Characterization of intestinal brush border guanylate cyclase activation by Escherichia coli heat-stable enterotoxin

Intestinal brush border guanylate cyclase was previously reported to be activated by the Escherichia coli enterotoxin (STa). This system was reexamined in order to develop a hypothesis for the mechanism of activation. The extent of activation was previously underestimated, since by using sodium azide to inhibit competing reactions and ethylene glycol bis(beta-aminoethyl ether) N,N-tetraacetic acid to chelate Ca2+, which is inhibitory, maximal activations of 30- to 50-fold were obtained. Ca2+ inhibition was only partially relieved by the calmodulin inhibitor calmidazolium. Inhibitors of the O2-dependent activation of soluble guanylate cyclase had no effect on STa activation; hence, it was concluded that STa activation did not involve arachidonate release and oxidation. STa was able to further increase activity already elevated by the nonionic detergent Lubrol PX. The membrane-active agent filipin, which was previously reported to inhibit both basal and agonist-stimulated adenylate cyclase, did not inhibit STa activation of guanylate cyclase. Digitonin, another cholesterol binder, inhibited STa activation at low concentrations, which disappeared at higher concentrations. Both of these agents stimulated basal activity. Dimethyl sulfoxide produced a concentration-dependent inhibition of STa activation, while increasing basal activity 7-fold. Ethanol inhibited both basal and STa-stimulated activity, with the former being more affected. Benzyl alcohol, like ethanol, a "fluidizer" of cell membranes, also inhibited both basal and activated enzymes. We concluded that STa directly activates this guanylate cyclase and, because of the differential effects of inhibitors on basal and STa-stimulated activity, propose a receptor-mediated mechanism.     

The increase of cGMP by atrial natriuretic factor correlates with the distribution of particulate guanylate cyclase

We have demonstrated previously that atrial natriuretic factor (ANF) augments urinary, plasma and kidney cGMP levels but has no significant effect upon cAMP. Using cGMP as a marker, we searched for specific target sites involved in the action of ANF in the dog kidney, and observed no change of cGMP in the proximal tubules, a 2-fold increase over basal levels in the thick loop of Henle and a 3-fold elevation in the collecting duct. The most striking action on cGMP occurred in the glomeruli with a rise of up to 50-fold being evident at 1-2 min. after the addition of ANF. The results obtained in the absence or presence of a phosphodiesterase inhibitor support the notion that the effects of ANF were exerted at the level of guanylate cyclase stimulation rather than cGMP phosphodiesterase inhibition. The action of sodium nitroprusside (SNP), a direct stimulator of soluble guanylate cyclase, differed from that of ANF. The ability of the factor to enhance cGMP levels was correlated with the distribution of particulate guanylate cyclase. This study identifies the glomeruli and the distal part of the nephron as specific targets of ANF and implicates particulate guanylate cyclase as the enzyme targetted for the expression of its action.     

Activation of particulate guanylate cyclase by adrenomedullin in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells

We investigated the effects of adrenomedullin (ADM) on cGMP production in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. ADM increased cGMP accumulation in a time- and concentration- dependent manner. The peptide increased cGMP formation in the transformed cells by 405-fold as compared to 1. 6-fold in primary cultured CISM cells. The basal cGMP concentrations in both cell types were comparable. In addition, ADM increased cAMP accumulation in SV-CISM-2 cells and in primary cultured cells by 18. 9- and 5.8-fold, respectively. The ADM receptor antagonist, ADM(26-52), but not the atrial natriuretic peptide (ANP) receptor antagonist, anantin, inhibited ADM-induced cGMP formation. The phorbol ester, phorbol 12, 13-dibutyrate (PDBu), which inhibits particulate guanylate cyclases in smooth muscle, blocked ADM-stimulated cGMP accumulation. In contrast, inhibitors of the soluble guanylate cyclases, such as LY83583 and ODQ, and inhibitors of the nitric oxide cascade had little effect on ADM-stimulated cGMP production. The stimulatory effect of ADM on cGMP formation is due to activation of the guanylate cyclase system and not to a much reduced phosphodiesterase activity. ADM stimulated guanylate cyclase activity in membrane fractions isolated from SV-CISM-2 cells in a concentration-dependent manner with EC(50) value of 72 nM. Pertussis toxin, an activator of the G-protein, Gi, inhibited ADM-stimulated cGMP accumulation, whereas cholera toxin, a stimulator of the Gs G-protein and subsequently cAMP accumulation, had little effect. Pretreatment of the plasma membrane fraction with Gialpha antibody attenuated ADM-stimulated guanylate cyclase activity by 75%. We conclude that ADM increases intracellular cGMP levels in SV-CISM-2 cells through activation of the ADM receptor and subsequent stimulation of a Gi-mediated membrane-bound guanylate cyclase.     

