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.     

Cimetidine partially blocks gastrin's activation of gastric mucosal guanylate cyclase

The objective of the present investigation was to determine if gastrin at physiological concentrations has part of its mechanism(s) of action through stimulation of guanylate cyclase (EC 4.6.1.2). Human gastrin (I), pentagastrin, tetragastrin, and gastrin-related tetrapeptide all increased cyclic GMP levels and guanylate cyclase activity in rat gastric mucosa, whole stomach, and duodenum. Maximal stimulation was seen at 1 microM with all of the above. There was no further enhancement of guanylate cyclase with increasing the concentration to the millimolar range. The ED50 for human gastrin and pentagastrin was 0.01 microM, whereas the ED50 was 0.1 microM for tetragastrin and the tetrapeptide. No enhancement of guanylate cyclase activity was seen with decreasing the concentration to 1 nM of the respective gastrins. Cimetidine utilized at 1 microM or 1 mM concentrations partially blocked the augmentation by gastrin suggesting that part of this enhancement was through the histamine 2 receptor which has been shown to be important in pentagastrin-stimulated gastric acid release. Since the block was only partial these data would also indicate that some part of gastrin's activation of this enzyme is not mediated through the histamine 2 receptor.     

Inhibition of growth and guanylate cyclase activity of an undifferentiated prostate adenocarcinoma by an extract of the balsam pear (Momordica charantia abbreviata).

We have recently described the presence of a guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] inhibitor (GCI) in an aqueous extract of the balsam pear (Momordica charantia abbreviata). Because the guanylate cyclase-cyclic GMP system is though to be involved in cell growth, DNA and RNA synthesis, and possible malignant transformation, we examined the effect of the aqueous extract containing GCI on an undifferentiated adenocarcinoma of the rat prostate and concanavalin-A-stimulated [3H]thymidine incorporation into cultured splenic lymphocytes, a process thought to be mediated by cyclic GMP. The results demonstrate that the extract of the balsam pear blocks both the growth of the rat prostatic adencarcinoma in vitro and [3H]thymidine incorporation into DNA. DNA histograms from flow cytometry indicated that the extract containing GCI inhibited in the G2 + M phase of the cell cycle, a presumed locus of cyclic GMP effects. In addition, guanylate cyclase activity was significantly greater in the tumor than normal prostate tissue and was decreased by the extract containing GCI. Cyclic GMP levels in the tumor in culture wer also decreased by addition of the extract. It remains to be determined whether or not the anti-tumor agent and GCI are the same substance.     

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.     

Estrogens and progesterone increase fetal and maternal guanylate cyclase activity

Since both estrogens and cyclic guanosine 3',5'-monophosphate stimulate protein synthesis, the objective of the present investigation was to determine if estrogens and their precursors might have part of their mechanism of action through stimulation of guanylate cyclase (E.C.4.6.1.2), the enzyme that catalyzes the conversion of guanosine triphosphate to cyclic guanosine 3',5'-monophosphate. The precursors of estrogen synthesis originate from cholesterol. Cholesterol itself had no effect on guanylate cyclase activity. The precursors of estrogen synthesis generated from cholesterol, namely, progesterone, 17 alpha-OH-progesterone, androstenedione, pregnenolone, 17 alpha-OH-pregnenolone, and dehydroepinandrosterone, however, caused a 2- to 3-fold enhancement of fetal and maternal guinea pig hepatic and uterine guaynlate cyclase activity at a concentration of 1 microM. In comparative studies, similar effects were seen on immature female Sprague-Dawley rat hepatic and uterine guanylate cyclase activity. Estrone, estradiol-17 beta, estriol, and the synthetic estrogen, diethylstilbestrol, enhanced guanylate cyclase activity in the same tissues 2- to 3- fold at the 1 microM concentration. Dose-response relationships revealed that these estrogens and their precursors had their maximal effect at 0.001 microM. Estradiol-17 alpha also enhanced uterine guanylate cyclase activity, but a 1000-fold greater concentration compared to the other estrogens was necessary to show any significant effect. The data in this investigation suggest that guanylate cyclase may play a role in the mechanism of action of estrogens and their precursors.     

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.     

Activation of guanylate cyclase by superoxide dismutase and hydroxyl radical: a physiological regulator of guanosine 3'',5''-monophosphate formation.

