone produced an early cAMP peak followed by depression. ACTH plus corticosterone stimulated tyrosine activity coincident with the early cAMP peak followed by a drop in tyrosinase activity which was subsequently elevated. cGMP levels were not altered by any hormone treatment. The results indicate that cAMP is not the sole modulator of tyrosinase activity and suggest the interaction of ACTH, corticosterone and cAMP in the regulation of melanoma tyrosinase activity.     

Renal and systemic effects of synthetic atrial natriuretic factor

A synthetic peptide corresponding to a sequence of 26 amino acids contained in endogenous rat atrial natriuretic factor (ANF), was infused into one renal artery of anesthetized dogs for a comprehensive in vivo evaluation of the renal and systemic effects of pure ANF. The results proved conclusively that ANF acted directly on the kidney since urine volume and fractional excretion of sodium, potassium, chloride and calcium were elevated in a dose-related manner in the ANF-treated kidney, but were not significantly affected in the contralateral saline-infused organ. The maximum effects achieved with the synthetic ANF were higher than any reported following intravenous administration of crude extracts of rat atria and were similar to those produced by thiazide diuretics. In four of the five dogs studied, renal vascular resistance fell progressively as doses of ANF were increased. Glomerular filtration rate was not significantly elevated during ANF infusion, but was correlated with sodium excretion rates. Even though mean arterial pressure was progressively reduced, there was no significant change in heart rate and no stimulation of renin secretion. Arterial cyclic GMP concentration was higher in the basal state and rose more rapidly than did renal venous levels, indicating that increases in circulating concentrations of arterial cyclic GMP originated from an extrarenal source. Dose-related elevations in urinary cyclic GMP excretion could be explained by increased cyclic GMP filtration, by enhanced production in tubular cells, or by renal tubular secretion. Especially in the saline-infused kidney, there was a clear dissociation between excretion of cyclic GMP and fractional sodium excretion. We conclude that the synthetic ANF increased electrolyte excretion via a direct renal action which was not solely dependent upon changes in renal vasculature, renin secretion or cyclic GMP levels.     

Cyclic nucleotide metabolism and reactive oxygen production by macrophages

The production of reactive oxygen species by elicited rat peritoneal macrophages was assessed by $\text{in vitro}$ measurement of chemiluminescence in the presence of luminol. The divalent ion ionophore A23187 stimulated the production of reactive oxygen species. This action was inhibited by monobutyryl and dibutyryl derivatives of cyclic AMP but was not affected by derivates of cyclic GMP. Cyclic AMP and cyclic GMP concentrations increased rapidly in macrophages exposed to A23187 or zymosan. Indomethacin (20 μmol/1) inhibited the increase in cyclic AMP concentration but not the increase in cyclic GMP concentration. Neither A23187 nor zymosan stimulated adenylate cyclase activity in broken cell preparations of macrophages. The observations are consistent with the hypothesis that PGE produced by macrophages after phagocytotic stimuli may inhibit certain macrophage functions and perform a regulatory role in these cells. This action of PGE may be mediated by cyclic AMP.     

Stimulation of guanosine 3',5'-cyclic monophosphate accumulation in rat anterior pituitary gland in vitro by synthetic somatostatin

Synthetic somatostatin stimulated cyclic GMP accumulation with dose dependency (10 ng/ml – 10 μg/ml in a dose examined) in rat anterior pituitary gland in vitro. The stimulation of cyclic GMP levels in the gland was observed after 2 min incubation with somatostatin. Cyclic AMP production induced by TRH or PGE₁ was supressed by this GH release inhibiting factor, while cyclic GMP concentration in the gland was elevated. The present results seem to suggest that inhibitory effect on GH release by somatostatin in anterior pituitary gland is mediated through change in concentration of cyclic AMP and cyclic GMP in the target cells.     

On the role of cyclic AMP and cyclic GMP in steroid production by bovine cortical cells

The time course of corticotropin-induced steroidogenesis and changes in intracellular cyclic AMP and cyclic GMP levels were investigated in isolated bovine adrenocortical cells prepared by trypsin digestion. Corticotropin produced a pea a peak rise in cyclic AMP during the first 5 min of stimulation and enhanced steroid production after 15 min. Corticotropin also caused a decrease in cortical cyclic GMP at 5 min; this decrease in cyclic GMP reverted to a 2–3 fold increase at 15–30 min which gradually subsided by 60 min. A steroidogenic concentration of prostaglandin E₂ also produced an elevation in the levels of both nucleotides, but the rise in cyclic GMP preceded the rise incyclic AMP. These results suggest that the relative amount of cyclic AMP and cyclic GMP, rather than the absolute levels of cyclic AMP, may be a key factor in the regulation of steroidogenesis.     

On the role of cyclic AMP and cyclic GMP in steroid production by bovine cortical cells.