Fluid secretory responses to enterotoxin STa and 8-bromo-cyclic GMP in fed and nutrionally-deprived gerbils: jejunum, ileum and colon in vivo

Fluid transport was measured gravimetrically in vivo in the jejunum, ileum and colon of fed, fasting (four days) and undernourished (50 % of control food intake for 21 days) gerbils (Gerbillus cheesmani). The effects of luminal enterotoxin Escherichia coli STa (50 ng/ml) and luminal 8-bromo-cyclic GMP (cGMP 1 mM) on fluid transport across jejunum, ileum and colon were also assessed. Fasting and undernourishment reversed the normal basal fluid absorption measured in fed ileum and colon into secretion. Neither fasting nor undernourishment had any effect on jejunal basal fluid absorption. In jejuna, ilea and colons of fed animals as well as in jejuna from fasting and undernourished gerbils STa (50 ng) reversed the normal absorptive "tone" to secretion but it had no significant effects on fluid secretion in either the ileum or colon from fasted gerbils. STa increased significantly the fluid secretion in ileum from undernourished gerbils. Luminal cGMP had no effect on basal absorptive tone in the jejunum of fed and fasted gerbils, but reversed absorption into secretion in the jejuna from undernourished gerbils. In the ilea taken from fed animals the small basal absorption was reversed to secretion by luminal cGMP. Although cGMP produced no significant changes in fluid secretion in the ilea taken from fasted gerbils, yet it caused a significant increase in those from undernourished gerbils. In the colon taken from fed animals cGMP decreased the basal fluid absorption significantly, but it had no significant effect on fluid secretion in the colon of fasted or undernourished gerbils. We conclude that fasting and undernourishment have no significant effects on fluid transport across the gerbil jejunum but reversed basal absorption in the fed ileum and colon into secretion. cGMP mimic the effects of STa in the jejunum taken from undernourished gerbils, in the ileum obtained under the three nutritional states and in the colon taken from fasting animals.     

Developmental changes in the effects of carbachol and morphine on cGMP contents of plasma, heart, and lung of mice

It is considered that carbachol increases plasma cGMP levels by acting on muscarinic receptors and morphine increases these levels by acting on opioid receptors, followed by stimulation of muscarinic receptors. We investigated the ability of carbachol and morphine to increase cGMP contents of plasma, heart, and lung and the guanylate cyclase activity of heart and lung homogenate in 1-, 2-, 3-, and 7-week-old mice. The increase in plasma cGMP levels induced by carbachol showed a peak at 2 and 3 weeks of age. The basal cGMP contents in heart and lung and their rise induced by carbachol, as well as the guanylate cyclase activity of these organs, were decreased in 7-week-old mice. The effects of morphine on the cGMP contents showed a similar developmental change, except for no effect in 1-week-old mice. These changes in the effects of carbachol and morphine may be the result of developmental changes of the muscarinic receptor--guanylate cyclase system and opioid receptors.     

Activation of intestinal CFTR Cl- channel by heat-stable enterotoxin and guanylin via cAMP-dependent protein kinase.

