Role of cyclic nucleotides in the inhibition of growth-hormone secretion by somatostatin

The effects of somatostatin on GH secretion, cyclic AMP and cyclic GMp concentrations in dispersed bovine anterior pituitary cells were studied following activation of adenylate cyclase with cholera toxin and inhibition of phosphodiesterase with isobutylmethylxanthine (IBMX). Cholera toxin (10(-5)M) increased intracellular cyclic AMP concentration 10-fold and cyclic GMP concentration 3-fold relative to control, and stimulated the secretion of GH. IBMX (10(-4) M) als increased intracellular concentrations of both cyclic AMP and cyclic GMP and the secretion of GH and potentiated the actions of cholera toxin particularly in raising intracellular cyclic AMP concentrations which were elevated 40-fold in the presence of cholera toxin and IBMX. Somatostatin (5 X 10(-7) M) completely prevented GH secretion elicited by cholera toxin and/or IBMX. Somatostatin was without effect on control cyclic AMP and cyclic GMp concentrations and on the increases in both cyclic AMP and cyclic GMP caused by cholera toxin and by IBMX alone, or in combination. The data suggest that bovine GH secretion is increased when concentrations of either or both cyclic nucleotides are elevated within the cells, although incubation of cells with extracellular concentrations of cyclic AMP and cyclic GMP derivatives up to 2 x 10(-3) M caused only small changes in GH release. We suggest that somatostatin inhibits cholera toxin-induced bovine GH secretion by preventing activation of the secretory process by either cyclic AMP or cyclic GMP.     

Effect of hypophysectomy on cyclic 3',5'-nucleotidemetabolizing enzymes in the rat thyroid gland

Adenylate cyclase and cyclic AMP phosphodiesterase activities in the thyroid gland were significantly reduced after hypophysectomy, followed by a gradual restoration of the enzyme activities to the levels seen in sham-operated rats whereas a slight and persistent reduction was evident in guanylate cyclase and cyclic GMP phosphodiesterase activities in the same tissue. These changes in enzyme activities were restored by TSH administration but not by ACTH. The recovery of activity produced by TSH administration was inhibited by cycloheximide. Hypophysectomy, or TSH and cycloheximide administration, did not produce any significant changes in the concentrations of calmodulin, suggesting that the alteration of these enzyme activities is not induced by a decrease in the concentration of calmodulin. Since forskolin activation of adenylate cyclase did not restore the reduced activity in the hypophysectomized rat thyroid to the level found in the sham-operated control rat thyroid, we conclude that there is a reduction of the amount of enzyme after hypophysectomy rather than a change of the active site on adenylate cyclase. The spontaneous restoration of adenylate cyclase and cyclic AMP phosphodiesterase activities after hypophysectomy implies that cyclic AMP-metabolizing enzymes are responsive to an autoregulatory mechanism in thyroid follicular cells.     

Testosterone and its precursors and metabolites enhance guanylate cyclase activity.

Both testosterone and cyclic GMP stimulate DNA synthesis. Because cyclic GMP and testosterone seem to have similar actions, the objective of this investigation was to determine if testosterone and its precursors might have part of their mechanism of action through stimulation of guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2], the enzyme that catalyzes the formation of cyclic GMP from GTP. The precursors--namely, progesterone, pregnenolone, 17 alpha-progesterone, 17 alpha-hydroxypregnenolone, androstenedione, and dehydroepiandrosterone--caused a 2- to 3 1/2-fold enhancement of guanylate cyclase activity in rat liver, kidney, skeletal muscle, and ventral prostate at a concentration of 1 microM. These precursors are generated from cholesterol, which had no effect itself on guanylate cyclase activity. Testosterone, 19-nortestosterone, 17-methyltestosterone, and 5 alpha-dihydrotestosterone enhanced guanylate cyclase activity 2- to 5-fold in the same tissues at 1 microM. Etiocholanolone, androsterone, and epiandrosterone, metabolites of testosterone metabolism, enhanced guanylate cyclase activity 1 1/2- to 2-fold at this same concentration. Dose-response relationships revealed that testosterone and its precursors and metabolites had their maximal effect at 1 microM but still had some effect at 0.001 microM. The data in this investigation suggest that the guanylate cyclase-cyclic GMP system plays a role in the mechanism of action of testosterone and its precursors.     

