Particulate Guanylate Cyclase Activity Research Articles

Alteration of mesangial response to ANP and angiotensin II by glucose

To test the hypothesis that the function of glomerular mesangial cells is impaired in diabetes, we examined the responsiveness of mesangial cells cultured under high concentrations of glucose to atrial natriuretic peptide (ANP1) and angiotensin II (Ang II). The ANP-induced accumulation of cGMP was enhanced in mesangial cells cultured under high glucose conditions, possibly due to the activation of particulate guanylate cyclase. Ang II action in mesangial cells was evaluated by measuring the ability of Ang II to inhibit ANP-induced cGMP accumulation through both activating phosphodiesterase (initial phase) and inhibiting guanylate cyclase (maintenance phase). The inhibition of both ANP-induced cellular cGMP accumulation and particulate guanylate cyclase activity by Ang II was significantly reduced in mesangial cells cultured under high concentrations of glucose. Moreover, in the cells exposed to high concentrations of glucose, both basal and Ang II-stimulated levels of inositol 1,4,5-trisphosphate (IP3) were significantly reduced. These results indicate that, in high glucose conditions, the actions of ANP and Ang II are modulated differently, resulting in the impairment of contractile responsiveness of mesangial cells.

Agonistic and antagonistic properties of angiotensin analogs at the AT2 receptor in PC12W cells.

Despite some recent reports describing the effects of AT2 receptor selective ligands in vitro and in vivo, the physiological function of this receptor is still a matter of debate. This problem stems amongst others from the difficulty in interpreting results from in vivo experiments with drugs of which it is not known whether they act as agonists or antagonists. We reported earlier that angiotensin II inhibits basal and atrial natriuretic peptide stimulated particulate guanylate cyclase activity through AT2 receptors in PC12W cells. We have used this parameter in intact PC12W cells in order to determine the pharmacological properties of different widely used angiotensin receptor ligands. We found CGP 42112 to behave as a full agonist in this system, whereas PD 123319 and Sar Ile angiotensin II act as antagonists. As expected, the AT1 antagonist losartan did not affect this response.

1,25-Dihydroxycholecalciferol rapidly activates rat colonic particulate guanylate cyclase via a protein kinase C-dependent mechanism.

The present studies were performed to determine whether the major biologically active metabolite of vitamin D3, 1,25-dihydroxycholecalciferol [1,25(OH)2D3], could influence the activities of rat colonic particulate guanylate cyclase and adenylate cyclase. To address these issues, colonocytes were harvested from Sprague-Dawley rats and suspended in Krebs-Ringer bicarbonate buffer. The cells were then treated with 1,25(OH)2D3 or other agents (see below) and crude membranes were prepared and analyzed for particulate guanylate cyclase and adenylate cyclase activities. The results of these studies demonstrated that: 1) 1,25(OH)2D3, in a concentration-dependent manner, rapidly (within minutes) stimulated guanylate, but not adenylate cyclase activity; 2) preincubation of the cells with staurosporine, a protein kinase inhibitor, or U73122, an inhibitor of phosphoinositide-phospholipase C-dependent processes, blocked the increase in guanylate cyclase activity induced by 1,25(OH)2D3; and 3) 12-O-tetradecanoyl phorbol 13-acetate and 1,2-dioctanoyl-sn-glycerol, known activators of protein kinase C, also rapidly stimulated rat colonic particulate guanylate cyclase activity. Taken together, these results demonstrate that 1,25(OH)2D3 rapidly stimulates together, these results demonstrate that 1,25(OH)2D3 rapidly stimulates rat colonic particulate guanylate cyclase, at least in part, via a protein kinase C-dependent mechanism.

Modulation of barrier function of bovine aortic and pulmonary artery endothelial cells: dissociation from cytosolic calcium content.