Heat-stable enterotoxins (STa) produced by pathogenic bacteria induce profound salt and water secretion in the gut, leading to diarrhea. Recently, guanylin, an endogenous peptide with properties similar to STa, was identified. While STa and guanylin bind to the same receptor guanylyl cyclase and raise cell cGMP, the signaling mechanism distal to cGMP remains controversial. Here we show that STa, guanylin and cGMP each activate intestinal Cl- secretion, and that this is abolished by inhibitors of cAMP-dependent protein kinase (PKA), suggesting that PKA is a major mediator of this effect. These agents induce Cl- secretion only in cells expressing the wild-type CFTR, indicating that this molecule is the final common effector of the signaling pathway. The involvement of CFTR suggests a possible cystic fibrosis heterozygote advantage against STa-induced diarrhea.     

Biochemical mechanisms of atrial natriuretic factor action

Since atrial natriuretic factor (ANF) is a natriuretic and vasodilatory hormone, its mechanisms of action expectedly involve so-called negative pathways of cell stimulation, notably cyclic nucleotides. Indeed, the guanylate cyclase-cyclic GMP (cGMP) system appears to be the principal mediator of ANF's action. Specifically, particulate guanylate cyclase, a membrane glycoprotein, transmits ANF's effects, as opposed to the activation of soluble guanylate cyclase such agents as sodium nitroprusside. The stimulation of particulate guanylate cyclase by ANF manifests several characteristics. One of them is the functional irreversibility of stimulation with its apparent physiological consequences: the extended impact of ANF on diuresis and vasodilation in vivo lasts beyond the duration of increased plasma ANF levels and is accompanied by a prolonged elevation of cGMP. Another characteristic is the parallelism between guanylate cyclase stimulation and increases of cGMP in extracellular fluids. cGMP egression appears to be an active process, yet its physiological implications remain to be uncovered. In heart failure, cGMP continues to reflect augmented ANF levels, suggesting that in this disease, the lack of an ANF effect on sodium excretion is due to a defect distal to cGMP generation. In hypertension, where ANF levels are either normal or slightly elevated, probably secondary to high blood pressure, the ANF responsiveness of the particulate guanylate cyclase-cGMP system, the hypotensive effects, diuresis and natriuresis are exaggerated. The implications of this exaggerated responsiveness of the ANF-cGMP system in the pathophysiology of hypertension and its potential therapeutic connotations remain to be evaluated.     

Phosphodiesterase type 2 and the homeostasis of cyclic GMP in living thalamic neurons

The ubiquitous second messenger cyclic GMP (cGMP) is synthesized by soluble guanylate cyclases in response to nitric oxide (NO) and degraded by phosphodiesterases (PDE). We studied the homeostasis of cGMP in living thalamic neurons by using the genetically encoded fluorescence resonance energy transfer sensor Cygnet, expressed in brain slices through viral gene transfer. Natriuretic peptides had no effect on cGMP. Basal cGMP levels decreased upon inhibition of NO synthases or soluble guanylate cyclases and increased when PDEs were inhibited. Single cell RT-PCR analysis showed that thalamic neurons express PDE1, PDE2, PDE9, and PDE10. Basal cGMP levels were increased by the PDE2 inhibitors erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and BAY60-7550 but were unaffected by PDE1 or PDE10 inhibitors. We conclude that PDE2 regulates the basal cGMP concentration in thalamic neurons. In addition, in the presence of 3-isobutyl-1-methylxanthine (IBMX), cGMP still decreased after application of a NO donor. Probenecid, a blocker of cGMP transporters, had no effect on this decrease, leaving PDE9 as a possible candidate for decreasing cGMP concentration. Basal cGMP level is poised at an intermediate level from which it can be up or down-regulated according to the cyclase and PDE activities.     