Effects of cholera enterotoxin, glucagon, and dibutyryl cyclic AMP on rat liver alkaline phosphatase, bile flow, and bile composition.

Cholera enterotoxin, 45 mug per 250 g body weight, administered intravenously to rats, caused a 6-fold rise in the activity of liver alkaline phosphatase in 12 hr. There was no change in bile volume or in the concentration or total bile content of Na+, K+, HCO3-, or Cl- for 36 hr after the administration of cholera toxin. However, bile phospholipid output fell markedly from a control level of 15.0 +/- 1.0 mumol per 6 hr to a low level of 4.0 +/- 1.2 mumol per 6 hr in the 12- to 18-hr collection, P less than 0.001. There was a similar fall in bile acid secretion, from a control value of 9.8 +/- 0.4 mumol per 6 hr to 4.1 +/- 0.9 mumol in the 12- to 18-hr period, P less than 0.01. The cholera effect was prolonged. Bile acid and phospholipid secretion rates did not return to control values until 30 to 36 hr after the administration of cholera enterotoxin. The cholera toxin-induced reductions in bile acid and phospholipid secretion into bile did not appear to be mediated by adenyl cyclase or cyclic AMP because neither glucagon, a known stimulator of liver adenyl cyclase, nor dibutyryl cyclic AMP had any effect on the secretion into bile of bile acids or phospholipid. The administration of cholera toxin was not associated with any increase in the secretion of free choline into bile. Glucagon and dibutyryl cyclic AMP, two other substances known to increase the activity of rat liver alkaline phosphatase, also had no stimulatory effect on the secretion of free choline into bile. The results do not support the hypothesis that the main function of rat liver alkaline phosphatase is to facilitate the excretion of free choline into bile.     

Effect of nipradilol, a new beta-blocker, on leukotriene D4-induced contraction in guinea pig tracheal smooth muscle

The relaxant effect on smooth muscle of nitro compounds is suggested to be linked with the increase in the tissue level of cyclic GMP by activating guanylate cyclase. In this study, we investigated the effects of nipradilol, a new beta-blocker, which has NO2 residue in the molecular structure, and isosorbide dinitrate (ISDN) on guinea pig tracheal smooth muscle in comparison with the effect of propranolol. Nipradilol and ISDN showed dose-dependent relaxant effects on leukotriene (LT) D4-induced contraction of tracheal smooth muscle, though propranolol had no effect. 8-Bromo-cyclic GMP also showed a relaxant effect dose dependently. Nipradilol and ISDN elevated cyclic GMP levels in tracheal tissue dose dependently; however, propranolol caused no change in cyclic GMP levels. From these results, it is suggested that nipradilol relaxes LTD4-induced contraction of tracheal smooth muscle by increasing the tissue level of cyclic GMP.     

Modulation of cyclic AMP in isolated rat uterine tissue slices by porcine relaxin

Porcine relaxin produced a rapid, dose-related rise of cyclic AMP values in rat uterine tissue incubated in vitro. In time-course experiments, peak cyclic AMP concentrations were observed in the uterine slices at 5 min; subsequently the values fell, at first rapidly and then more slowly with the tissue concentration remaining significantly raised at 15 min. Levels of cyclic GMP in the same tissue slices were not significantly altered by relaxin. Furthermore, no increase in basal cyclic AMP values was measured in control slices prepared from the rat heart or jejunum. An increase in cyclic AMP concentration comparable to that found in the rat uterus was observed in slices of porcine uterus and cervix but not of vagina when they were stimulated with porcine relaxin. Our results suggest that the hormonal action of relaxin on the uterus and cervix is mediated through receptors linked to the enzyme, adenylate cyclase.     

