Cyclic GMP phosphodiesterase and guanylate cyclase activities in rabbit ovaries and the effect of in-vivo stimulation with LH

The activities of guanylate cyclase and cyclic GMP (cGMP) phosphodiesterase, enzymes that are responsible for maintaining tissue levels of cGMP, were determined in the ovaries of rabbits killed without treatment or 4 h after administration of LH. Ovarian activities of the two enzymes were determined in the 100 000 g supernatant fraction (cytosol) and the resulting pellet (particulate fraction). Significant phosphodiesterase and cyclase activities were detected in both the cytosol and particulate fractions. Administration of LH had no significant effect on phosphodiesterase activity in either of the tissue fractions. On the other hand, LH caused a significant drop in guanylate cyclase activity in the cytosol and particulate fractions. This drop in the cyclase activity may be the cause of the decreased rabbit ovarian concentrations of cGMP that we have previously observed after LH stimulation.     

Regulation of hepatic nuclear guanylate cyclase.

In immunohistochemical studies of rat liver tissue slices and purified nuclei, adenosine 3':5'-cyclic monophosphate (cAMP) and guanosine 3':5'-cyclic monophosphate (cGMP) immunofluorescence on the nuclear membrane are sequentially increased after glucagon administration. An explanation for the increased cGMP immunofluorescence was sought in experiments in which guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2]activity of hepatic subcellular fractions was determined. The results showed that a nuclear guanylate cyclase exists which can be distinguished from the soluble and crude particulate guanylate cyclases. The activity of the nuclear enzyme was increased by 35% in nuclei isolated from rats 30 min after glucagon injection, the time at which maximal nuclear membrane cGMP immunofluorescence is observed. Because glucagon altered both cAMP location and levels prior to the observed changes in nuclear cGMP metabolism, the hypothesis that cAMP acted as the second messenger was tested. In vitro incubation of nuclei isolated from control rats with 10(-5) M cAMP produced a 25% increase in nuclear guanylate cyclase activity. With nuclei isolated from glucagon-treated rats, no significant increase in enzyme activity was observed; this indicates that maximal stimulation of nuclear guanylate cyclase by cAMP occurred at levels that are obtained in vivo after glucagon administration. These findings suggest that hepatic nuclear cGMP content may be regulated by a specific organelle guanylate cyclase and that cAMP may be one of the determinants of this enzyme's activity.     

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

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

Increased particulate and decreased soluble guanylate cyclase activity in regenerating liver, fetal liver, and hepatoma.

We determined the activities of soluble and particulate guanylate cyclase [GTP pyrophosphatelyase (cyclizing); ?EC 4.6.1.2] IN REGENERATING RAT LIVER, FETAL AND NEONATAL RAT LIVER, AND HEPATOMA. TIn these tissues we found increased particulate and decreased soluble enzyme activities compared to normal adult rat liver. The particulate activity increased 12 hr after partial hepatectomy, reached maximal activity at 48 hr, and then declined. The soluble enzyme activity decreased within 8 hr and continued to decline. The activity of homogenates did not change. Guanylate cyclase activity was increased in plasma membrane and microsome fractions from regenerating liver. The increase in particulate activity was prevented with cycloheximide. Decreased soluble and increased particulate enzyme activities were found in fetal liver. After birth the soluble activity increased and the particulate activity decreased. Seven to 14 days after birth the activities of soluble and particulate fractions were similar to those of adult rat liver. In hepatoma 3924A, the activity of particulate guanylate cyclase was 9-fold greater and that of the soluble enzyme was 50% that of normal liver. These studies suggest that guanylate cyclase activity and its subcellular distribution may be related to liver growth through some unknown mechanism.     

Studies on oxidative phosphorylation and steroidogenesis by ovarian mitochondria after gonadotropic stimulation.

No differences in oxidative phosphorylation or in the per cent of [4-14C]progesterone were found in ovarian mitochondria of immature rats after treatment with 20 IU of pregnant mare serum gonadotropin (PMSG) iv 30 min before killing. However, treatment of immature rats with 20 IU of PMSG sc 54 h prior to killing decreased the ADP:O ratio and increased the per cent of [4-14C]cholesterol conversion. Electron microscopic studies showed that mitochondria with lamellar cristae were prominent in ovaries of untreated rats, while large pleomorphic mitochondria and mitochondria with tubulovesicular cristae dominated in ovaries of PMSG-treated rats. Ovarian homogenates separated by zonal centrifugation showed three peaks od cytochrome oxidase activity which shifted to the heavier end of the gradient after PMSG treatment. These studies suggest that PMSG treatment influences ovarian mitochondria, possibly by stimulating the synthesis of additional functional components and/or the biogenesis of new mitochondria. Aminoglutethimide addition to bovine luteal mitochondria decreased steroidogenesis by 60% when succinate was used as substrate. However, there was a 16% increase in the ADP:O ratio, apparently due to a decrease in oxygen utilization. When oligomycin was added to luteal mitochondria, there was a 30% decrease in the ACP:O ratio but a 300% increase in [4-14C]cholesterol conversion. Dinitrophenol also decreased mitochondrial steroidogenesis. These results suggest that energy obtained from succinate oxidation can be diverted from phosphorylation to support steroidogenesis.     

