Stimulation Of cGMP Synthesis Research Articles

Changes in expression of cGMP selective phosphordiesterses 2,5 and 9 in the rat brain during aging

Nitric oxide (NO)-stimulated cGMP synthesis is present in the adult rat brain in close proximity to the NO-synthase containing structures [1]. Intracellular cGMP concentration is under very precise control and depends on parameters of its synthesis by guanylyl cyclase and degradation by phosphodiesterases (PDE). It is known that in the senescent brain the concentration of cGMP decreased [2]. When brain slices were incubated in the presence of isobutylmethylxanthine (IBMX), a nonselective phosphodiesterase inhibitor, sildenafil as a selective PDE5 inhibitor and BAY 60-7550 as a selective PDE2 inhibitor and DEANONOate as an NO donor, we have previously found that old brains are unresponsive to sildenafil treatment. Therefore in this study we wanted to investigate how the expression of cGMP selective phosphodiesterases changes in the brain during aging by using mRNA in situ hybridization.

INSECT MOLECULAR OXYGEN SENSOR FOUND

Insects exchange respiratory gases via tracheal systems –highly branched, gas-filled tubes. Numerous experiments over the past 50 years have shown that insects use information about concentrations of oxygen and carbon dioxide in the tracheal system to respond – behaviourally,morphologically, and physiologically – to changing metabolic demand or environmental oxygen levels. Although a few studies have suggested that the oxygen-sensing mechanism resides in the central nervous system, the molecular details of the mechanism have remained elusive. Now work by David Morton appears to have solved the mystery.A clue came from recent work on Caenorhabditis elegans showing that nematodes lacking a functional copy of an atypical guanylyl cyclase(GCY-35) no longer congregated at preferred oxygen concentrations. Guanylyl cyclases are enzymes that synthesize cGMP, a ubiquitous and important intracellular messenger. GCY-35 and others are termed `atypical' because,unlike most soluble guanylyl cyclases, they bind oxygen and are not stimulated by nitric oxide. The mutant-nematode experiment directly implicated atypical cyclases as oxygen sensors.Building on these findings, Morton decided to see if atypical cyclases in insects could also sense oxygen levels. He transfected mammalian cells with combinations of three atypical guanylyl cyclases from Drosophila and measured cellular cGMP content after exposing the cells to different oxygen levels. In anoxia, cells containing all combinations of atypical cyclases showed rapid several-fold increases in cGMP compared with cells in normal oxygen levels. By contrast, anoxia did not stimulate cGMP synthesis in cells transfected with a conventional cyclase. These results show that in Drosophila the atypical, but not the conventional, guanylyl cyclases can sense oxygen and are activated by low oxygen levels.What additional characteristics should an oxygen sensor have, and do atypical guanylyl cyclases exhibit them? In engineering terms, a reasonable all-purpose sensor would give off a signal that is linearly proportional to the variable sensed – because highly nonlinear outputs would provide little discrimination over some portions of the sensed variable's range and too much over others. Morton showed that cells transfected with atypical cyclases exhibit just this sort of linear behaviour in response to graded oxygen concentrations. Cells accumulated cGMP at low levels in normoxia (21%oxygen) and at progressively higher levels in increasingly severe hypoxia. The response was not all-or-nothing.A final experiment suggested that oxygen interacts with atypical cyclases by binding to their haem groups, the iron-containing groups that, in another incarnation, lug oxygen around our circulatory systems. Morton abolished haem function by incubating transfected cells with a soluble guanylyl cyclase inhibitor that oxidizes iron in the haem group. When he exposed these cells to anoxia, Morton found no accumulation of cGMP. These data suggest that, in normoxia, oxygen bound to the haem group inhibits guanylyl cyclase activity;in hypoxia or anoxia, oxygen dissociation from the group activates guanylyl cyclase activity.Much remains to be done, not least demonstrating that atypical cyclases work in vivo in insects. Nonetheless, in this insect respiratory whodunit, the apprehension of a molecular suspect is an arresting development indeed.