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)     

Effects of mammalian gonadotropins on progesterone release and cyclic nucleotide production by isolated avian granulosa cells

Cyclic AMP and, to a lesser extent, cyclic GMP have been implicated as mediators of gonadotropin-induced steroidogenesis in ovarian tissue and cells of mammals The functional role of these cyclic nucleotides in steroidogenesis in avian granulosa cells has not yet been investigated. In the present study the release of progesterone and levels of cyclic nucleotides were measured in granulosa cells isolated by a nonenzymatic procedure from the largest preovulatory follicle of laying hens 22–24 hr prior to ovulation. Bovine LH(NIH-B10) promoted progesterone production in a dose-related manner. Although steroidogenesis was maximally stimulated by 0.1–0.2 μg/ml bLH, cyclic nucleotide levels remained unaffected even at 5 μg/ml bLH at which concentration progesterone release was significantly inhibited when compared to maximal responses. Similarly, oFSH while stimulating steroidogenesis, although less effectively than bLH, failed to significantly increase cyclic AMP production. Theophylline potentiated the steroidogenic effect of gonadotropins and raised basal levels of the cyclic nucleotides. Dibutyryl cyclic AMP (BU₂cAMP) induced a dose-dependent release of progesterone while dibutyryl cyclic GMP (BU₂cGMP) had an inhibitory effect. Agonists of adenylate cyclase such as isoproterenol and cholera toxin in combination with theophylline stimulated progesterone production while heparin, an inhibitor of adenylate cyclase, completely blocked the steroidogenic effect of bLH. Moreover, imidazole, a phosphodiesterase activator, suppressed both progesterone and cyclic AMP production. It is suggested therefore that a small fraction of intracellular cyclic AMP, but not cyclic GMP, which cannot be accurately detected by the RIA method employed, plays an important role in LH-promoted steroidogenesis in chicken granulosa cells.     

Guanylin: an endogenous activator of intestinal guanylate cyclase.

Intestinal guanylate cyclase mediates the action of the heat-stable enterotoxin to cause a decrease in intestinal fluid absorption and to increase chloride secretion, ultimately causing diarrhea. An endogenous ligand that acts on this guanylate cyclase has not previously been found. To search for a potential endogenous ligand, we utilized T84 cells, a human colon carcinoma-derived cell line, in culture as a bioassay. This cell line selectively responds to the toxin in a very sensitive manner with an increase in intracellular cyclic GMP. In the present study, we describe the purification and structure of a peptide from rat jejunum that activates this enzyme. This peptide, which we have termed guanylin, is composed of 15 amino acids and has the following amino acid sequence, PNTCEICAYAACTGC, as determined by automated Edman degradation sequence analysis and electrospray mass spectrometry. Analysis of the amino acid sequence of this peptide reveals a high degree of homology with heat-stable enterotoxins. Solid-phase synthesis of this peptide confirmed that it stimulates increases in T84 cyclic GMP levels. Guanylin required oxidation for expression of bioactivity and subsequent reduction of the oxidized peptide eliminated the effect on cyclic GMP, indicating a requirement for cysteine disulfide bond formation. Synthetic guanylin also displaces heat-stable enterotoxin binding to cultured T84 cells. Based on these data, we propose that guanylin is an activator of intestinal guanylate cyclase and that it stimulates this enzyme through the same receptor binding region as the heat-stable enterotoxins.     