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

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

Correlation of nitric oxide and atrial natriuretic peptide changes with altered cGMP homeostasis in liver cirrhosis.

BACKGROUND: Cyclic GMP (cGMP) concentration is increased in plasma of patients with liver cirrhosis. Three possible mechanisms may contribute: increased cGMP synthesis by soluble (activated by nitric oxide), or particulate (activated by atrial natriuretic peptide (ANP)) guanylate cyclase or increased release from cells. AIM: The aim of this work was to analyze the possible contributors to increased plasma cGMP and to assess whether changes in the parameters of the system vary with the degree of liver disease (Child Pugh score) or by the presence of ascites. METHODS: We measured cGMP in plasma and lymphocytes, soluble guanylate cyclase activation by nitric oxide in lymphocytes, nitrates and nitrites and ANPs (activator of particulate guanylate cyclase) in plasma. We analyzed the correlation between changes in different parameters to discern which parameters contribute to increased plasma cGMP. RESULTS: The plasma content of nitrates+nitrites, ANP and cGMP are increased. Activation of soluble guanylate cyclase by nitric oxide is increased in patients while basal cGMP in lymphocytes is decreased. CONCLUSIONS: Both increased ANP and increased activation of soluble guanylate cyclase by nitric oxide contribute to increased plasma cGMP in patients. The concentrations of ANP and cGMP in plasma increase with the degree of disease and are higher in patients with ascites.     

Ovarian and peripheral blood inhibin concentrations increase with gonadotropin treatment in immature rats

The effects of PMSG treatment on ovarian and circulating inhibin concentrations in immature female rats has been examined. Sixty-four hours after injection of 10, 20 or 40 IU of PMSG the animals were anesthetized with ether; ovaries, uteri and blood from the abdominal aorta were collected. Steroid-free extracts of ovary and serum samples were prepared and assayed quantitatively for inhibin activity in an in vitro bioassay system. PMSG treatment elevated (p less than 0.001) both uterine and ovarian wt, and ovarian and peripheral concentrations of inhibin. A dose-related increase occurred ovarian wt, and in peripheral and ovarian content of inhibin. Ovarian inhibin concentration increased with dose of PMSG until the highest dose, where a significant decline and luteinization were seen. Peripheral FSH levels were significantly lowered at all doses of PMSG treatment; in contrasts, LH was significantly elevated, due to cross-reaction of PMSG in the LH assay. These results show that both ovarian and circulating levels of inhibin are related to the degree of gonadotropic stimulation, supporting the view that inhibin is involved in folliculogenesis and in the feedback regulation of FSH.     

Exit of inhibin-like bioactivity from gonadotropin-stimulated prepubertal pig ovaries

Inhibin-like bioactivity (IBA) was assessed by bioassay in the follicular fluid, ovarian venous plasma, and peripheral plasma of immature gilts in which follicular development had been synchronized by administration of pregnant mare serum gonadotropin (PMSG) and hCG. IBA was detectable in the pooled follicular fluid of control animals, but the concentration as well as the total content increased markedly with the growth of the follicle induced by PMSG. About 36-72 h after PMSG treatment, IBA was found consistently in the venous plasma of both ovaries, although in controls as well as after ovulation (i.e. +48 h after hCG) IBA was not detected. The amount of IBA exiting the ovary by way of venous circulation was only a small fraction (less than 1%) of the total activity calculated to be in each ovary at a given time. IBA was undetectable in the peripheral plasma at any sampling time. On the basis of these results we could speculate that other routes of entry of inhibin into peripheral circulation may exist in the immature pig and/or that the predominant action of IBA might be within the follicle itself.     