Calcium-dependent Dephosphorylation Mediates the Hyperosmotic and Lysophosphatidic Acid-dependent Inhibition of Natriuretic Peptide Receptor-B/Guanylyl Cyclase-B

C-type natriuretic peptide binding to natriuretic peptide receptor-B (NPR-B) stimulates cGMP synthesis, which regulates vasorelaxation, cell proliferation, and bone growth. Here, we investigated the mechanistic basis for hyperosmotic and lysophosphatidic acid-dependent inhibition of NPR-B. Whole cell cGMP measurements and guanylyl cyclase assays indicated that acute hyperosmolarity decreased NPR-B activity in a reversible, concentration- and time-dependent manner, whereas chronic exposure had no effect. Acute hyperosmolarity elevated intracellular calcium in a concentration-dependent fashion that paralleled NPR-B desensitization. A calcium chelator, but not a protein kinase C inhibitor, blocked both calcium elevations and desensitization. Hyperosmotic medium stimulated NPR-B dephosphorylation, and the receptor was rapidly rephosphorylated and resensitized when the hypertonic media was removed. Lysophosphatidic acid also inhibited NPR-B in a calcium- and phosphorylation-dependent process, consistent with calcium being a universal regulator of NPR-B. The absolute requirement of dephosphorylation in this process was demonstrated by showing that a receptor with glutamates substituted at all known NPR-B phosphorylation sites is unresponsive to hyperosmotic stimuli. This is the first study to measure the phosphorylation state of an endogenous guanylyl cyclase and to link intracellular calcium elevations with its dephosphorylation.

Nitric oxide inhibition of IGF-1 stimulated proteoglycan synthesis: role of cGMP

Insulin-like growth factor (IGF-1) is critical for normal development and maintenance of cartilage, however arthritic cartilage responds poorly to IGF-1; part of this insensitivity is mediated by nitric oxide (NO). These studies test if cGMP is responsible for NO dependent insensitivity to IGF-1 in chondrocytes in situ in organ culture and in monolayer culture. Lapine cartilage and chondrocytes in monolayer culture and cartilage from osteoarthritic human knees were used. Tissues were exposed to NO from iNOS induced by IL-1, and proteoglycan synthesis in response to IGF-1 was evaluated in the presence and absence of cGMP dependent protein kinase (PKG) inhibitors. PKG activators inhibited IGF-1 responses in cartilage but not chondrocytes in monolayer. IL-1 stimulated cGMP synthesis in both monolayer and organ cultures. However, PKG inhibitors in cartilage slices but not in monolayer cultures restored response to IGF-1. PKG activity was detected in both fresh and monolayer chondrocytes, confirming this part of the cGMP signal cascade is intact in both of the preparations evaluated. Arthritic cartilage response to IGF-1 was restored by both N G-monomethyl- l-arginine inhibition of NO synthesis and PKG inhibitors. The data suggests that cGMP mediated effects are critical to NO actions on chondrocytes in situ in the cartilage matrix and supports a role for cGMP in the pathophysiologic effects of NO in osteoarthritis.

Estrogen Decreases the Sensitivity of Anterior Pituitary to the Inhibitory Effect of Nitric Oxide on Prolactin Release

Objective: We investigated the effect of chronic estrogen treatment on the inhibitory action of nitric oxide (NO) on prolactin release. Methods: The effect of NO on prolactin release was studied in anterior pituitaries of female Wistar rats, intact at random stages, ovariectomized (OVX), and OVX treated for 15 days with 17β-estradiol (OVX-E₂). Results: Sodium nitroprusside (NP, 0.5 mM), a NO donor, inhibited prolactin release from anterior pituitaries and was able to stimulate cGMP synthesis in intact and OVX rats. Only a high, supraphysiological concentration of NP (2 mM) inhibited prolactin release from anterior pituitaries of OVX-E₂ rats and increased cGMP synthesis in OVX-E₂ rats. 8-Br-cGMP, a cGMP analogue, decreased prolactin release from anterior pituitaries of OVX rats but did not affect it in OVX-E₂ rats. Conclusion: Our results suggest that estrogen may modify the sensitivity of the anterior pituitary to the inhibitory effect of NO on prolactin release by affecting guanylyl cyclase activity and the cGMP pathway.