Demonstration of Dopamine-sensitive Adenylate Cyclase in Malignant Neuroblastoma Cells and Change in Sensitivity of Adenylate Cyclase to Catecholamines in “Differentiated” Cells

Adenylate cyclase (EC 4.6.1.1) activity was stimulated by low concentrations of dopamine and apomorphine, but not by low concentrations of norepinephrine in homogenates of malignant mouse neuroblastoma cells. In cyclic AMP-induced "differentiated" cells, dopamine concentration required for a maximal increase in adenylate cyclase activity was about 10-fold less than that required for a similar increase in control cells, and norepinephrine-sensitive adenylate cyclase activity became apparent at low norepinephrine concentrations. The pharmacological properties of dopamine-sensitive adenylate cyclase were different from those of norepinephrine-sensitive enzyme. For example, dopamine-stimulated adenylate cyclase activity was markedly reduced by low concentrations of haloperidol and phentolamine, whereas norepinephrine-stimulated enzyme activity required higher concentrations of these blocking agents for a similar amount of inhibition. Norepinephrine-stimulated enzyme activity was markedly blocked by low concentrations of propranolol, whereas dopamine-stimulated enzyme activity required a much higher concentration of this blocking agent for a similar amount of inhibition. Low concentrations of isoproterenol increased adenylate cyclase activity in malignant cells, but in "differentiated" cells even a high concentration failed to do so. The fact that dopamine and norepinephrine produced an additive stimulatory effect on adenylate cyclase activity suggests that they interact at different receptor sites. This suggestion is further supported by the observation that the combination of prostaglandin E(1) and norepinephrine produced an additive stimulatory effect of enzyme activity. The observation that the effects of dopamine and prostaglandin E(1) are not additive, coupled with the observation that a low concentration of phentolamine blocked the effect of prostaglandin E(1), suggests that these two agents may interact at a common site.     

Regulation of nucleoside cyclic 3':5'-monophosphate phosphodiesterase activity from rat brain by a modulator and Ca2+

Gel filtration of the 40,000 rpm supernatant fraction of a homogenate of rat cerebral cortex on a Sepharose 6B column yielded two fractions: fraction II with the "Ca(2+) plus Mg(2+)-dependent" phosphodiesterase activity and fraction III containing its modulator. The activity of fraction II was stimulated by micromolar concentrations of Ca(2+) and the modulator when present together; the modulator stimulated the activity of fraction II only when the Ca(2+) concentration was above a threshold value (about 2 muM with 0.4-1 muM substrate), and the stimulatory effect of Ca(2+) was dependent upon the presence of the modulator. A possibility is discussed that the modulator may reversibly bind to the enzyme, which by itself is inactive, to form an active enzyme-modulator complex and that Ca(2+) stimulates the activity of phosphodiesterase by shifting the equilibrium between these three species towards the formation of the active enzyme-modulator complex. Although fraction II hydrolyzed both cyclic AMP and cyclic GMP, hydrolysis of the latter was more significantly influenced by Ca(2+) and the modulator than that of the former, and the "Ca(2+) plus Mg(2+)-dependent" phosphodiesterase is likely to be a cyclic GMP enzyme. This conclusion is based on the following evidence: (a) Ca(2+) stimulated hydrolysis of cyclic GMP by fraction II more than that of cyclic AMP. (b) In the presence of Ca(2+) and the modulator, fraction II hydrolyzed cyclic GMP about 8 times faster than cyclic AMP when incubated with 0.4 muM substrate. (c) Half-maximal stimulation of hydrolysis of cyclic GMP was attained at a lower concentration of Ca(2+) (4 muM) than that of cAMP (8 muM). (d) Increase in the concentration of Ca(2+) from 0.06 muM to 12 muM in the presence of the modulator caused a decrease in the K(m) value of cyclic GMP hydrolysis by fraction II from 20 muM to 2 muM accompanied by 4-fold increase in the V(max) value. Under similar conditions, there was only a slight decrease in the K(m) value of cylic AMP hydrolysis (90 muM --> 50 muM), although the V(max) value increased 7-fold. The anomalous shape of the kinetic plot of cyclic GMP hydrolysis became linear when the Ca(2+) concentration was increased in the presence of the modulator. The modulator seems to be a protein, but it is heat stable. It is probably identical to the protein activator of phosphodiesterase first described by Cheung.     

NG-methyl-L-arginine causes endothelium-dependent contraction and inhibition of cyclic GMP formation in artery and vein.