Human chorionic gonadotrophin-induced suppression of serum inhibin involves reduction in ovarian inhibin alpha-subunit messenger RNA levels in the pregnant mare serum gonadotrophin-primed female rat

We have investigated the effect of administration of human chorionic gonadotrophin (hCG) to immature female rats pretreated with pregnant mare serum gonadotrophin (PMSG) on ovarian inhibin alpha-subunit mRNA, serum inhibin and circulating gonadotrophin levels. PMSG stimulation alone caused a 5-fold increase in relative inhibin alpha-subunit mRNA levels and a 12-fold increase in serum inhibin by 48 h. However, injection of hCG at 40 h suppressed PMSG-stimulated ovarian inhibin alpha-subunit mRNA and serum inhibin to levels at 48 h not statistically significantly different from controls. Serum follicle-stimulating hormone (FSH) fell after PMSG treatment, but rose after combined PMSG-hCG treatment. Serum luteinizing hormone (LH) was unchanged after PMSG alone but also rose after combined PMSG and hCG therapy. In conclusion, hCG suppresses ovarian inhibin synthesis in PMSG-stimulated immature female rats with preservation of the reciprocal relationship between inhibin and FSH. This immature female rat model therefore provides further insight into the effects of LH or hCG on granulosa cell inhibin production just prior to ovulation.     

In vivo estradiol production in postovulatory ovaries is enhanced by administration of testosterone but not by 5α-dihydrotestosterone

Pre- and postovulatory states of ovaries were induced by the injection of PMSG and PMSG + hCG treatments, respectively, to immature rats. The concentration of ovarian estradiol measured by radioimmunoassay decreased significantly following hCG treatment to PMSG-pretreated rats. Subcutaneous administration of testosterone in soybean oil-glycerol mixture (9:1, v/v) restored the decreased concentration of the ovarian estradiol markedly in the PMSG + hCG treated rats, but not in the group treated with PMSG alone or in the control group treated with no gonadotropin. On the other hand, 5α-dihydrotestosterone showed no increase in the ovarian estradiol of any group. When an ethanol solution of testosterone was administered s.c. to the PMSG + hCG treated rats, the ovarian estradiol level was maximally enhanced from 0.5 to 1.0 h after the injection. On the other hand, 5α-dihydrotestosterone, dehydroepiandrosterone and 17α-hydroxyprogesterone in ethanol showed no effect l h after the injection. These results indicate that the drastic decrease in ovarian estradiol production due to the hCG administration is caused by an acute decrease in the supply of aromatizable androgens to ovarian aromatase.     

Atrial natriuretic peptide and sodium nitroprusside stimulate cyclic GMP accumulation by avian skin fibroblasts and epiphyseal growth-plate chondroprogenitor cells

Chondroprogenitor cells derived from avian tibia epiphyseal growth plate, and skin fibroblasts were cultured in vitro. In the fibroblasts, human (1-28) and rat (5-28) atrial natriuretic peptide (ANP) stimulated cyclic GMP (cGMP) production in a dose-dependent manner without affecting cAMP. Sodium nitroprusside also stimulated cGMP accumulation by chondroprogenitor cells and fibroblasts, but the maximum cGMP accumulation elicited by sodium nitroprusside was much lower than that obtained with ANP. The effects of ANP and sodium nitroprusside on chondroprogenitor cells and skin fibroblasts were additive. Human ANP increased cGMP production by the particulate fraction prepared either from chondroprogenitor cells or fibroblasts. Sodium nitroprusside, at concentrations of up to 1 mmol/l, did not affect cGMP production by the particulate fraction prepared from either cell type. The present study provides additional evidence that avian growth-plate chondroprogenitor cells and skin fibroblasts are targets for ANP. ANP and nitroprusside activate different guanylate cyclase isoenzymes--the particulate and soluble forms of the enzyme respectively. The data suggest that most of the guanylate cyclase activity in these cells is localized in the particulate fraction.     

The preovulatory increase in ovarian collagenase activity in the rat is independent of prostaglandin production