Partially purified soluble rat liver guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] was activated by superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1). This activation was prevented with KCN or glutathione, inhibitors of superoxide dismutase. Guanylate cyclase preparations formed superoxide ion. Activation by superoxide dismutase was further enhanced by the addition of nitrate reductase. Although guanylate cyclase activity was much greater with Mn2+ than with Mg2+ as sole cation cofactor, activation with superoxide dismutase was not observed when Mn2+ was included in incubations. Catalase also decreased the activation induced with superoxide dismutase. Thus, activation required the formation of both superoxide ion and H2O2 in incubations. Activation of guanylate cyclase could not be achieved by the addition of H2O2 alone. Scavengers of hydroxyl radicals prevented the activation. It is proposed that superoxide ion and hydrogen peroxide can lead to the formation of hydroxyl radicals that activate guanylate cyclase. This mechanism of activation can explain numerous observations of altered guanylate cyclase activity and cyclic GMP accumulation in tissues with oxidizing and reducing agents. This mechanism will also permit physiological regulation of guanylate cyclase and cyclic GMP formation when there is altered redox or free radical formation in tissues in response to hormones, other agents, and processes.     

Neurotransmitter receptors mediate cyclic GMP formation by involvement of arachidonic acid and lipoxygenase.

Evidence is presented that has led us to abandon the hypothesis that receptor-mediated cyclic GMP formation in cultured nerve cells occurs via the influx of extracellular calcium ions and an increase in the cytosolic free calcium ion concentration. While the cyclic GMP response is absolutely dependent on the presence of Ca2+, there is no increase in free intracellular Ca2+ subsequent to agonist stimulation. Instead, we have found that muscarinic or histamine H1 receptor stimulation elicits the release of arachidonic acid through a quinacrine-sensitive mechanism, possibly phospholipase A2. Inhibition of the release or metabolism of arachidonate by the lipoxygenase pathway prevents receptor-mediated cyclic GMP formation. We hypothesize that neurotransmitter receptors that mediate cyclic GMP synthesis function by releasing arachidonic acid and that an oxidative metabolite of arachidonic acid then stimulates soluble guanylate cyclase.     

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.     

Immunohistochemical localization of guanylate cyclase within neurons of rat brain.

The immunohistochemical localization of guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] has been examined in rat neocortex, caudate-putamen, and cerebellum by using specific monoclonal antibodies. Immunofluorescence could be seen within somata and proximal dendrites of neurons in the these regions. A nuclear immunofluorescence reaction to guanylate cyclase was characteristically absent. The staining pattern for guanylate cyclase was coincident with previously described localizations of cyclic GMP immunofluorescence within medium spiny neurons of the caudate-putamen and pyramidal cells of the neocortex. Cerebellar guanylate cyclase immunoreactivity was primarily confined to Purkinje cells and their primary dendrites, similar to the pattern reported for cyclic GMP-dependent protein kinase localization. Guanylate cyclase immunofluorescence was abolished when the monoclonal antibodies were exposed to purified enzyme prior to incubation of the tissue slices or when control antibody was substituted for the primary antibody. Immunohistochemical localization of cyclic AMP in these same tissues was readily distinguished from that of guanylate cyclase or cyclic GMP, showing uniform fluorescence throughout the cell bodies of neurons and glial elements.     

Stimulation of human platelet guanylate cyclase by unsaturated fatty acid peroxides.

Guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] activity of human platelet homogenates was stimulated by the addition of phospholipase A2 or unsaturated fatty acids such as oleic, vaccenic, linoleic, linolenic, eicosenoic, eicosadienoic, and arachidonic acids. The addition of lipoxidase potentiated the fatty acid-induced stimulation of guanylate cyclase purified by DEAE-cellulose column chromatography. The extent of the stimulation was dependent on the concentration of the oxidized form of these fatty acids (peroxides). Saturated fatty acids such as stearic and arachidic acids had no effect on the guanylate cyclase activity in the presence or absence of lipoxidase, indicating that human plateletguanylate cyclase is stimulated by unsaturated fatty acid peroxides rather than by fatty acids.Hemoglobin prevented the enzyme stimulation produced by low concentrations of fatty acid peroxides, but enhanced stimulation of the enzyme activity with high concentrations of fatty acid peroxides. 2-Mercaptoethanol, dithiothreitol, and N-ethylmaleimide inhibited the guanylate cyclase activities both in the presence and absence of unsaturated fatty acidperoxide. The stimulation of guanylate cyclase activity by unsaturated fatty acid peroxidesis attributed to oxidation of sulfhydryl residues of the enzyme protein.     