The objective of this study was to determine whether the vascular smooth muscle contractile effect of NG-methyl-L-arginine (NMA) is endothelium dependent and attributed to a decline in smooth muscle levels of cyclic GMP. Vascular smooth muscle levels of cyclic GMP are severalfold greater in endothelium-intact than in endothelium-denuded preparations because of the continuous formation and release of a lipophilic endothelium-derived chemical factor that diffuses into the underlying smooth muscle and activates cytosolic guanylate cyclase. This chemical substance, believed to be nitric oxide (NO) or a labile nitroso precursor, appears to account for the biological actions of endothelium-derived relaxing factor. NMA inhibits the formation of NO from endogenous L-arginine in endothelial cells. In the present study, NMA caused marked endothelium-dependent contraction of isolated rings of bovine pulmonary artery and vein, and this was similar to the contraction elicited by hemoglobin, an inhibitor of the relaxant action of NO. Both NMA and hemoglobin caused endothelium-dependent potentiation of contractile responses to phenylephrine in artery and vein. NMA caused endothelium-dependent decreases in the resting or basal levels of cyclic GMP in artery and vein to levels that were characteristic of those in endothelium-denuded vessels. Finally, NMA inhibited endothelium-dependent relaxant responses and cyclic GMP formation stimulated by acetylcholine and bradykinin. These observations reveal that interference with the continuous or basal generation of endothelium-derived NO in artery and vein can cause marked increases in vascular smooth muscle tone as a result of inhibition of cyclic GMP formation.     

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.     

C-type natriuretic peptide inhibits thrombin- and angiotensin II-stimulated endothelin release via cyclic guanosine 3',5'-monophosphate.

We examined the inhibitory effect of porcine C-type natriuretic peptide (CNP) on endothelin-1 secretion stimulated by thrombin and angiotensin II (Ang II) in cultured porcine endothelial cells. The results were compared with the effects of atrial (ANP) and brain (BNP) natriuretic peptides. Thrombin and Ang II produced a concentration-dependent stimulation of immunoreactive endothelin-1 secretion, and porcine CNP-22 potently inhibited this stimulated secretion in a concentration-dependent manner. CNP-22 had a stronger inhibitory effect than either porcine ANP(1-28) or porcine BNP-26. In addition, CNP potently increased the cellular level of cyclic guanosine 3',5'-monophosphate (GMP), with the inhibition of immunoreactive endothelin-1 secretion in response to thrombin and Ang II being paralleled by the increase in the cyclic GMP level. The increase of cyclic GMP produced by CNP was also greater than that due to porcine ANP(1-28) or porcine BNP-26. The immunoreactive endothelin-1 in the culture medium had two components on high-performance liquid chromatography; the major one corresponded to endothelin-1 (1-21) and the minor one to big endothelin-1 (porcine 1-39). Treatment with CNP did not affect this profile. Our results suggest that CNP probably inhibits the endothelin-1 secretion stimulated by thrombin and Ang II through a cyclic GMP-dependent process. The increase of cyclic GMP levels and the inhibition of immunoreactive endothelin-1 secretion produced by CNP appear to be greater than those produced by ANP or BNP.     

Escherichia coli enterotoxin (STa) binds to receptors, stimulates guanyl cyclase, and impairs absorption in rat colon.

To determine the contribution of the colon in Escherichia coli heat-stable enterotoxin-mediated diarrheal disease, toxin binding, guanyl cyclase activation, and toxin-induced water flux in the rat colon and ileum were compared. Scatchard analysis suggested a single class of heat-stable enterotoxin receptors with an affinity constant of binding of 10(9) L/mol in both colonocytes and ileocytes; however, the number of toxin receptors per cell was 3.5-fold greater in coloncytes than ileocytes (8.32 +/- 1.33 x 10(5) vs. 2.33 +/- 0.28 x 10(5) receptors per cell; P = 0.02). Heat-stable enterotoxin stimulated guanyl cyclase activation in an identical dose-dependent manner in proximal colonic and ileal membranes, with similar sensitivity and maximum response. Heat-stable enterotoxin also inhibited net water flux to a similar degree in both colon and ileum (-47.8 vs. -48.4 microL.cm-1.h-1, respectively) at a dose of 8 nmol/L. At this dose in the colon, because of a higher baseline of absorption, absorption continued, but at a diminished level. At this dose in the ileum, heat-stable enterotoxin induced net secretion. These data are consistent with the concept that heat-stable enterotoxin-induced diarrheal disease results from a decreased absorptive capacity in the colon in the face of increased small intestinal fluid secretion.     