1. Barrier function and cytosolic free calcium content [Ca2+]i was measured in monolayers of bovine pulmonary artery endothelial cells (BPAEC) and bovine aortic endothelial cells (BAEC). 2. Thrombin (1 u ml-1) increased albumin transfer across monolayers of BPAEC but not BAEC, yet induced biphasic increases in [Ca2+]i in both endothelial cell types, consisting of a rapid, initial phasic component which decayed to a lower, more sustained plateau phase. 3. 4 beta-Phorbol 12-myristate 13-acetate (PMA; 0.3-3000 nM) increased albumin transfer across monolayers of BPAEC and BAEC, but had no effect on basal levels of [Ca2+]i in either endothelial cell type. 4. Treatment of BPAEC and BAEC with forskolin (30 microM), an activator of adenylate cyclase, had no effect on resting transfer of albumin, but inhibited that stimulated by PMA (600 nM). It also inhibited the thrombin (1 u ml-1)-induced increase in albumin transfer across monolayers of BPAEC, but enhanced the plateau phase of the associated increase in [Ca2+]i. 5. Treatment of BPAEC and BAEC with either atriopeptin II (100 nM), an activator of particulate guanylate cyclase, or 8 bromo cyclic GMP (30 microM) had no effect on resting or PMA (600 nM)-stimulated transfer of albumin. Both agents did, however, inhibit the thrombin (1 u ml-1)-induced increase in albumin transfer across monolayers of BPAEC, but had no effect on the associated increase in [Ca2+]i. 6. These data suggest a dissociation between the ability of agents that increase or decrease albumin transfer and their effects on [Ca2+]i. Consequently, activation of protein kinase C may be the major stimulus for trans-endothelial transfer of macromolecular solutes. Endothelial barrier function is enhanced by elevation of either cyclic AMP or cyclic GMP content. Cyclic AMP appears to act by inhibiting the actions of protein kinase C, while cyclic GMP may act to inhibit a key step proximal to activation of this enzyme.

Biphasic 24-hour variations in cyclic GMP accumulation in the rat pineal gland are due to corresponding changes in the activity of cytosolic and particulate guanylate cyclase.

Various parameters of the rat pineal gland display a 24-h rhythm. However, nothing is known about possible 24-h variations in cyclic GMP (cGMP) metabolism. In the present study, 24-h variations in pineal gland cGMP accumulation were investigated by determining the increase in cGMP level with and without inhibitors of phosphodiesterase at different time points over a light/dark cycle (12/12 h). Furthermore, the activity of guanylate cyclase (GC) was determined under substrate-saturated conditions regarding the cytosolic and particulate forms of the enzyme. It has been found that cGMP accumulation and GC activity display biphasic 24-h variations with two peaks--one approximately 7 h after lights "on" and the other approximately 7 h after lights "off." The activity of cytosolic GC remains unchanged in the presence of the nitric oxide (NO) synthesis inhibitor N-monomethyl-L-arginine, indicating that 24-h variations in the activity do not reflect changes in the synthesis of the GC stimulator NO.

Effect of age on activation of porcine intestinal guanylate cyclase and binding of Escherichia coli heat-stable enterotoxin (STa) to porcine intestinal cells and brush border membranes

Summary Development of age-dependent resistance to enterotoxigenic Escherichia coli was studied, using isolated enterocytes and brush border membranes (bbm) from 7-day-old and 7-week-old pigs. Binding of 125I-labeled heat-stable (125I-STa) enterotoxin to enterocytes and bbm was specific, temperature- and time-dependent, saturable, and partially reversible. Scatchard analysis revealed a single class of receptors. Mean ± sd avidity of binding (apparent affinity constant, Ka) of 125I-STa to enterocytes from 7-day-old and 7-week-old pigs was 2.14 ± 0.29 × 108 and 2.72 ± 0.25 × 108 L/mol, respectively. Numbers of STa receptors were calcuated to be 64,903 ± 2,900/enterocyte for 7-day-old pigs and 53,029 ± 3,117/enterocyte for 7-week-old pigs. Numbers of STa receptors expressed per milligram of bbm protein from 7-day-old pigs were 2.66 × 1011, compared with 2.29 × 1011 for bbm from 7-week-old pigs. By 5 minutes after addition of STa to reaction mixtures, intracellular cyclic guanosine monophosphate concentration increased 13.9-fold in enterocytes from 7-day-old pigs and 8.7-fold in enterocytes from 7-week-old pigs. The particulate guanylate cyclase activity associated with bbm from 7-week-old pigs was slightly more sensitive to low amounts of STa, compared with bbm from 7-day-old pigs; however, differences were not observed at intermediate and high amounts. These data indicate that lack of a secretory response to STa by older pigs is not attributable either to decreased numbers of STa receptors or to decreased signal response between the STa receptor and membrane-bound guanylate cyclase. Development of age-dependent resistance by porcine small intestine to STa appears to be attributable to steps in the secretory pathway that respond to increased concentration of cyclic guanosine monophosphate.