Pituitary Adenylate Cyclase-activating Polypeptide Stimulates Nitric-oxide Synthase Type I Expression and Potentiates the cGMP Response to Gonadotropin-releasing Hormone of Rat Pituitary Gonadotrophs

Nitric-oxide synthase type I (NOS I) is expressed primarily in gonadotrophs and in folliculo-stellate cells of the anterior pituitary. In gonadotrophs, the expression and the activity of NOS I are stimulated by gonadotropin-releasing hormone (GnRH) under both experimental and physiological conditions. In the present study, we show that pituitary adenylate cyclase-activating polypeptide (PACAP) is twice as potent as GnRH at increasing NOS I levels in cultured rat anterior pituitary cells. The action of PACAP is detectable after 4-6 h and maximal at 24 h, this effect is mimicked by 8-bromo-cAMP and cholera toxin and suppressed by H89 suggesting a mediation through the cAMP pathway. Surprisingly, NADPH diaphorase staining revealed that these changes occurred in gonadotrophs exclusively although PACAP and cAMP, in contrast to GnRH, have the potential to target several types of pituitary cells including folliculo-stellate cells. There was no measurable alteration in NOS I mRNA levels after cAMP or PACAP induction. PACAP also stimulated cGMP synthesis, which was maximal within 15 min and independent of cAMP, however, only part resulted from NOS I/soluble guanylate cyclase activation implying that in contrast to GnRH, PACAP has a dual mechanism in cGMP production. Interestingly, induction of NOS I by PACAP markedly enhanced the capacity of gonadotrophs to produce cGMP in response to GnRH. The fact that PACAP may act on gonadotrophs to alter NOS I levels, generate cGMP, and potentiate the cGMP response to GnRH, suggests that cGMP could play important cellular functions.

Cytokines decrease sGC in pulmonary artery smooth muscle cells via NO-dependent and NO-independent mechanisms

Exposure of rat pulmonary artery smooth muscle cells (rPASMC) to cytokines leads to nitric oxide (NO) production by NO synthase 2 (NOS2). NO stimulates cGMP synthesis by soluble guanylate cyclase (sGC), a heterodimer composed of alpha(1)- and beta(1)-subunits. Prolonged exposure of rPASMC to NO decreases sGC subunit mRNA and protein levels. The objective of this study was to determine whether levels of NO produced endogenously by NOS2 are sufficient to decrease sGC expression in rPASMC. Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) increased NOS2 mRNA levels and decreased sGC subunit mRNA levels. Exposure of rPASMC to IL-1beta and TNF-alpha for 24 h decreased sGC subunit protein levels and NO-stimulated sGC enzyme activity. L-N(6)-(1-iminoethyl)lysine (NOS2 inhibitor) or 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (sGC inhibitor) partially prevented the cytokine-mediated decrease in sGC subunit mRNA levels. However, cytokines also decreased sGC subunit mRNA levels in PASMC derived from NOS2-deficient mice. These results demonstrate that levels of NO and cGMP produced in cytokine-exposed PASMC are sufficient to decrease sGC subunit mRNA levels. In addition, cytokines can decrease sGC subunit mRNA levels via NO-independent mechanisms.

L-Arginine administration prevents reperfusion-induced cardiomyocyte hypercontracture and reduces infarct size in the pig.

