Calcitonin gene-related peptide stimulates cyclic AMP formation in rat aortic smooth muscle cells

In rat aortic smooth muscle cells in culture, calcitonin gene-related peptide stimulated cAMP formation in a dose-dependent manner, half-maximally effective at 0.5 to 1 nM. There was no effect on formation of cGMP, which was increased 300-fold in the same experiments by atriopeptin or sodium nitroprusside. The vasodilator effect of CGRP in rat aorta requires an intact endothelium, indicating that increase in vascular smooth muscle cAMP is not in itself sufficient to bring about relaxation. cAMP is probably a mediator of CGRP action in vascular smooth muscle.     

Acidic reoxygenation protects against endothelial dysfunction in rat aortic rings submitted to simulated ischemia

Ischemia-reperfusion causes endothelial dysfunction. Prolongation of acidosis during initial cardiac reperfusion limits infarct size in animal models, but the effects of acidic reperfusion on vascular function are unknown. The present work analyzes the effects of acidic reoxygenation on vascular responses to different agonists in rat aortic rings. Arterial rings obtained from Sprague-Dawley rat aorta were placed in organ baths containing a Krebs solution oxygenated at 37 degrees C (pH 7.4). After equilibration (30 mN, 1 h), the effects of acidosis (pH 6.4) on aortic responses to acetylcholine and norepinephrine were initially assessed under normoxic conditions. Thereafter, the effects of acidosis during hypoxia (1 h) or reoxygenation on aortic responses to acetylcholine, norepinephrine, or sodium nitroprusside were analyzed and compared with those observed in control rings. Acidosis did not modify aortic responses to acetylcholine or adrenaline during normoxia. In contrast, rings submitted to hypoxia and reoxygenated at pH 7.4 showed a reduction in vasodilator responses to acetylcholine and in contractions to norepinephrine with no change in responses to sodium nitroprusside. Reoxygenation at pH 6.4 did not modify the depressed response to norepinephrine but enhanced the recovery of acetylcholine-induced vasorelaxation. Cumulative concentration-response curves to acetylcholine showed an increased responsiveness to this drug in rings reoxygenated at a low pH. This functional improvement was associated with the preservation of aortic cGMP content after stimulation of reoxygenated rings with acetylcholine. In conclusion, acidic reoxygenation preserves endothelial function in arterial rings submitted to simulated ischemia, likely through the preservation of cGMP signaling.     

Dendroaspis natriuretic peptide-like immunoreactivity and its regulation in rat aortic vascular smooth muscle

Dendroaspis natriuretic peptide (DNP) is a recently isolated 38 amino acid peptide that shares structural and functional properties with the other members of the natriuretic peptide family. The present study demonstrates the presence of DNP-like immunoreactivity in sections of rat aorta, carotid artery and renal vasculature and tubules. DNP-like immunoreactivity was detected in culture aortic vascular smooth muscle cells and medium and is regulated by endothelin-1, angiotensin II and sodium nitroprusside but not by transforming growth factor-beta. Our observations indicate that DNP elicits a marked inhibitory effect on DNA synthesis in culture rat aortic vascular smooth muscle cells.     

Requirement for bivalent cations in the actions of insulin and sodium nitroprusside on metabolism in rat adipocytes

The requirement for Ca2+ and Mg2+ in the actions of insulin and sodium nitroprusside on rat adipocyte metabolism was investigated: sodium nitroprusside, but not insulin, increased cGMP levels in cells incubated in the absence of Ca2+ and/or Mg2+; sodium nitroprusside and insulin are unable to increase the incorporation of [14C]glucose into triglycerides and [14C]leucine into proteins in the absence of Ca2+ and Mg2+; sodium nitroprusside and insulin showed antilipolytic actions in Ca2+- and Mg2+-free medium. We conclude that in the absence of Ca2+ and Mg2+, sodium nitroprusside and insulin have very similar regulatory properties on triglyceride, protein synthesis and adrenaline-stimulated lipolysis, but not on cGMP levels in rat adipocytes. This could provide evidence that omission of bivalent cations was inhibitory at more than one site, or that sodium nitroprusside mimics insulin's actions by another mechanism that does not involve cGMP.     

Role of cGMP signals in tetramethylpyrazine induced relaxation of the isolated rat aortic strip

In the present study, role of guanosine-3',5'-cyclic monophosphate (cGMP) in the vasodilatation of tetramethylpyrazine (TMP), one of the active ingredients of the Chinese herb Chuang-xion, was investigated. We found that the TMP could decrease the vascular tone of isolated rat aorta precontracted with phenylephrine (10(-8) M) in a concentration-dependent manner from 10(-5) M to 10(-3) M. Also, the TMP-induced relaxation was reduced by 1H-(1,2,4)-oxadiazol-(4,3-a)-quinoxalin-1-one (ODQ) or methylene blue, the inhibitor of soluble guanylyl cyclase. Moreover, the vasodilative response to TMP was enhanced significantly in the presence of sildenafil, a well-known inhibitor of phosphodiestrase type 5 that is sensitive to cGMP. In addition, TMP could increase the cGMP level in the isolated aortic rings and TMP-induced vasodilatation was deleted by cGMP-dependent protein kinases (PKG) blockade. These results suggest that relaxation of rat aortic strip by TMP is induced in the cGMP-dependent manner.     

In vivo and in vitro studies of a putative inhibitor of cyclic guanosine 3',5'-monophosphate production.