Ovarian cyclic GMP concentration and guanylate cyclase activity in immature rats after treatment with pregnant mare serum gonadotrophin

We have previously reported that the LH-induced decrease in the concentration of ovarian cyclic GMP (cGMP) in the rabbit was accompanied by a drop in ovarian guanylate cyclase activity. The present experiments were carried out to see if the increase in cGMP concentration that occurs in immature rat ovaries after stimulation with pregnant mare serum gonadotrophin (PMSG) is also accompanied by changes in guanylate cyclase activity. Total ovarian cGMP, along with ovarian weight, was found to be increased at 16 h after PMSG treatment. Ovarian concentrations of cGMP, however, increased only after that period (at 20, 24 and 48 h) and the increase was progressive. Guanylate cyclase activity was found in both the cytosol and 100 000 g particulate fractions of the immature rat ovaries. Forty-three hours after PMSG treatment, activity in the particulate fraction was found to be significantly increased. This increase in guanylate cyclase activity was also found at 20 h but not at 16 h. Thus, the increase in ovarian cGMP concentration in immature rats after PMSG treatment was accompanied by increased guanylate cyclase activity.     

Atrial natriuretic factor receptor heterogeneity and stimulation of particulate guanylate cyclase and cyclic GMP accumulation

Since the seminal discovery by deBold that atria contain factors that produce diuresis and natriuresis, the biologic effects attributed to ANF have expanded to the point where the name "atrial natriuretic factor" seems inappropriate. In addition to promoting diuresis and natriuresis, ANF has been shown to produce vascular smooth muscle relaxation and to inhibit the secretion of aldosterone from the adrenal cortex, renin from the juxtaglomerlular apparatus, vasopressin from the hypothalamus, and salt and water intake after central administration. ANF also promotes intestinal secretion and stimulates testosterone synthesis in Leydig cells. However, the cellular mechanisms whereby ANF elicits these diverse effects are poorly understood. ANF has been reported to inhibit adenylate cyclase in a number of tissues. However, the significance of ANF inhibition of adenylate cyclase is unknown. This effect cannot be associated with vascular relaxation since decreased cyclic AMP would be expected to promote contraction rather than relaxation. ANF inhibition of adenylate cyclase may mediate the inhibitory effects of ANF on hormone secretion from the anterior pituitary gland. The inhibition of adenylate cyclase could also explain the inhibitory effect of ANF on aldosterone synthesis, since agents that stimulate cyclic AMP increase aldosterone synthesis. However, ANF also inhibits the dibutyryl-cyclic AMP-induced stimulation of aldosterone secretion, suggesting that an inhibition of adenylate cyclase cannot account fully for the inhibitory effects of ANF on aldosterone synthesis. There is no evidence to support a role for cyclic AMP in the diuretic and natriuretic action of ANF. An inhibition of membrane phosphoinositide breakdown by ANF and the subsequent formation of IP3 and intracellular calcium release could explain the inhibitory effects of ANF on vascular contraction and steroid synthesis. However, there is very little evidence to suggest that ANF regulates phosphoinositide metabolism, while some recent studies suggest that ANF may regulate calcium fluxes in vascular tissue. Clearly, cyclic GMP has emerged as the most likely intracellular mediator of ANF effects. ANF increases cyclic GMP in a wide range of tissues by selectively activating particulate guanylate cyclase. However, it is not known which effects of ANF are mediated by cyclic GMP. The discovery that ANF increases cyclic GMP in vascular tissue clearly suggests that cyclic GMP mediates the vascular relaxation effect of ANF, since other classes of vasodilators also increase cyclic GMP. There is preliminary evidence that cyclic GMP may inhibit renin secretion and sodium transport in kidney cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Bromocriptine enhances guanylate cyclase activity

Bromocriptine and its parent compound alpha-ergocryptine were investigated with respect to their ability to interact with the guanylate cyclase (E.C.4.6.1.2)-cyclic GMP system in vitro in the rat pituitary and ovary. Both bromocriptine and alpha-ergocryptine enhanced guanylate cyclase two- to threefold in both of these tissues over a concentration range of 1 nM to 1 microM. Since bromocriptine is thought to be a dopamine agonist in the pituitary, dopamine's effects on guanylate cyclase were also tested. Dopamine caused a twofold enhancement of guanylate cyclase activity in the pituitary and ovary. When bromocriptine and dopamine were used in combination, bromocriptine had to be in equal or a greater concentration with respect to dopamine in vitro to enhance guanylate cyclase activity. These findings suggest that bromocriptine's effect at the level of the pituitary and ovary may be mediated through enhancement of guanylate cyclase activity.     