Stimulation of cGMP synthesis protects cardiomyocytes against reoxygenation-induced hypercontracture. The purpose of this study was to determine whether L-arginine supplementation has a protective effect against reperfusion-induced hypercontracture and necrosis in the intact animal. Twenty-four Large-White pigs were randomized to receive either 100 mg/kg of L-arginine i.v. or vehicle 10 min before 48 min of coronary occlusion and 2 h of reperfusion. Hemodynamic variables, coronary blood flow and myocardial segment length changes (piezoelectric crystals) were monitored. Postmortem studies included quantification of myocardium at risk (in vivo fluorescein), infarct size (triphenyltetrazolium reaction), myocardial myeloperoxidase activity and histological analysis. Systemic, coronary vein, and myocardial cGMP concentration were measured in additional animals. Administration of L-arginine had no significant effect in hemodynamics or coronary blood flow. During reperfusion, myocardial cGMP content was reduced in the LAD as compared to control myocardium (P=0.02). L-Arginine increased myocardial cGMP content and caused a transient increase in plasma cGMP concentration during the initial minutes of reperfusion (P=0.02). The reduction in end-diastolic segment length induced by reperfusion, reflecting hypercontracture, was less pronounced in the L-arginine group (P=0.02). Infarct size was smaller in pigs receiving L-arginine (47.9+/-7.2% of the area at risk) than in controls (62.9+/-4.9%, P=0.047). There were no differences between groups in leukocyte accumulation in reperfused myocardium (P=0.80). L-Arginine supplementation reduces myocardial necrosis secondary to in situ ischemia-reperfusion by a direct protective effect against myocyte hypercontracture.

Nitric oxide inhibits aldosterone synthesis by a guanylyl cyclase-independent effect.

To investigate the mechanism of nitric oxide (NO) inhibition of aldosterone release, this study compared the effects of type A natriuretic peptide and heat-stable enterotoxin to a nitric oxide donor, deta nonoate, on cGMP production and angiotensin II-stimulated aldosterone synthesis ill primary cultures of bovine adrenal zona glomerulosa cells. Type A natriuretic peptide (10(-10)-10(-6) M) and deta nonoate (10(-6)-10(-3) M) stimulated concentration-related increases in cGMP production. Heat-stable enterotoxin (10(-6) M) failed to stimulate cGMP synthesis in zona glomerulosa cells. Type A natriuretic peptide and deta nonoate attenuated angiotensin II-stimulated aldosterone production over the same concentration range that stimulated cGMP production. Heat-stable enterotoxin (10(-6) M) was without effect on aldosterone release. To further test the hypothesis that cGMP mediated the inhibition of aldosterone synthesis, the selective inhibitor of soluble guanylyl cyclase, 1H-(1,2,4)oxadiazolo [4,3-a]quinoxalin-1-one (ODQ) was used. ODQ pretreatment (10(-5) M) completely prevented deta nonoate-stimulated cGMP production without altering the inhibitory effect of deta nonoate on angiotensin II-stimulated steroidogenesis. Consistent with its selectivity for inhibiting soluble guanylyl cyclase, ODQ did not block type A natriuretic peptide-stimulated cGMP synthesis or type A natriuretic peptide inhibition of steroidogenesis. Deta nonoate completely blocked 25-hydroxycholesterol- and progesterone-stimulated aldosterone synthesis in zona glomerulosa cells and inhibited the conversion of 25-hydroxycholesterol to pregnenolone in mitochondrial fractions from bovine adrenal cortex. Deta nonoate-derived NO gave an absorbance maximum of the mitochondrial cytochrome P450 of 453 nm and inhibited the absorbance at 450 nm caused by carbon monoxide binding to the enzyme. These results suggest that deta nonoate reduces steroidogenesis independent of guanylyl cyclase activation and that NO has a direct effect to inhibit the activity of cytochrome P450, probably by binding to the heme groups of the cytochrome.