Interaction of atrial natriuretic peptide-stimulated guanylate cyclase and vasopressin-stimulated calcium signaling pathways in the glomerular mesangial cell

Receptors for atrial natriuretic peptide (ANP) have been demonstrated in renal mesangial cells as well as other cell types in the glomerulus. The biochemical basis for the effects of ANP on glomerular hemodynamics remains undefined. Using cultured rat glomerular mesangial cells, we demonstrated a concentration-dependent stimulation of cGMP production in intact cells, and of guanylate cyclase in membranes. Despite the presence of a guanylate cyclase response, ANP had no inhibitory effect on basal inositol trisphosphate production nor on basal cytosolic calcium. Arginine vasopressin stimulated IP3 production, caused a rise in cytosolic calcium as measured using the calcium-sensitive fluorescent probe Indo-1, and caused mesangial cell contraction. ANP caused a slight but significant enhancement of vasopressin-stimulated IP3 production, but had no effect on the cytosolic calcium response nor on the contractile response. 8-Bromo-cGMP likewise had no effect on the generation of the calcium signal. These results indicate that the effects of ANP on glomerular hemodynamics are not mediated by an alteration in the generation of the calcium signal in mesangial cells. In contrast, addition of calcium inhibited ANP stimulated guanylate cyclase activity.     

Solubilization and reprecipitation from intestinal brush border membranes of a complex containing guanylate cyclase activatable by the heat-stable enterotoxin.

Extraction of pig intestinal brush border membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (Chaps) in the presence of 0.5 M KCl yielded a solution which contained 60-70% of the receptor for the Escherichia coli heat-stable enterotoxin (STa) and of the Lubrol PX-activated guanylate cyclase activity present in the membrane. When the supernatant solution was diluted fivefold with 10 mM Hepes buffer (pH 7.4) and kept at 4 degrees C overnight, a precipitate formed. Centrifugation yielded a pellet (P2) which contained 25-30% of both the cyclase and the receptor in the original membranes, with a 2.5- to 3-fold enrichment of both. The process could be repeated for further enrichment (P4). The addition of MgCl2 to the diluted extract affected both basal and STa-stimulated activity of P2; 1 mM was optimal. P2 resembled membranes with respect to competitive inhibition of 125I-STa binding by STa, and the concentration-dependent activation of cyclase by STa. Guanylate cyclase in resolubilized P2 was also activated by STa. Most of the enzymes interfering with guanylate cyclase determinations were removed, as were the brush border marker enzymes sucrase and gamma-glutamyltransferase, and a GTP-binding protein that is a pertussis toxin substrate. Specific cross-linking of 125I-STa to receptors in the membrane was preserved in P2 and P4, the three proteins showing the strongest radioactivity having relative molecular masses of 55,000-60,000, 70,000-80,000, and 135,000-140,000. P2 and P4 appear to contain a complex of membrane proteins with certain functional properties intact.     

Atrial natriuretic factor activates membrane-bound guanylate cyclase of chief cells

The present study examined the effect of atrial natriuretic factor (ANF) on cGMP generation by dispersed chief cells from guinea pig stomach. ANF caused a rapid dose-dependent increase in cGMP, a 7-fold increase in cGMP caused by 1 microM ANF, with or without 3-isobutyl-1-methylxanthine present. Methylene blue reduced cGMP in response to nitroprusside but not ANF. Guanylate cyclase activity of a chief cell membrane fraction doubled in response to ANF, but was not affected by nitroprusside. ANF had no effect on guanylate cyclase activity of the soluble fraction of lysed chief cells. Dose-response curves for whole cell cGMP production and membrane guanylate cyclase activity in response to ANF were closely related. These data indicate that ANF increases chief cell cGMP production by activating particulate guanylate cyclase, providing functional evidence that chief cells possess surface membrane receptors for ANF.     