Oestrogen-induced increase in uterine cGMP content in vitro: effects of inhibitors of protein and RNA synthesis

We have shown previously that oestradiol elevates the cGMP content od isolated uterine horns incubated for 2 h with the hormone. Cycloheximide (30 micrograms/ml) or actinomycin D (100 micrograms/ml), at concentrations which markedly inhibit protein and RNA synthesis, blocked the oestrogen-induced increase in cGMP. These agents do not inhibit the rise in uterine cGMP content provoked by sodium nitroprusside, thus arguing against a direct toxic effect on the enzyme guanylate cyclase. alpha-Amanitin, even at very high concentrations (80 micrograms/ml), interfered much less efficiently with total RNA and protein synthesis and also failed to prevent the oestrogen-induced increase in cGMP content. Taken together, these observations indicate that oestrogen action on uterine cGMP concentration in vitro depends on an RNA and/or a protein biosynthetic event that takes places in the uterus. This therefore confirms and extends analogous observations made previously under conditions in vivo.     

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)     

Cyclic GMP affecting the tracheal nonadrenergic noncholinergic inhibitory system

An association between guanosine 3',5'-monophosphate (cyclic GMP) and the nonadrenergic noncholinergic inhibitory system (NANCIS) has been demonstrated in the isolated bovine tissue (Bowman and Drummond, 1984). In order to investigate this association in the guinea pig trachea, we used cyclic GMP derivatives, guanylate cyclase activators (N-methylhydroxylamine (NMH) and nitroglycerin (NG)] and inhibitors [oxyhemoglobin (HbO2) and methylene blue (MB)]. Under general anesthesia paralysis, the animals were ventilated and hourly injected with atropine (0.2 mg/kg) and propranolol (1 mg/kg). Cervical segment of the trachea was converted to a closed tracheal pouch and then filled with Kreb's solution augmented with atropine (1 microM) and propranolol (3.5 microM). A decrease in the pouch pressure (Pp) reflected NANCIS nerve transmural stimulation (TS)--or drug-induced relaxation. Pharmacological agents were applied intravenously. At 2-11 min after injection, NMH and NG decreased baseline Pp and reduced TS-induced relaxation. NMH, which is more potent than NG in activating particulate guanylate cyclase activity, potentiated the TS-induced relaxation at high frequencies, but NG did not. HBO2 inhibited the TS-induced relaxation at high but not at low frequencies. In contrast, MB inhibited the relaxation at low but not high frequencies. The results suggest that activation of particulate or membrane bound guanylate cyclase potentiates NANCIS-induced decrease in Pp. Therefore, there is a possible association between cyclic GMP and the NANCIS in the guinea pig trachea.     

Stimulation of soluble guanylate cyclase by an acetylcholine-induced endothelium-derived factor from rabbit and canine arteries

The present study was designed to investigate the hypothesis that, during acetylcholine-induced endothelium-dependent relaxation, a factor(s) is released from endothelial cells which directly activates soluble guanylate cyclase. We attempted to determine what similarities or differences existed between this factor and endothelium-derived relaxing factor. The study was performed on segments of rabbit aorta and canine femoral artery. Purified soluble guanylate cyclase was injected into the lumen of these vascular segments, together with its substrate, for intraluminal incubation of the enzyme. In endothelium-intact vascular segments, the activity of guanylate cyclase was enhanced over control values obtained by incubation in test tubes. The stimulation was further increased by acetylcholine in concentrations which caused relaxation of the vascular segments. The stimulating principle could not be transferred from the vessel lumen to an external solution of guanylate cyclase, indicating a short life-time. Removal of the endothelium prevented formation and release of the guanylate cyclase stimulating factor(s). Atropine, mepacrine, or nordihydroguaiaretic acid, which inhibit acetylcholine-induced endothelium-dependent relaxations, also inhibited acetylcholine-induced endothelium-mediated activation of guanylate cyclase. The results support the hypothesis that acetylcholine-induced endothelium-derived relaxing factor increases cyclic guanosine monophosphate levels of vascular smooth muscle by a stimulation of soluble guanylate cyclase.     

Phosphoprotein Phosphatase Activity Associated with Estrogen-Induced Protein in Rat Uterus

Estrogen-induced protein was purified from rat uteri and assayed for several enzymatic activities involved in the metabolism and action of cyclic nucleotides. No adenylate and guanylate cyclase (EC 4.6.1.1 and 4.6.1.2, respectively), protein kinase (EC 2.7.1.33), and cyclic nucleotide binding activities could be demonstrated in three independent preparations of the protein. However, all three preparations exhibited significant phosphoprotein phosphatase activity (EC 3.1.3.16) on phosphorylated protamine and histones F1. This activity is optimal at neutral pH, inhibited by Zn(++), and unaffected by cyclic AMP or cyclic GMP.