Ultraviolet B Radiation Acts through the Nitric Oxide and cGMP Signal Transduction Pathway to Stimulate Melanogenesis in Human Melanocytes

Ultraviolet B (UVB) radiation is the main physiological stimulus for human skin pigmentation; however, the molecular mechanisms underlying this process are still unclear. Recently, nitric oxide (NO) and cGMP have been involved in mediation of skin erythema induced by UVB. Therefore, we investigated the role of NO and cGMP in UVB-induced melanogenesis. In this study, we demonstrated that UVB stimulation of melanogenesis was mimicked by exogenous NO donors. Additionally, we showed that NO stimulated cGMP synthesis and that cGMP was also a potent stimulator of melanogenesis. Furthermore, the inhibition of the melanogenic effect of NO by guanylate cyclase inhibitor demonstrated that NO mediated its effect through the activation of guanylyl cyclase. Interestingly, 1 min after UVB irradiation, we observed a significant increase in cGMP content in melanocytes. The effects of UVB on cGMP production and on melanogenesis were blocked by both guanylate cyclase and NO synthase inhibitors. Additionally, inhibition of cGMP-dependent kinase also prevented the stimulation of melanogenesis by UVB and NO. Therefore, we concluded that NO and cGMP production is required for UVB-induced melanogenesis and that cGMP mediated its melanogenic effects mainly through the activation of cGMP-dependent kinase.

Role of adenosine on glucagon-induced cAMP in a human cortical collecting duct cell line

The hormonal responsiveness profile of the cortical collecting duct varies from one species to another. To identify the hormones and agonists that modulate the functions of this tubule segment in the human species, we generated a cell line (HCD) immortalized by SV40 virus. The tubular origin of this cell line was assessed by the expression of collecting duct-specific antigens and the ability of vasopressin to increase by nine-fold cAMP synthesis. Glucagon and adenosine stimulated cAMP synthesis, and atrial natriuretic peptide stimulated cGMP synthesis in a concentration-dependent manner. Bradykinin, adenosine and angiotensin increased intracellular calcium concentration ([Ca2+]i). Because adenosine can regulate tubular functions, we examined its role on glucagon-induced cAMP synthesis. Using adenosine analogs, we demonstrated that HCT cells both expressed adenosine type-2 (A2) receptors which stimulated cAMP production, and adenosine type-1 (A1) receptors linked to [Ca2+]i increase which inhibited glucagon-stimulated cAMP synthesis. The inhibitory effect was abolished by pertussis toxin, and was neither due to [Ca2+]i increase nor to protein kinase C activation, which indicated that some A1 adenosine receptors were directly negatively coupled to adenylyl cyclase. These results suggest that adenosine can modify human cortical collecting duct functions in opposite ways according to the adenosine receptor activated.

Natriuretic Peptide Receptor-B (Guanylyl Cyclase-B) Mediates C-type Natriuretic Peptide Relaxation of Precontracted Rat Aorta

The most potent known agonist for the natriuretic peptide receptor-B (NPR-B)/guanylyl cyclase-B is C-type natriuretic peptide (CNP). A homologous ligand-receptor system consists of atrial natriuretic peptide (ANP) and NPR-A/guanylyl cyclase-A. A third member of this family is NPR-C, a non-guanylyl cyclase receptor. Monoclonal antibodies were raised against NPR-B by immunizing mice with a purified receptor-IgG fusion protein consisting of the extracellular domain of NPR-B and the Fc portion of human IgG-gamma 1. One monoclonal antibody, 3G12, did not recognize NPR-A or NPR-C and bound to human and rat NPR-B. CNP binding to NPR-B and stimulation of cGMP synthesis were inhibited by 3G12. With cells isolated from either the media or adventitia layers of rat thoracic aorta, 3G12 did not interfere with ANP-stimulated cGMP synthesis, but it inhibited CNP-stimulated cGMP levels in cells from both layers. CNP (IC50 = 10 nM) and ANP (IC50 = 1 nM) caused relaxation of phenylephrine-contracted rat aortic rings. 3G12 caused a marked increase in the IC50 for CNP, from 10 nM to 140 nM, but failed to affect ANP-mediated relaxation. Therefore, our results for the first time demonstrate that CNP relaxes vascular smooth muscle by virtue of its binding to NPR-B.