Regulation of intracellular cyclic GMP concentration by light and calcium in electropermeabilized rod photoreceptors

This study examines the regulation of cGMP by illumination and by calcium during signal transduction in vertebrate retinal photoreceptor cells. We employed an electropermeabilized rod outer segment (EP-ROS) preparation which permits perfusion of low molecular weight compounds into the cytosol while retaining many of the features of physiologically competent, intact rod outer segments (ROS). When nucleotide-depleted EP-ROS were incubated with MgGTP, time- and dose- dependent increases in intracellular cGMP levels were observed. The steady state cGMP concentration in EP-ROS (0.007 mol cGMP per mol rhodopsin) approached the cGMP concentration in intact ROS. Flash illumination of EP-ROS in a 250-nM free calcium medium resulted in a transient decrease in cGMP levels; this occurred in the absence of changes in calcium concentration. The kinetics of the cGMP response to flash illumination of EP-ROS were similar to that of intact ROS. To further examine the effects of calcium on cGMP metabolism, dark-adapted EP-ROS were incubated with MgGTP containing various concentrations of calcium. We observed a twofold increase in cGMP steady state levels as the free calcium was lowered from 1 microM to 20 nM; this increase was comparable to the behavior of intact ROS. Measurements of guanylate cyclase activity in EP-ROS showed a 3.5-fold increase in activity over this range of calcium concentrations, indicating a retention of calcium regulation of guanylate cyclase in EP-ROS preparations. Flash illumination of EP-ROS in either a 50- or 250-nM free calcium medium revealed a slowing of the recovery time course at the lower calcium concentration. This observation conflicts with any hypothesis whereby a reduction in free calcium concentration hastens the recovery of cytoplasmic cGMP levels, either by stimulating guanylate cyclase activity or by inhibiting phosphodiesterase activity. We conclude that changes in the intracellular calcium concentration during visual transduction may have more complex effects on the recovery of the photoresponse than can be accounted for solely by guanylate cyclase activation.     

Evidence for the presence of cGMP-dependent protein kinase-II in human distal colon and in T84, the colonic cell line

Heat-stable enterotoxin (STa) stimulates intestinal Cl(-) secretion by activating guanylate cyclase C (GCC) to increase intracellular cyclic GMP (cGMP). In the colon, cGMP action could involve protein kinase (PK) G-II or PKA pathways, depending on the segment and species. In the human colon, both PKG and PKA pathways have been implicated, and, therefore, the present study examined the mechanism of cGMP-mediated Cl(-) transport in primary cultures of human distal colonocytes and in T84, the colonic cell line. Both cell preparations express mRNA for CFTR, Na(+)-K(+)-2Cl(-) cotransporter (NKCC1), GCC and PKG-II as detected by RT-PCR. The effects of STa and the PKG-specific cGMP analogues, 8Br-cGMP and 8pCPT-cGMP, on Cl(-) transport were measured using a halide-sensitive probe. In primary human colonocytes and T84 cells, STa, the cGMP analogues and the cAMP-dependent secretagogue, prostaglandin E(1) (PGE(1)), enhanced Cl(-) transport. The effects of 8Br-cGMP and 8pCPT-cGMP suggested the involvement of PKG, and this was explored further in T84 cells. The effects of 8pCPT-cGMP were dose-dependent and sensitive to the PKG inhibitor, H8 (70 microM), but H8 had no effect on PGE(1)-induced Cl(-) secretion. In contrast, a PKA inhibitor, H7 (50 microM), blocked PGE(1)-mediated but not 8pCPT-cGMP-induced Cl(-) transport. 8pCPT-cGMP enhanced phosphorylation of the PKG-specific substrate, 2A3, by T84 membranes in vitro. This phosphorylation was inhibited by H8. These results strongly suggest that cGMP activates Cl(-) transport through a PKG-II pathway in primary cells and in the T84 cell line of the human colon.     

Primary structure and functional expression of the human receptor for Escherichia coli heat-stable enterotoxin.