Retinal on-bipolar cells contain a nitric oxide-sensitive guanylate cyclase

Retinal on-bipolar cells possess specialized glutamate receptors which are coupled via a G-protein to the control of a cyclic GMP (cGMP) cascade. Whole-cell voltage clamp recordings were obtained from light-responsive on-bipolar cells in retinal slices of the dogfish. Inclusion of nitroprusside in the patch-pipette solution induced effects in on-bipolar cells which were consistent with a rise in intracellular cGMP and thus stimulation of guanylate cyclase (GC) activity. Conversely, the soluble GC inhibitors, methylene blue and ferricyanide, induced effects consistent with a fall in intracellular cGMP. Activators of particulate GC had no effect. We conclude that cGMP synthesis in on-bipolar cells is catalysed by a NO-sensitive cyclase.

Determinants of aortic cyclic guanosine monophosphate in hypertension induced by chronic inhibition of nitric oxide synthase.

Nitric oxide (NO) and atrial natriuretic factor (ANF) cause vascular relaxation by generating cyclic guanosine monophosphate (cGMP) via activation of the soluble and particulate guanylate cyclases, respectively. The chronic effects of NG-nitro-L-arginine methyl ester (L-NAME), an L-arginine antagonist and NO synthase inhibitor, on the blood pressure and plasma and aortic cGMP levels of rats were tested. Wistar rats (n = 10 per group) were given doses of L-NAME (0, 1, 5, 10, 20, 50, and 100 mg/kg.d) by gavage twice a day for 4 wk. Chronic L-NAME induced a time- and dose-dependent increase in blood pressure. The total heart weight/body weight ratio did not change in any group, despite the hypertension. The plasma levels of cGMP did not change significantly in any group, and were correlated with the plasma ANF levels (r = 0.51, P less than 0.0001). Aortic cGMP decreased in negative correlation with increasing L-NAME from 0 to 10 mg/kg.d, culminating in a 10-fold drop arterial wall cGMP. The aortic cGMP content of rats in the four highest dose groups (from 10 to 100 mg/d) tended to increase slightly and was positively correlated with endogenous ANF (r = 0.48, P less than 0.002, n = 40). Intravenous L-arginine decreased arterial blood pressure and reversed the decline in aortic cGMP. Exogenous ANF and sodium nitroprusside both significantly increased aortic cGMP. Neither the arterial wall concentrations of cGMP-dependent kinase nor cAMP was changed by L-NAME. Thus, chronic blockade of NO synthase with L-NAME induces a dose-dependent increase in blood pressure and decrease in aortic cGMP. The in vivo basal aortic cGMP seems to be mainly dependent on NO synthase: soluble guanylate cyclase activity and to a minor extent on particulate guanylate cyclase activity.