Platelet aggregation is inhibited by a nitric oxide-like factor released from human neutrophils in vitro

Thrombin-induced platelet aggregation was inhibited in vitro by washed human neutrophils. Aggregation was inhibited in a neutrophil concentration dependent manner but glutaraldehyde fixed neutrophils had no significant effect on platelet aggregation. The neutrophil-derived inhibitory factor had the pharmacological profile of nitric oxide. Its action was potentiated by both superoxide dismutase and M&B22, 948, a selective cyclic guanosine monophosphate (cyclic GMP) phosphodiesterase inhibitor. Haemoglobin lessened this inhibitory action of neutrophils. L-Arginine, the substrate for nitric oxide formation, enhanced inhibition, whereas, L-canavanine, a structural analogue of L-arginine, prevented it. Nitric oxide release by neutrophils antagonized platelet ATP secretion and thromboxane B2 release. Inhibition was mediated by nitric oxide activation of guanylate cyclase with a subsequent rise in cyclic GMP. When neutrophils were stimulated with formyl-met-leu-phe, there was a further increase in platelet cyclic GMP. This was enhanced by superoxide dismutase, but lessened by haemoglobin. Leukotriene B4 stimulation of neutrophils promoted inhibition of platelet aggregation. Leukotriene B4 alone had no direct effect on thrombin-induced aggregation of platelets. Platelets, when incubated with neutrophils and stimulated with calcium ionophore A23187, increased leukotriene B4 production by neutrophils in a platelet concentration dependent manner. Platelets alone were unable to release leukotriene B4. The action of platelets in haemostasis is modified as they come into contact with neutrophils. This may be an important physiological mechanism.     

Subcellular distribution of the enzymes related to the cellular action of vasopressin in renal medulla.

Subcellular distribution of the enzymes related to the cellular action of antidiuretic hormone was studied in bovine renal medulla. The highest activity of vasopressin-stimulated adenylate cyclase was found in plasma membranes. The basal activity increased two times above homogenate while vasopressin-stimulated and NaF-stimulated activities both increased five times. Adenylate cyclase activity was present also in other particulate fractions, but it was not significantly stimulated by vasopressin. Cyclic AMP phosphodiesterase was predominantly located in the cytosol when assayed with 0.5 mM cyclic AMP or with 5 muM cyclic AMP. However, with the latter concentration of cyclic AMP more activity remained associated with the particulate fractions and was more inhibited by theophylline. The highest cyclic AMP-stimulated protein kinase activity occurred in the cytosol. Protein kinase activity present in other subcellular fractions was not markedly stimulated by cyclic AMP. Protein phosphatase activity was highest in cytosol when assayed using 32P-histones, 32P-plasma membrane proteins, and 32P-cytoslic proteins. The activity was unaffected by 10-6M to 10-4M cyclic AMP or cyclic GMP. The activity was completely inhibited by 10mM ZnSO4 and 10mM CuSO4; 10mM NaF inhibited the activity by approximately 14%. The enzymes related to the cellular action of vasopressin are predominatly localized in the cytosol except for the vasopressin-sensitive adenylate cyclase which is plasma membrane bound. To mediate the effect of antidiuretic hormone and act on the luminal plasma membrane these soluble enzymes and their substrates should be compartmentalized, possibly by a system of cytoplasmic microtubules.     