Heat-stable enterotoxin (STa) produced by Escherichia coli induces intestinal secretion in mammals by binding to the brush border membrane of the small intestine and activating guanylyl cyclase. We report here the cloning and expression of a cDNA encoding the human receptor for STa. The receptor contains both an extracellular ligand binding site and a cytoplasmic guanylyl cyclase catalytic domain, making it a member of the same receptor family as the natriuretic peptide receptors. Stable mammalian cell lines over-expressing the STa receptor specifically bind 125I-STa (Kd approximately 1.0 nM) and respond to STa by dramatically increasing (approximately 50-fold) cellular cGMP levels. Sequence comparisons between the human and the rat STa receptors show less conservation in the extracellular domain than similar comparisons of natriuretic peptide receptors. This divergence may indicate important species differences in ligand-receptor interaction.     

17β-Estradiol inhibits soluble guanylate cyclase activity through a protein tyrosine phosphatase in PC12 cells

Besides its involvement in reproductive functions, estrogen protects against the development of cardiovascular diseases. The guanylate cyclase/cGMP system is known to exert potent effects on the regulation of blood pressure and electrolyte balance. We examined whether 17β-estradiol can affect soluble guanylate cyclase in PC12 cells. The results indicate that 17β-estradiol decreases cGMP levels in PC12 cells. 17β-Estradiol decreases sodium nitroprusside (SNP)-stimulated, but not atrial natriuretic factor-stimulated cGMP formation in PC12 cells, indicating that 17β-estradiol decreases cGMP levels by inhibiting the activity of soluble guanylate cyclase. 17β-Estradiol also stimulates protein tyrosine phosphatase activities in PC12 cells and dephosphorylates at least three proteins. Addition of sodium vanadate, a protein tyrosine phosphatase inhibitor, blocks the inhibitory effects of 17β-estradiol on soluble guanylate cyclase activity in PC12 cells. Furthermore, transfection of SHP-1, a protein tyrosine phosphatase, into PC12 cells inhibits both basal and SNP-stimulated guanylate cyclase activity. Amino acid analysis also reveals that the 70-kDa subunit of soluble guanylate cyclase contains the SHP-1 substrate consensus sequence. These results suggest that 17β-estradiol inhibits soluble guanylate cyclase activity through SHP-1.     

Effects of oxytocin and methacholine on cyclic nucleotide levels of rabbit myometrium

The effects of oxytocin and methacholine on cyclic nucleotide levels in estrogen-primed rabbit myometrium were studied in the presence and absence of 1-methyl-3-isobutyl xanthine (MIX), a phosphodiesterase inhibitor. In the absence of MIX, methacholine increased guanosine 3',5'-cyclic monophosphate (cGMP) levels at a time when contraction was decreasing, but had no influence on adenosine 3',5'-cyclic monophosphate (cAMP) levels. In contrast, oxytocin did not elevate cGMP, but rapidly decreased cAMP levels. MIX (1 mM) increased both cAMP and cGMP levels. Oxytocin or methacholine further increased cGMP, indicating activation of guanylate cyclase. Oxytocin- but not methacholine-induced stimulation of guanylate cyclase was abolished in Ca2+-free solution. Oxytocin increased cAMP over the levels produced by MIX alone, whereas methacholine decreased cAMP below the MIX control values; these effects were insensitive to indomethacin. Tissue levels of cGMP and cAMP did not directly correlate with isometric tension. The results also indicate that both oxytocin and methacholine stimulate guanylate cyclase but have opposing effects on adenylate cyclase of rabbit myometrium.     

Opossum kidney contains a functional receptor for the Escherichia coli heat-stable enterotoxin

The Escherichia coli heat-stable enterotoxin (ST1 or STa) binds to specific receptors on mammalian intestinal brush border membranes, and stimulates guanylate cyclase in those membranes. We have found a similar signal transduction system in brush border membranes prepared from kidney cortex of the American opossum (Didelphis virginiana, and in a cell line (OK cell) derived from that tissue. Activation of guanylate cyclase by ST1 is therefore not limited to intestinal cells. Furthermore, since it is unlikely that ST1 which is produced in the intestinal lumen, would have access to kidney receptors, this suggests the existence of an endogenous peptide resembling ST1, at least in marsupials.     

Regulation of glucagon-stimulated production of glucose in rat liver by guanosine 3',5'-cyclic phosphate.