The time course of corticotropin-induced steroidogenesis and changes in intracellular cyclic AMP and cyclic GMP levels were investigated in isolated bovine adrenocortical cells prepared by trypsin digestion. Corticotropin produced a peak rise in cyclic AMP during the first 5 min of stimulation and enhanced steroid production after 15 min. Corticotropin also caused a decrease in cortical cyclic GMP at 5 min; this decrease in cyclic GMP reverted to a 2-3 fold increase at 15-30 min which gradually subsided by 60 min. A steroidogenic concentration of prostaglandin E2 also produced an elevation in the levels of both nucleotides, but the rise in cyclic GMP preceded the rise in cyclic AMP. These results suggest that the relative amounts of cyclic AMP and cyclic GMP, rather than the absolute levels of cyclic AMP, may be a key factor in the regulation of steroidogenesis.     

Atrial Natriuretic Factors Stimulate Accumulation and Efflux of Cyclic GMP in C6–2B Rat Glioma and PC12 Rat Pheochromocytoma Cell Cultures

Atrial natriuretic factors (ANFs) were tested for their effects on cyclic GMP production in two neurally derived cell lines, the C6-2B rat glioma cells and the PC12 rat pheochromocytoma cells. These cell lines were selected because both are known to possess high amounts of the particulate form of guanylate cyclase, a proposed target of ANF in peripheral organs. Previous studies from our laboratory have shown that ANF selectively activates particulate, but not soluble, guanylate cyclase in homogenates of a variety of rat tissues and that one class of ANF receptor appears to be the same glycoprotein as particulate guanylate cyclase. In the present study we found that four analogs of ANF stimulate cyclic GMP accumulation in both C6-2B and PC12 cells with the rank order of potency being atriopeptin III = atriopeptin II greater than human atrial natriuretic polypeptide greater than atriopeptin I. Atriopeptin II (100 nM) for 20 min elevated cyclic GMP content in C6-2B cells fourfold and in PC12 cells 12-fold. Atriopeptin II (100 nM) for 20 min also stimulated the efflux of cyclic GMP from both C6-2B cells (47-fold) and PC12 cells (12-fold). Accumulation of cyclic GMP in both cells and media was enhanced by preincubation with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (250 microM). After 20 min of exposure to atriopeptin II, cyclic GMP amounts in the media were equal to or greater than the amounts in the cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of the pentose phosphate pathway from 14CO2 data. Fallibility of a classic equation when applied to non-homogeneous tissues.

Two cyclic nucleotide phosphodiesterase (PDE) activities were identified in pig aortic endothelial cells, a cyclic GMP-stimulated PDE and a cyclic AMP PDE. Cyclic GMP-stimulated PDE had Km values of 367 microM for cyclic AMP and 24 microM for cyclic GMP, and low concentrations (1 microM) of cyclic GMP increased the affinity of the enzyme for cyclic AMP (Km = 13 microM) without changing the Vmax. This isoenzyme was inhibited by trequinsin [IC50 (concn. giving 50% inhibition of substrate hydrolysis) = 0.6 microM for cyclic AMP hydrolysis in the presence of cyclic GMP; IC50 = 0.6 microM for cyclic GMP hydrolysis] and dipyridamole (IC50 = 5 microM for cyclic AMP hydrolysis in the presence of cyclic GMP; IC50 = 3 microM for cyclic GMP hydrolysis). Cyclic AMP PDE exhibited a Km of 2 microM for cyclic AMP and did not hydrolyse cyclic GMP. This activity was inhibited by trequinsin (IC50 = 0.2 microM), dipyridamole (IC50 = 6 microM) and, selectively, by rolipram (IC50 = 3 microM). Inhibitors of cyclic GMP PDE (M&B 22948) and of low Km (Type III) cyclic AMP PDE (SK&F 94120) only weakly inhibited the two endothelial PDEs. Incubation of intact cells with trequinsin and dipyridamole induced large increases in cyclic GMP, which were completely blocked by LY-83583. Rolipram, SK&F 94120 and M&B 22948 did not significantly influence cyclic GMP accumulation. Dipyridamole enhanced the increase in cyclic GMP induced by sodium nitroprusside. Cyclic AMP accumulation was stimulated by dipyridamole and trequinsin with and without forskolin. Rolipram, although without effect alone, increased cyclic AMP in the presence of forskolin, whereas M&B 22948 and SK&F 94120 had no effects on resting or forskolin-stimulated levels. These results suggest that cyclic GMP-stimulated PDE regulates cyclic GMP levels and that both endothelial PDE isoenzymes contribute to the control of cyclic AMP.     