Developmental changes in the effect of atrial natriuretic peptide on tissue cyclic GMP content and particulate guanylate cyclase activity of aorta, kidney and lung of rats

To investigate possible developmental changes in the physiological effect of atrial natriuretic peptide (ANP) after birth, we studied the effect of ANP on the slice cGMP content and the particulate guanylate cyclase activity of aorta, kidney and lung in neonate, 2-week-old and adult rats of both sexes. Incubation with human ANP(99–126) (hANP) increased significantly the slice cGMP content of aorta, kidney and lung in three ages of rats. The hANP-stimulated fraction of cGMP contents of kidney decreased, that of lung increased with development, whereas that of aorta showed no significant change. Consistently, the hANP-responsive particulate guanylate cyclase activity decreased in kidney, increased in lung during development, without significant developmental change in aorta. These results indicate a differential change in the effect of hANP on the slice cGMP content among tissues during development. The developmental change in the effect of hANP on slice cGMP content is probably caused by the ontogenetic change in activation of ANP receptor-linked guanylate cyclase.

Atriopeptins and Escherichia coli enterotoxin STa have different sites of action in mammalian intestine

Studies with Escherichia coli-induced heat-stable enterotoxin (STa), an activator of intestinal particulate guanylate cyclase, have established an independent role for cyclic guanosine monophosphate (cGMP) as an intracellular mediator of intestinal salt and water secretion. The present study addressed whether atriopeptins (APs), known activators of particulate guanylate cyclase in other tissues, function as physiological agonists for cGMP-linked Cl−1 secretion in intestine. APs, in contrast to STa, caused no or only minor changes in cGMP levels in freshly isolated rat intestinal villus and crypt cells and in cultured human colon carcinoma cell lines (HT29glc−, CaCo-2, and T84). Conversely, APs, but not STa, induced a large increase in intracellular cGMP levels in the undifferentiated small intestinal cell lines IEC-6, IEC-18, and INT407. Addition of APII (atrial natriuretic peptide fragment 5–27) to stripped mucosa of rat proximal colon in Ussing chambers caused a transient increase in the transepithelial potential difference (PD), which most likely represents an increase in Cl− secretion. In contrast, a sustained increase in PD was observed in response to STa or 8Br-cGMP. The AP II-provoked increase in PD was blocked by the neurotoxin tetrodotoxin. Immunohistochemical detection of cGMP in this tissue provided evidence for a different localization pattern of cells responding with an increase in cGMP levels to STa (colonocytes and goblet cells) or AP (specific cells in the submucosa) in rat proximal colon. This indicates that APs, unlike STa, do not directly stimulate the colonic epithelial cells but possibly provoke Cl− secretion by release of a neurotransmitter in the submucosa.

Atriopeptins and Escherichia coli enterotoxin STa have different sites of action in mammalian intestine

Studies with Escherichia coli-induced heat-stable enterotoxin (STa), an activator of intestinal particulate guanylate cyclase, have established an independent role for cyclic guanosine monophosphate (cGMP) as an intracellular mediator of intestinal salt and water secretion. The present study addressed whether atriopeptins (APs), known activators of particulate guanylate cyclase in other tissues, function as physiological agonists for cGMP-linked Cl- secretion in intestine. APs, in contrast to STa, caused no or only minor changes in cGMP levels in freshly isolated rat intestinal villus and crypt cells and in cultured human colon carcinoma cell lines (HT29glc-, CaCo-2, and T84). Conversely, APs, but not STa, induced a large increase in intracellular cGMP levels in the undifferentiated small intestinal cell lines IEC-6, IEC-18, and INT407. Addition of AP II (atrial natriuretic peptide fragment 5-27) to stripped mucosa of rat proximal colon in Ussing chambers caused a transient increase in the transepithelial potential difference (PD), which most likely represents an increase in Cl- secretion. In contrast, a sustained increase in PD was observed in response to STa or 8Br-cGMP. The AP II-provoked increase in PD was blocked by the neurotoxin tetrodotoxin. Immunohistochemical detection of cGMP in this tissue provided evidence for a different localization pattern of cells responding with an increase in cGMP levels to STa (colonocytes and goblet cells) or AP (specific cells in the submucosa) in rat proximal colon. This indicates that APs, unlike STa, do not directly stimulate the colonic epithelial cells but possibly provoke Cl- secretion by release of a neurotransmitter in the submucosa. (Gastroenterology 1992 Apr;102(4 Pt 1):1161-9)