Follicle-stimulating hormone-induced prostaglandin E formation in isolated rat ovarian theca

The aim of this study was to search for direct biochemical effects of highly purified FSH on isolated ovarian follicular theca in vitro. Granulosa cells (GC; approximately 1 x 10(5) cells per follicle) were flushed from isolated follicles of pro-oestrous rats. The remaining theca layer and the isolated GC were incubated with highly purified ovine FSH. Prostaglandin E (PGE) accumulation was measured by radioimmunoassay. Follicle-stimulating hormone induced a 15-fold increase in PGE accumulation over the basal level in the follicular theca, the stimulated rate exceeding threefold that observed in the GC fraction derived from the same follicle. Follicle-stimulating hormone caused no significant increase in cyclic AMP level or steroidogenesis in the theca layer, but was active on these parameters in the GC. In contrast, LH increased the accumulation of cyclic AMP, progesterone and testosterone, as well as of PGE, in follicular theca. Exogenous 8-bromo cyclic AMP or cyclic GMP also stimulated PGE production in follicular theca or GC, but FSH was without any effect on the level of endogenous cyclic GMP in GC or follicular theca. Antibodies to FSH prevented the effect of FSH (but not that of LH) on PGE formation by follicular theca and GC, while antibodies to the beta-subunit of LH blocked the effect of LH but not of FSH. We conclude that highly purified FSH has a stimulatory effect on PGE formation by the follicular theca.     

Calcium and calcium slow channel antagonists on cyclic nucleotide levels in the isolated rat heart

An investigation has been carried out into the effects of extracellular calcium on cyclic nucleotide levels in rat ventricle. Increasing extracellular calcium, over the range which directly affected contractility, produced a concentration related increase in the level of cyclic AMP. Positive correlations were found between extracellular calcium and cyclic AMP; and between cyclic AMP and isometrically developed tension. No reciprocal relationships were found for cyclic GMP over the same calcium concentration range. Increasing extracellular calcium above the maximal inotropic concentration did not produce a further increase in cyclic AMP, although increases in cyclic GMP were noted. Calcium slow channel antagonists, nifedipine and manganese both depressed developed tension at the concentrations used, but this effect was not associated with any significant change in cyclic nucleotide levels. Verapamil depressed developed tension and cyclic AMP levels but the effect on cyclic AMP was not concentration related. No calcium slow channel antagonist significantly affected cyclic GMP levels. In electrically paced hearts, increasing rate of stimulation depressed developed tension but had no significant effect on cyclic AMP.     

An L-arginine/nitric oxide pathway present in human platelets regulates aggregation.

Aggregation of human washed platelets with collagen is accompanied by a concentration-dependent increase in cyclic GMP but not cyclic AMP. NG-Monomethyl-L-arginine (L-MeArg), a selective inhibitor of nitric oxide (NO) synthesis from L-arginine, reduces this increase and enhances aggregation. L-Arginine, which has no effect on the basal levels of cyclic GMP, augments the increase in this nucleotide induced by collagen and also inhibits aggregation. Both of these effects of L-arginine are attenuated by L-MeArg. The anti-aggregatory action of L-arginine is potentiated by prostacyclin and by M&B22948, a selective inhibitor of the cyclic GMP phosphodiesterase, but not by HL725, a selective inhibitor of the cyclic AMP phosphodiesterase. L-Arginine also inhibits platelet aggregation in whole blood in a similar manner, although the concentrations required are considerably higher. L-Arginine stimulates the soluble guanylate cyclase and increases cyclic GMP in platelet cytosol. This stimulation is dependent on NADPH and Ca2+ and is associated with the formation of NO. Both the formation of NO and the stimulation of the soluble guanylate cyclase induced by L-arginine are enantiomer specific and abolished by L-MeArg. Thus, human platelets contain an NO synthase which is activated when platelets are stimulated. The consequent generation of NO modulates platelet reactivity by increasing cyclic GMP. Changes in the activity of this pathway in platelets may have physiological, pathophysiological, and therapeutic significance.