REGULATION OF GUANOSINE 3'',5'' CYCLIC MONOPHOSPHATE IN THE RAT PINEAL AND POSTERIOR PITUITARY GLANDS

Potassium and norepinephrine stimulate the accumulation of cyclic AMP and cyclic GMP in rat pineal glands and their efflux into the medium. The efflux of both cyclic nucleotides was blocked by probenecid. The accumulation and efflux of cyclic GMP, but not of cyclic AMP, depends upon the presence of intact nerve endings and extracellular calcium. The calcium-dependent release of norepinephrine caused by veratridine was accompanied by the efflux of both cyclic AMP and cyclic GMP. In contrast, the calcium-independent release of norepinephrine caused by tyramine was accompanied by the efflux of cyclic AMP but not cyclic GMP. Changes in cyclic GMP therefore, may be related to exocytosis from the sympathetic nerve endings in the gland. High concentrations of potassium also increased tissue levels of cyclic GMP in the posterior pituitary gland. Veratridine and potassium, but not norepinephrine, stimulated the efflux of cyclic GMP from this neurosecretory gland. Thus, the relationship between cyclic GMP and exocytosis may extend beyond sympathetic nerve endings. The enhanced accumulation of cyclic GMP in the pineal gland after potassium does not appear to be mediated by extracellular (released) norepinephrine. Desmethylimipramine blocked the norepinephrine-stimulated changes in cyclic GMP, but not those caused by potassium. Investigation of the possible relationship between cyclic GMP and release of neurotransmitters is complicated by the apparent seasonal variation in the response of pineal cyclic GMP to potassium or norepinephrine.     

In vitro studies of the testis: the effect of LH on cyclic nucleotides.

This study evaluated the relationship between LH, cyclic AMP, cyclic GMP, and testosterone using in vitro incubation of decapsulated rat testes and sampling incubation medium. With added LH (1.0, 5.0, 100, and 500 mIU/ml) there were statistically significant increases in cyclic AMP at 5 mIU/ml or more LH, and progressively greater titers of this nucleotide were produced as LH was increased. For cyclic GMP all levels of added LH caused significant increments in titers of nucleotide; however, peak cyclic GMP concentrations occurred with 5 mIU/ml of LH. The addition of 10(-3) and 10-(4)M 8-bromo-cyclic AMP caused significant increases in testosterone production, while no changes in production of this androgen were found with 10(-3), 10(-4), or 10(-5)M 8-bromo-cyclic GMP. Neither cyclic AMP nor cyclic GMP titers were altered by the addition of 1 to 50 micrograms/ml of testosterone to medium bathing the rat testes. The dose response curves of cyclic AMP and cyclic GMP to LH are different. Progressive increments in added LH cause parallel increases of cyclic AMP and a biphasic change of cyclic GMP, 8-bromo-cyclic GMP does not cause testosterone generation, suggesting that cyclic GMP does not result in androgen synthesis. However, cyclic GMP may be involved in other Leydig cell functions.     

Detection of regional ischemia in perfused beating hearts by phosphorus nuclear magnetic resonance.

Rat Graafian follicles isolated intact responded to 8-Br-cyclic GMP (0.3 and 1.0 mM) with increased prostaglandin E (PGE) production (4-fold and 8-fold, respectively) during a 6 h incubation. The effect of 8-Br-cyclic GMP was noted after a lag period of 2–4 h. 8-Br-cyclic AMP (1.0 mM) also stimulated PGE production (4-fold increase), while 8-Br-cyclic IMP, 8-Br-5′GMP and 8-Br-5′AMP were inactive in this respect. Actinomycin D (10 μg/ml) and cycloheximide (10 μg/ml) given simultaneously with 8-Br-cyclic GMP prevented the stimulatory effect of the cyclic nucleotide. The results suggest that cyclic GMP induces de novo synthesis of a macromolecular component of the ovarian prostaglandin synthetase system, and that this cyclic nucleotide, along with cyclic AMP, may play a role in the known stimulatory action of luteinizing hormone on follicular prostaglandin production.     

Stimulation of rat liver cyclic 3':5'-nucleotide phosphodiesterase by cyclic GMP is dependent on enzyme concentration

A high-speed supernatant of rat liver extract displayed multiple forms of cyclic nucleotide phosphodiesterase (EC 3.1.4.17). One of the forms catalyzed the hydrolysis of cyclic AMP and cyclic GMP, with approximately comparable facility. One salient feature of the enzyme is that at micromolar concentrations, cyclic GMP stimulated the hydrolysis of cyclic AMP, but not vice versa. Another is that the activity of phosphodiesterase varied as a function of enzyme concentration in the assayed system: the enzyme activity was higher at low than at high enzyme concentrations. A concentrated enzyme was not stimulated by cyclic GMP but was stimulated by cyclic GMP upon dilution of the enzyme. Conversely, stimulation of the enzyme by cyclic GMP could be reversed by increasing the enzyme concentration. The cyclic GMP-stimulated cyclic AMP phosphodiesterase was partially purified by a continuous sucrose density gradient. The apparent change of phosphodiesterase activity as a function of enzyme concentration was also observed after partial purification by the sucrose density gradient. High enzyme concentrations favored the aggregated form of phosphodiesterase, whereas low concentrations favored the dissociated form. Dilution of the enzyme shifted the equilibrium toward the dissociated form, which presumably exposed the cyclic GMP regulatory site on the enzyme molecule.