Exogenous cGMP can inhibit both basal and glucagon-stimulated production of glucose in liver slices from fed rats. Thus, cAMP and cGMP have opposite effects on the production of glucose in rat liver. Acetylcholine, an activator of guanylate cyclase (EC 4.6.1.2) in other systems, also inhibits the glucagon-stimulated production of glucose. No effect on glucose production was observed with secretin or exogenous GTP.     

Are cyclic nucleotides involved in the initiation of mitogenic activation of human lymphocytes?

In order to obtain more insight into the possible role of cyclic AMP or cyclic GMP in modulating the initial cellular processes following activation of lymphocytes, we measured the effects of the T-cell mitogen concanavalin A and other substances including hormones on the cyclic nucleotide levels in human peripheral blood lymphocytes. The enzyme activities of the corresponding nucleotide cyclases, adenylate cyclase and guanylate cyclase were measured in both isolated plasma membranes or the cytosol of resting or concanavalin A stimulated rabbit thymocytes. Concanavalin A in a mitogenic concentration of about 5-10 micrograms/ml caused small, but consistent increases in cAMP but no changes in cGMP levels during the first hour of activation. Concomitantly, the specific activity of plasma membrane-bound adenylate cyclase was always increased at least 1.5-fold 30 min after stimulation of rabbit thymocytes with concanavalin A, but no effect could be detected on the specific activities of plasma membrane-bound or soluble guanylate cyclase. At high, supraoptimal concentrations of concanavalin A (more than 20 micrograms/ml) cAMP levels dramatically increased in human lymphocytes within minutes, but cGMP levels again were unaffected. Forskolin and beta-adrenergic hormones elevated cAMP in human lymphocytes, whereas cGMP levels were increased by the addition of sodium nitroprusside or alpha-adrenergic hormones. Sodium nitroprusside, in concentrations which elevated cGMP in human lymphocytes, had no influence on the incorporation of [3H]uridine into RNA of resting or concanavalin A stimulated human lymphocytes. Addition of forskolin resulted in an increase of cAMP levels and a dose-dependent decrease of [3H]uridine incorporation into RNA of concanavalin A-stimulated lymphocytes with no effect on resting lymphocytes. The data suggest that cGMP does not play a role in the initial phase of mitogenic activation of lymphocytes, whereas cAMP may be involved in the blast transformation process as an inhibitory signal.     

Effects of maitotoxin on atrial natriuretic factor-mediated accumulation of cyclic GMP in PC12 cells

Maitotoxin (MTX) activates calcium channels and stimulates phosphoinositide breakdown in pheochromocytoma PC12 cells, while having no effect on basal levels of the cyclic nucleotides cAMP and cGMP. Atrial natriuretic factor (ANF) induces a dose-dependent accumulation of cGMP in PC12 cells through the activation of a membrane bound guanylate cyclase. Effects of ANF on cGMP are independent of extracellular concentrations of calcium. Since agents that activate phosphoinositide breakdown can indirectly affect cyclic nucleotide formation, the effects of MTX on ANF-mediated accumulation of cGMP was studied. MTX induces a dose-dependent inhibition of ANF-mediated accumulation of cGMP. The inhibition by MTX requires the presence of extracellular calcium, but is unaffected by the calcium channel blocker nifedipine. The inhibitory effect of MTX is not mimicked by the calcium ionophore ionomycin. A phorbol ester, PMA, which stimulates protein kinase C, also inhibits ANF-mediated accumulation of cGMP. Sodium nitroprusside induces large accumulations of cGMP in PC12 cells through the stimulation of a soluble guanylate cyclase. Neither MTX nor PMA inhibit nitroprusside-mediated accumulation of cGMP. The results indicate that in PC12 cells, protein kinase C activation, either directly with PMA, and indirectly with MTX through phosphoinositide breakdown and formation of diacylglycerol, leads to inhibition of ANF-mediated, but not nitroprusside-mediated accumulation of cGMP.