The angiotensin AT 2 receptor stimulates protein tyrosine phosphatase activity and mediates inhibition of particulate guanylate cyclase

The signalling mechanism and cellular targets of the AT 2 receptor are still unknown. We report that angiotensin II (Ang II) inhibits basal and atrial natriuretic peptide stimulated particulate guanylate cyclase (pGC) activity through AT 2 receptors in rat adrenal glomerulosa and PC12W cells. This inhibition is blocked by the phosphotyrosine phosphatase (PTPase) inhibitor orthovanadate but not by the Ser Thr phosphatase inhibitor okadaic acid, suggesting the involvement of a PTPase in this process. Moreover, Ang II induces a rapid, transient and orthovanadate sensitive dephosphorylation of phosphotyrosine containing proteins in PC12W cells. Our findings suggest that AT 2 receptors signal through stimulation of a PTPase and that this mechanism is implicated in the regulation of pGC activity. This observation is also the first example of hormonal inhibition of basal pGC activity.

Effects of somatostatin analog SMS 201-995 on enterotoxigenic diarrhea

Somatostatin analog SMS 201-995 causes a dose-related suppression of the release and/or action of several gastrointestinal hormones and impairs several anterior pituitary functions. Some patients with illness involving abnormal hormonal activity have responded to treatment with SMS 201-995, including resolution of severe secretory diarrhea. This study examined SMS 201-995 inhibition of Escherichia coli heat-stable enterotoxin STa (STa) effects and effects of the analog in the rabbit RITARD model with enterotoxigenic Escherichia coli. SMS 201-995 did not alter STa binding to its receptor on piglet brush border membranes. The analog, at concentrations of 0.1 micrograms/ml (0.1 microM) and 1 microgram/ml (1 microM) did not significantly alter STa activation of intestinal epithelial cell particulate guanylate cyclase. At maximal dosing the analog significantly reduced intestinal fluid secretion in suckling mice that was induced by either 8-bromo cyclic GMP or STa. In piglets, the analog reduced by 37-44% the amount of diarrhea induced by STa. However, even with maximal dosing, the piglets still had significant diarrhea, although of a lesser amount. In the rabbit RITARD model the drug failed to alter the severe diarrheal response seen when dosing the animals with enterotoxigenic Escherichia coli. Overall, SMS 201-995 had a significant but incomplete effect in reducing the STa effects seen in the various assays. Additionally, in the RITARD model the analog did not alter the clinical responses to various enterotoxigenic bacteria. SMS 201-995 should be useful as a probe into the mechanisms involved in intestinal fluid secretion, but a clinical role in enterotoxigenic gastrointestinal disease was not supported by this study.

Identification and characterization of a new family of high-affinity receptors for Escherichia coli heat-stable enterotoxin in rat intestinal membranes

Novel high-affinity, low-capacity binding sites in intestinal membranes for the heat-stable toxin produced by Escherichia coli have been defined. The appearance of these sites is observed in the presence of physiological concentrations of NaCl in binding reactions. Scatchard analyses of equilibrium binding in the absence of NaCl demonstrated a single class of binding sites with KD = 1.9 x 10(-9) M and Bmax = 0.75 pmol/mg of protein. In contrast, similar experiments in the presence of NaCl demonstrated, in addition to the previously described low-affinity site, a high-affinity site with a KD of 2.1 x 10(-11) M and a Bmax of 73 fmol/mg of protein. Confirmation of the presence of high- and low-affinity sites was obtained in studies of the kinetics of ST binding. These sites exhibited similar dissociation but markedly different association kinetics. Determination of the association and dissociation constants permitted calculation of the KD's for the high- and low-affinity sites, which were 1.15 x 10(-11) M and 1.89 x 10(-9) M, respectively. These data agree closely with those obtained in studies of equilibrium binding. Furthermore, similar values for the KD's of these sites were obtained in experiments of competitive displacement of labeled ST, confirming the presence of two receptors for this toxin. Binding of ST to high-affinity sites is completely reversible and does not appear to be coupled to activation of particulate guanylate cyclase. In contrast, binding of ST to low-affinity sites appears to be partially reversible and may be coupled to activation of guanylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)