In the present study, we have examined the role of gonadotropins and prostaglandins in the preovulatory increase of ovarian collagenase activity in the rat. Whole ovaries of immature PMSG-primed rats (20 IU) were removed before and 8 h after the rats were treated with human (h) CG, Nembutal, and/or indomethacin. The ovaries were homogenized in a solution containing Triton X-100 (0.25%) and centrifuged. Collagenase was extracted by resuspending the pellets in buffer containing 100 mM CaCl2, heating to 60 C for 6 min, and centrifuging. The supernatants were treated with dithiothreitol (2 mM) and iodoacetamide (5 mM) to inactivate collagenase inhibitors. Collagenase activity was measured as the percent digestion of 3H-type I collagen/100 microliters aliquot of ovarian sample. At zero time (52 h after PMSG), ovarian collagenase activity was 4.2 +/- 1.2% digestion (mean +/- SEM, n = 3). In ovaries collected 8 h after the endogenous LH surge or 8 h after the administration of 10 IU hCG at time zero, collagenase activity rose to 19.6 +/- 2.1 (n = 6) and 22.5 +/- 1.7% digestion (n = 11), respectively. Indomethacin (1.5 mg/100 g BW) administered 30 min after hCG, produced no change in collagenase activity (24.8 +/- 2.5% digestion, n = 7) although the expected increase in ovarian prostaglandin E after hCG treatment was blocked. When the endogenous LH surge was blocked with Nembutal (3 mg/100 g BW), collagenase activity in 8-h ovaries was 6.8 +/- 1.1% digestion (n = 10). The Nembutal block of the preovulatory collagenase increase was overcome by administration of hCG (8-h ovarian enzyme activity = 22.7 +/- 3.2% digestion, n = 8). These observations demonstrate that hCG stimulates ovarian collagenase activity and that this stimulation is not dependent on prostaglandin synthesis.     

Gonadotropin-releasing hormone induces ovulation in hypophysectomized rats: studies on ovarian tissue-type plasminogen activator activity, messenger ribonucleic acid content, and cellular localization

GnRH and its agonists are known to induce ovulation in hypophysectomized rats by acting directly at the ovary. Because tissue-type plasminogen activator (tPA) has been implicated in the gonadotropin induction of ovulation, we examined the effect of an ovulatory dose of GnRH on ovarian tPA activity, mRNA content, and cellular localization. Hypophysectomized immature rats were injected sc with 20 IU PMSG and a single dose of a GnRH agonist (GnRHa; des-Gly10,DLeu6(N alpha Me)Leu7,Pro9NHEt-GnRH) 58 h later. At different times after treatment, ovaries were prepared for morphological analysis. Using a fibrin overlay method, tPA activities were measured in ovarian homogenates and cumulus-oocyte complexes, whereas granulosa cells were cultured for 24 h to estimate tPA secretion. Total ovarian RNA was prepared for hybridization analysis of tPA message levels, and tPA localization was studied by immunohistochemistry of ovarian sections. GnRHa induced ovulation in PMSG-primed hypophysectomized rats 14-16 h after injection in a dose-dependent manner, and the GnRHa action was blocked by concomitant treatment with a GnRH antagonist. GnRHa stimulated the induction of tPA, but not urokinase-type PA, activity in ovarian homogenates and granulosa cell-conditioned medium in a time-dependent manner, reaching a maximum before ovulation. tPA activity in cumulus-oocyte complexes was also increased before ovulation, but this increase was sustained. Hybridization analysis of steady state tPA mRNA levels was performed using a rat cRNA probe. Northern blot analysis of total ovarian RNA demonstrated that GnRHa stimulated tPA mRNA levels 12 h after treatment, with a subsequent decrease 24 h after treatment. Immunohistochemistry indicated substantial increases in tPA staining in granulosa cells and oocytes of preovulatory follicles before ovulation. Thus, GnRHa acts through specific receptors to increase ovarian tPA enzyme activity, mRNA content, as well as immunostaining in granulosa cells and oocytes. Like gonadotropins, GnRH may induce ovulation by directly stimulating tPA levels in the ovary.     

Stage-specific expression of pituitary adenylate cyclase-activating polypeptide type I receptor messenger ribonucleic acid during ovarian follicle development in the rat

Expression of pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide with considerable homology to vasoactive intestinal peptide, has been shown to be stimulated by gonadotropins in the ovary. The present studies further evaluated the cell-type specific expression and gonadotropin regulation of PACAP type I receptor (PACAPR) messenger RNA in immature rat ovaries and in cultured preovulatory follicles. Northern blot analysis of ovaries obtained from prepubertal rats revealed the increased expression of PACAPR during prepubertal development. The major cell types expressing PACAPR messenger RNA were granulosa cells of large preantral follicles. Treatment of immature rats with PMSG caused a decrease in ovarian PACAPR expression. In contrast, treatment with human (h) CG at 2 days after PMSG treatment stimulated ovarian PACAPR messenger RNA within 3-6 h in granulosa cells of preovulatory follicles. Treatment of cultured preovulatory follicles in vitro with LH further confirmed the time- and dose-dependent stimulation of PACAPR by gonadotropins in granulosa cells of preovulatory follicles. Moreover, RNase protection assay revealed that the short variant of ovarian PACAPR was the predominant form stimulated during prepubertal development and by gonadotropins. These results demonstrate the expression of PACAPR messenger RNA in granulosa cells of growing follicles and of preovulatory follicles stimulated by gonadotropins, and suggest that PACAP may play a role in the growth of developing follicles and in ovulation as an autocrine/paracrine factor.     