Paracrine function of endothelium-derived nitric oxide.

We explored whether endothelium-derived nitric oxide (EDNO) acts in an autocrine or paracrine fashion to activate the soluble guanylate cyclase by measuring the elevations of cGMP levels. Comparisons were made between two culture models in which EDNO can stimulate cGMP synthesis within the same cells (endothelial cells) where it is formed, or in the neighboring cells (smooth muscle cells). The basal amount of EDNO showed no difference in cGMP levels when the endothelial cells (EC) were cultured either alone (iso-culture) or together with the smooth muscle cells (SMC, co-culture). However, cGMP levels were synergistically increased by the BK-stimulated EDNO when EC were co-cultured with SMC. The synergistical increase was significantly inhibited by 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186), an extracellular radical scavenger. These findings suggest that the tonic formed basal EDNO has an autocrine effect on cGMP levels of EC themselves. In contrast, BK-stimulated phasic formed EDNO has not only an autocrine effect, but also a paracrine effect on cGMP levels of EC themselves and the adjacent SMC.

Calcium controls light-triggered formation of catalytically active rhodopsin.

Background light reduces the gain of phototransduction in retinal rods so that the ability to register changes in light intensity is not prevented by saturation of the cell's response. The gain is reduced by a light-induced fall in the intracellular calcium concentration which results from blockage of Ca2+ entry through the channels closed by light and continued Ca2+ extrusion by the Na:Ca,K exchanger. Calcium seems to exert several coordinated effects on the cyclic GMP cascade: a fall in [Ca2+] stimulates cGMP synthesis, increases the affinity of the cGMP-gated channel for cGMP and accelerates rhodopsin deactivation by phosphorylation. We now report that lowering intracellular [Ca2+] reduces the catalytic rhodopsin activity produced by light. The effect is operationally equivalent to a fourfold reduction in the number of rhodopsin molecules available for activation. The reduction in gain is cooperative and half-maximal at about 35 nM Ca2+, suggesting that it is mediated by a specific Ca(2+)-binding protein. Reduced rhodopsin activity in low Ca2+ should contribute to adaptation in background light.

Immunocytochemistry of cGMP in the Cerebellum of the Immature, Adult, and Aged Rat: the Involvement of Nitric Oxide. A Micropharmacological Study.

In this study we describe the localization of formaldehyde-fixed cGMP-immunoreactivity (cGMP-IR) in rat cerebellar tissue slices incubated in vitro. In the absence of phosphodiesterase inhibition, cGMP-immunofluorescence was of low intensity in tissue slices prepared from immature cerebella. Addition of isobutylmethylxanthine (IBMX) to the incubation medium resulted in the appearance of cGMP-IR in clusters of astrocytes in the internal granular layer. Addition of N-methyl-d-aspartate (NMDA), kainic acid, atrial natriuretic factor (ANF), or sodium nitroprusside (SNP) gave an intense cGMP-IR in Bergmann fibres, Bergmann cell bodies, and astrocytes in the internal granular layer. Astrocytes in the white matter showed cGMP-IR after incubation of the slice in the presence of ANF or nitroprusside, but not after NMDA or kainic acid. In addition, after SNP stimulation of cGMP production, cGMP-IR was found in fibres which were not positive for glial fibrillary acidic protein (GFAP). In the adult cerebellar slice, intense basal cGMP-immunostaining was observed in Bergmann fibres, Bergmann cell bodies, and astrocytes in the granular layer. No cGMP-IR was observed in Purkinje cells. Stimulation of the cGMP-content in the glial structures by NMDA, ANF, or SNP, was suggested by the immunocytochemical results. However, when measured biochemically, only the effect of SNP was statistically significant, and immunocytochemistry showed that SNP clearly stimulated cGMP synthesis in neuronal cell structures. In the cerebellum of the aged rat a reduced cGMP-IR was found compared to the adult, in the same structures which showed cGMP-IR in the adult. Basal cGMP-immunostaining was reduced in the presence of haemoglobin, methylene blue, by inhibiting nitric oxide synthesis with NG-monomethyl-l-arginine (NGMAr), or by depletion of external Ca2+. Also the stimulatory effect of NMDA and of ANF (partly) on the cGMP-IR was inhibited by these compounds. cGMP-IR after stimulation of guanylate cyclase by SNP was reduced by the concomitant presence of haemoglobin or methylene blue, but not by NGMAr, or by omission of Ca2+. Our results point to an important role for cGMP in the functioning of glial tissue in the cerebellum and also suggest a role for nitric oxide as an intercellular mediator in the functioning of glutamate and ANF in the cerebellum.