Dual mechanism of angiotensin II inhibits ANP-induced mesangial cGMP accumulation

To evaluate an interaction between vasoconstrictive (Ang II) and vasodilating (ANP) peptides, we examined the effect of Ang II on ANP-induced accumulation of cGMP in cultured glomerular mesangial cells. ANP rapidly increased intracellular cGMP levels, with a peak stimulation at one minute in the absence of IBMX and at ten minutes in the presence of IBMX. The ANP-induced cGMP accumulation was significantly inhibited when the cells were treated with Ang II simultaneously with ANP for one minute in the absence of IBMX. This inhibitory effect of Ang II was completely abolished by IBMX and significantly reduced in calcium-free media or by W7, but not affected by H7. Similar inhibitory effect was observed when cells were treated with A23187 but not with TPA for one minute. In the presence of IBMX, Ang II inhibited ANP-induced cGMP accumulation when cells were treated with Ang II for 15 minutes prior to the stimulation by ANP. This inhibition by Ang II was blocked by H7. ANP-induced increase in particulate guanylate cyclase activity was significantly reduced in the cells treated with Ang II or TPA. This reduction of enzyme activity was also prevented by H7. These results indicate that Ang II inhibits ANP-induced cGMP accumulation in cultured glomerular mesangial cells through at least two mechanisms; one is the activation of calcium-dependent, calmodulin-stimulated cyclic nucleotide phosphodiesterase in the initial phase, and the other is the inhibition of guanylate cyclase resulting from protein kinase C activation in the maintenance phase.

C-Type natriuretic peptide is a growth inhibitor of rat vascular smooth muscle cells

C-type natriuretic peptide (CNP) which potently stimulates particulate guanylate cyclase activity in cultured rat vascular smooth muscle cells (VSMC) inhibited serum-induced DNA synthesis of the cells 10-fold more effectively than α-human atrial natriuretic peptide (α-hANP). The inhibitory effect of CNP was mimicked by 8-bromo-cGMP. The proliferation of VSMC was also suppressed by CNP more potently than α-hANP, while the peptide was less active for cGMP augmentation and for vasorelaxation than α-hANP in isolated rat aorta. These results suggest that CNP may be a growth regulating factor of VSMC rather than a vasodilator.

Activation of particulate guanylate cyclase by Escherichia coli heat-stable enterotoxin is regulated by adenine nucleotides.

Guanylate cyclase is regulated by adenine nucleotides in membranes of intestinal mucosal cells. Basal guanylate cyclase was activated about twofold by adenine nucleotides. Activation was specific for adenine, as compared with the pyrimidine nucleotides UTP and CTP. In addition, enzyme activation was obtained in the presence of saturating concentrations of GTP, the substrate for guanylate cyclase. The most potent adenine nucleotide was the nonhydrolyzable analog of ATP, adenosine 5'-O-(3-thiotriphosphate). Adenine nucleotide activation was specific for the particulate form of guanylate cyclase, as compared with the soluble form. Also, adenine nucleotides potentiated the activation of guanylate cyclase by the heat-stable enterotoxin produced by Escherichia coli. Indeed, enzyme activation by adenine nucleotides and toxin was greater than the sum of individual activations by these agents. Adenine nucleotides regulate guanylate cyclase by increasing the maximum velocity of the enzyme without altering its affinity for substrate or its cooperativity. In addition to stimulating guanylate cyclase, adenine nucleotides decreased the specific binding of the heat-stable enterotoxin to receptors in intestinal membranes. The coordinated regulation of the toxin-receptor interaction and guanylate cyclase activity by a process utilizing nonhydrolyzable analogs of a purine nucleotide is similar to the mechanisms involved in the hormone regulation of adenylate cyclase by guanine nucleotide-binding proteins. These data suggest that an adenine nucleotide-dependent protein may couple the toxin-receptor interaction to the regulation of particulate guanylate cyclase in intestinal membranes.