Adenylate and guanylate cylase system function in human hyperplastic adrenals in Itsenko-Cushing disease

The content of cyclic nucleotides and the activity of adenylate and guanylate cyclases as well as of cAMP phosphodiesterase in the human hyperplastic adrenals were determined after one- and two-step bilateral adrenalectomy for Itsenko-Cushing's disease. A decrease in cGMP concentration and a corresponding increase in cAMP/cGMP correlation were seen in the 2nd hyperplastic adrenal comparatively to those in the 1st one. An enhanced basal activity of adenylate cyclase and its lowered sensitivity to ACTH were found in the 1st and the 2nd adrenals. These changes correlate with a rise in the blood ACTH concentration in patients after the ablation of one adrenal. It is suggested that the augmented adenylate cyclase activity leads to an increased activation of steroidogenesis enzymes in the rest adrenal, ensuring the elevated rate of corticosteroid secretion. The nature of changes in guanylate cyclase activity is contrary to that of adenylate cyclase; namely, guanylate cyclase basal activity of the 2nd hyperplastic adrenal was shown to be lower than that of the 1st one. The cAMP phosphodiesterase activity in the 1st and the 2nd adrenals remained unchanged.     

Atrial natriuretic factor elicits an endothelium-independent relaxation and activates particulate guanylate cyclase in vascular smooth muscle.

A 26 amino acid synthetic peptide fragment of atrial natriuretic factor (ANF) relaxed isolated rabbit aortic segments in which the endothelium was either intact or functionally destroyed. The relaxations were temporally associated with increases in levels of cGMP with no change in the levels of cAMP. The ANF-induced increases in cGMP were also observed in aortic segments pretreated with calcium-free buffer or the cGMP phosphodiesterase inhibitor M&B 22,948. Qualitatively similar results were obtained for sodium nitroprusside. ANF selectively activated particulate guanylate cyclase, having no effect on the soluble form of the enzyme. Thus, the direct (endothelium-independent) vasodilator effect of ANF may be mediated via increased tissue levels of cGMP. ANF appears to increase vascular cGMP levels by activation of particulate guanylate cyclase.     

Adrenocorticotropic hormone-responsive guanylate cyclase in the particulate fraction of rat adrenal glands

Low concentrations of ACTH, 7 x 10(-12) M, caused a marked stimulation of the 100,000 x g particulate guanylate cyclase without any detectable change in the adenylate cyclase activity. The lowest concentration of the hormone that elicited adenylate cyclase stimulation was 7 x 10(-10) M, a concentration 100--fold higher than that required to stimulate the guanylate cyclase. Although calcium was found to be obligatory in the hormonally--dependent guanylate cyclase activity, calcium alone could not duplicate the ACTH effect. Sodium nitroprusside and ascorbic acid inhibited the particulate guanylate cyclase activity. While ACTH was unable to stimulate the soluble guanylate cyclase, sodium nitroprusside markedly stimulated this enzyme. From these data, we conclude that the adrenal guanylate cyclase exists in two forms, particulate and soluble. The particulate form is specifically responsive to ACTH, and calcium is one of the essential coupling factors of this hormonally--responsive guanylate cyclase.     

Activation of intestinal guanylate cyclase by heat-stable enterotoxin of Escherichia coli: studies of tissue specificity, potential receptors, and intermediates

Heat-stable enterotoxin (ST) of Escherichia coli increased guanylate cyclase activity in homogenates of rat and rabbit intestinal mucosa and stimulated intestinal fluid secretion in suckling mice. The ST effect on guanylate cyclase was dose-dependent, occurred without a time lag, and was confined to the particulate fraction. ST activation of guanylate cyclase was tissue-specific; ST did not alter activity of soluble or particulate rat liver, lung, heart, kidney, or cerebral cortex enzyme. The ST activity on guanylate cyclase and secretion was methanol-soluble and alkali-labile, and its effects were not altered by phentolamine, propranolol, or atropine. Monosialoganglioside did not reduce ST-induced secretion. However, indomethacin and butylated hydroxyanisole decreased the ST effect on both guanylate cyclase and secretion. Fluid secretion with ST sppears to result from specific activation of particulate intestinal guanylate cyclase. While adrenergic and cholinergic events are probably not involved in this process, the effects of ST may be mediated through prostaglandin synthesis or oxidative mechnanisms.