Ongoing protein synthesis needed for 1,25-(OH) 2D 3-mediated rapid increase of cyclic GMP in human skin fibroblasts

Recently we reported that 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2D 3) through interaction with its specific receptor rapidly (within 1 min) stimulated intracellular cGMP production in cultured human skin fibroblasts. Here we show that this effect of 100 nM 1,25-(OH) 2D 3 is prevented by brief (30 min) inhibition of RNA synthesis (with actinomycin D or α-amanitin) or by brief inhibition of protein synthesis (with cycloheximide or diphtheria toxin). The protein synthesis inhibitors also blocked stimulation of cGMP by other steroids (testosterone or dexamethasone at 100 nM) but did not block cGMP stimulation by sodium nitroprusside. Since the time for the 1,25-(OH) 2D 3 receptor to increase cGMP seems too short to require de novo protein synthesis, we conclude that the 1,25-(OH) 2D 3 receptor acts together with rapidly turning over protein(s) to stimulate cGMP synthesis.

Muscarinic receptors and receptor-mediated actions on rat thymocytes

Rat thymocytes possess a single class of saturable, high affinity binding sites for muscarinic antagonists of the benzilate type such as [ 3H]3-quinuclidinyl benzilate ([ 3H]3-QNB). The average number of receptors per cell is 3000 and the equilibrium dissociation constant of [ 3H]3-QNB on intact cells is 7.5 nM. In the work reported here we found that perturbation of the thymocyte membrane by addition of phytohemagglutinin (4 μg/ml) caused a transient increase in muscarinic antagonist binding, and hydrocortisone (100 mg/kg s.c.) treatment of rats for 2 days prior to sacrifice increased the average number of muscarinic receptor sites on thymocytes by 100%. Atropine treatment, which in other tissues causes increased muscarinic receptor concentration, did not alter the receptor number on thymocytes. Binding of carbachol to the receptor on intact cells resulted in inhibition of cAMP synthesis and stimulation of cGMP synthesis. These muscarinic agonist effects were each inhibited by the simultaneous addition of the muscarinic antagonist atropine (5 × 10 −5 M). No stimulation of phosphatidylinositol turnover by muscarinic agonists was observed.

Receptor-induced degradation of atrial natriuretic peptide by a rabbit carotid artery smooth muscle cell.