Adenine nucleotide regulation of particulate guanylate cyclase from rat lung

Adenine nucleotides activate basal particulate guanylate cyclase in rat lung membranes. Activation is specific for adenine and not guanine, cytidine or uridine nucleotides. The concentration of adenine nucleotides yielding half-maximum activation of particulate guanylate cyclase is 0.1 mM and this nucleotide activates the enzyme by increasing maximum velocity 11-fold without altering affinity for substrate. Activation is specific for particulate guanylate cyclase, since soluble enzyme is inhibited by adenine nucleotides. Similarly, activation is specific for magnesium as the enzyme substrate cation cofactor, since adenine nucleotides inhibit particulate guanylate cyclase when manganese is used. Adenine nucleotide regulation of particulate guanylate cyclase may occur by a different molecular mechanism compared to other activators, since the effects of these nucleotides are synergistic with those of detergent, hemin and atrial natriuretic peptides. Cystamine inhibits adenine nucleotide activation of particulate guanylate cyclase at concentrations having minimal effects on basal enzyme activity suggesting a role for critical sulfhydryls in mechanisms underlying nucleotide regulation of particulate guanylate cyclase. Purification and quantitative recovery of particulate guanylate cyclase by substrate affinity chromatography results in the loss of adenine nucleotide regulation. These data suggest that adenine nucleotides may be important in the regulation of basal and activated particulate guanylate cyclase and may be mediated by an adenine nucleotide-binding protein which is separate from that enzyme.

Particulate guanylate cyclase and adenylate cyclase activities after activation with various agents in rabbit platelets. An ultracytochemical study

The cytochemical localization of particulate guanylate cyclase and adenylate cyclase activities in rabbit platelets were studied after stimulation with various agents, at the electron microscope level. In the presence of platelet aggregating agents such as thrombin and ADP, the particulate reaction product of guanylate cyclase activity was detectable on plasma membrane and on membranes of the open canalicular system. In contrast, samples incubated with platelet-activating factor showed no activation of the cyclase activity. Atrial natriuretic factor stimulated the particulate guanylate cyclase. The ultracytochemical localization of this activated cyclase was the same as that of thrombin- or ADP-stimulated guanylate cyclase. Adenylate cyclase activity was studied in platelets incubated with prostaglandin E1 plus or minus insulin. The enzyme reaction product was found at the same sites where guanylate cyclase was detected. Therefore guanylate and adenylate cyclase activities do not seem to be preferentially localised in platelet membranes.

Distinct plasma atrial natriuretic factor, renin and aldosterone responses to prolonged high-salt intake in hypertensive and normotensive rats

Five weeks of high (8%) sodium intake, resulting in a decline of plasma atrial natriuretic factor (ANF) in normotensive Wistar-Kyoto (WKY) and Wistar rats, did not affect plasma ANF in spontaneously hypertensive rats (SHR) which became severely hypertensive. Regardless of salt consumption, SHR presented more pronounced glomerular particulate guanylate cyclase activation after large ANF doses in vitro than normotensive rats. In response to salt loading, plasma renin activity (PRA) and plasma aldosterone unexpectedly increased in SHR, in contrast to their decrease in the normotensive strains. Thus, SHR fail to react to prolonged high-salt intake as do normotensive rats, i.e. by a fall in plasma ANF, PRA and plasma aldosterone, and have higher stimulated glomerular particulate guanylate cyclase activity. Thus, ANF and its target response in SHR, as well as the PRA-plasma aldosterone reaction to prolonged salt loading, are distinct from those in normotensive strains. Since the relatively increased ANF and its target action in SHR appear to be a reactive antihypertensive defense rather than a primary event, systems other than ANF probably play an important role in the high salt-induced accelerated hypertension of SHR.