We have used a smooth muscle cell line isolated from rabbit carotid artery (RCA) as a model system with which to study the expression of atrial natriuretic peptide (ANP) receptors and, in addition, the receptor-mediated degradation of ANP. RCA cells bind rat alpha ANP-(1-28) reversibly at 37 C, apparently to a single class of high affinity (Kd approximately equal to 50 pM) binding sites (approximately equal to 20,000 sites per cell). Binding of rat alpha ANP-(1-28) elicits rapid accumulation of intracellular cGMP. However, the concentrations of rat alpha ANP-(1-28) and related peptides, abbreviated at the N- and C-terminals, required to stimulate cGMP synthesis are substantially greater than those required for binding. Analysis by HPLC of 125I-labeled rat alpha ANP-(1-28) bound to RCA cells at 37 C illustrates the rapid and continuous degradation of the radioiodinated rat alpha ANP-(1-28) to two radiolabeled products, one of which, 125I-labeled tyrosine is the major radiolabeled component that dissociates from the cells. Measurement of rat alpha ANP-(1-28) interaction with RCA cells by radioligand binding techniques therefore subsumes several processes. One of these processes is the rapid and continuous degradation of specifically bound ANP by these cells and perhaps also other target cells that respond to ANP.

Atrial natriuretic factor: Structural requirements of the peptide for receptor binding, biological activity, and cGMP stimulation

AbstractThe atrial natriuretic factor (ANF) family of peptides possesses diverse functional properties including smooth‐muscle relaxation, inhibition of aldosterone biosynthesis, and hypotensive activities. These separate activities are most likely mediated by the binding of the peptide to specific receptors located in the target organs. The present study was initiated to more closely examine the relationship between biological activity and the requirements of the various receptors for interaction with ANF and with ANF analogs. Binding affinities (Ki) were measured in plasma membranes isolated from rabbit kidney cortex, aorta, and adrenal, and in cultured rabbit aortic vascular smooth muscle cells (VSMC) using 125I‐Tyr‐28‐ANF (1–28) as the ligand. The VSMC were also utilized to quantitate stimulation of cGMP synthesis. Vasorelaxation (pD2) was measured in histamine‐contracted rabbit aortic rings. The activities of 19 ANF (5–28) analogs were compared, and a significant correlation between pD2 and Ki (r = −.83; p < 0.01) was obtained. The (D)Phe‐8 and Asn‐13 analogs were exceptions to this correlation; pD2 = 7.1 and 7.2, and pKi = 6.0 and 8.1, respectively. All analogs examined possessed comparable affinities in the four receptor assays. Conversely, comparison of vasorelaxant and cGMP stimulatory activities resulted in a much larger separation of activities. Tyr‐8 ANF (5–27) did not stimulate cGMP synthesis but relaxed smooth muscle (pD2 = 7.9), while ANF (7–23) was 500‐fold less active than ANF (5–28) in the vasorelaxant assay (pD2 = 6.1 and 8.9, respectively) but equipotent in the cGMP system. These and other experiments suggest the presence of ANF receptor subtypes and question the causative role of cGMP for inducing vasorelaxation of histamine‐constricted aortic rings.

Cyclic GMP accumulation by atriopeptins in cultured rat renal and vascular smooth muscle cells.

To evaluate the role of cyclic guanosine monophosphate (cGMP) in the vascular and renal action of atrial natriuretic peptide (ANP), we compared the effects of atriopeptins (APs) on cGMP accumulation in cultured cells from rat mesenteric vascular smooth muscle (VSM), glomerular mesangium (GM) and renal papillary collecting tubule (RPCT), and also evaluated the relationship between renal sodium or water excretion and urinary cGMP in AP-infused rats. Both AP I and AP III increased intracellular cGMP levels dose-dependently in all types of cells, while they did not affect intracellular cAMP levels or prostaglandin synthesis. AP III was 100 times more potent than AP I. The magnitude change in cGMP levels was largest in GM cells. The sensitivity of VSM and GM cells to AP III were greater than that of RPCT cells. There were significant positive relationships between urinary excretion of sodium or water and that of cGMP levels in AP-infused rats. These results may suggest that GM and VSM cells are the principal targets for ANP to stimulate cGMP synthesis and, possibly, to exert the renal sodium and water excretion, and also support the hypothesis that cGMP mediates the cellular action of ANP.