Our main objective was to test the efficacy of 6-anilino-5,8-quinolinedione (LY83583) in vivo, a putative inhibitor of cyclic guanosine 3',5'-monophosphate (cGMP) production. If the drug proved capable of lowering plasma, vascular, and kidney levels of cGMP and of inhibiting the hypotensive effect of sodium nitroprusside and methacholine, then LY83583 could be of potential use in exploring the contribution of cGMP to cardiovascular and renal physiology. We found that when administered to trained conscious rats, LY83583 (1-mg/kg bolus, followed by a 2-hr infusion of 3 mg/kg.hr) decreased plasma cGMP concentration by 36% (P less than 0.01). Doubling the dosage of drug (2-mg/kg bolus, 6 mg/kg.hr) decreased plasma cGMP by 46% (P less than 0.05). We next measured tissue levels of cGMP ex vivo from rats that had received LY83583 or vehicle for 2 hr. The cGMP content of aortic segments when LY83583 was infused at the low dose, or renal cortical tissue when LY83583 was infused at both doses, was not significantly different from the cGMP content of tissue from rats that had received vehicle. LY83583 in doses up to 10-mg/kg bolus, followed by 6 mg/kg.hr infusion also failed to attenuate the hypotensive response to sodium nitroprusside or methacholine in conscious rats. Last, we tested whether, in our hands, LY83583 could reduce cGMP of aortic segments and kidney cortical slices in vitro. We found that after 10 min of incubation, 10(-5) M LY83583 decreased intracellular cGMP by approximately 65% and 50% in aortic and kidney tissues, respectively. In order to ascertain whether LY83583 lowered cGMP by stimulating phosphodiesterase, we incubated tissues with 10(-4) M 3-isobutyl-1-methylxanthine to inhibit the enzyme. In the presence of 3-isobutyl-1-methylxanthine LY83583 still exerted an inhibitory effect on cGMP production by aortic and kidney tissues. In conclusion, although LY83583 is a useful agent to lower renal and vascular tissues levels of cGMP in vitro, its efficacy in vivo seems doubtful.     

Menadione induces endothelial dysfunction mediated by oxidative stress and arylation

Our previous studies showed that menadione causes endothelial dysfunction which results in decreased relaxation and increased contraction of blood vessels. This investigation examined the role of two possible mechanisms (oxidative stress and arylation) in menadione-induced endothelial dysfunction. Menadione increased superoxide anion generation in aortic rings in a dose-dependent manner. Superoxide dismutase (SOD), reversed the inhibitory effects of menadione on vascular relaxation. The relaxation induced by the NO donor, sodium nitroprusside, was inhibited by menadione pretreatment in a dose-dependent manner. Endothelial nitric oxide synthase activity (eNOS) was suppressed by menadione. Menadione resulted in a dose-dependent reduction of cGMP levels accumulated by acetylcholine. This reduction of cGMP levels was blocked by SOD treatment, suggesting that superoxide anion generated by menadione could play a role in the inhibition of the nitric oxide pathway. Evidence supporting a possible role for arylation in impaired vascular relaxation was suggested by the observation that benzoquinone, which does not induce oxidative stress in aortic rings, inhibited acetylcholine-induced vascular relaxation to the same extent as menadione. Collectively, these results suggest that menadione can cause endothelial dysfunction in blood vessels by the inhibition of the nitric oxide pathway via superoxide anion generation and that arylation activity may also be another important mechanism.     

Effects of acetylcholine and nitroprusside on cGMP-dependent protein kinase in the perfused rat heart

The effects of acetylcholine and sodium nitroprusside on the activity of cGMP-dependent protein kinase were studied in the perfused rat heart. Acetylcholine produced a dose-dependent increase in cGMP levels and cGMP-dependent protein kinase activity, and reduced the force of contraction. Both acetylcholine and sodium nitroprusside produced rapid increases in cardiac cGMP, with nitroprusside being the more potent agent. Only acetylcholine, however, raised the activity ratio of the cGMP-dependent protein kinase and decreased the force of contraction. Whereas acetylcholine and nitroprusside were slightly additive in their effects on total cGMP levels, the increase in the activity ratio of the cGMP-dependent protein kinase and the decrease in the force of contraction produced by acetylcholine were unchanged by nitroprusside. The results suggest that the cGMP produced by acetylcholine, but not nitroprusside, was coupled to protein kinase activation in this tissue.     

Possible involvement of nitroglycerin converting step in nitroglycerin tolerance.

Nitroglycerin (GTN) produces a dilation of vascular smooth muscle by releasing NO through a putative GTN-converting step. However, the response to GTN is markedly attenuated after prolonged or repeated exposure, resulting in tolerance. We investigated the mechanisms of GTN tolerance, employing exogenous and endogenous NO in rat aorta. In endothelium-denuded rat aortic strips, the GTN-induced relaxation response was attenuated by preceding exposure to either GTN or sodium nitroprusside (SNP). In contrast, the SNP-induced relaxation response was not affected by this protocol of GTN or SNP preexposure. Preincubation of aortic strips with lipopolysaccharide (LPS) +/- L-arginine for 12 h also caused attenuation of GTN-induced responses such as relaxation, cGMP production and nitrite/nitrate formation. The attenuating effect of LPS abolished in aortic strips co-incubated with LPS and cycloheximide or N(G)-nitro-L-arginine. These results suggest that GTN tolerance is predominantly associated with the reduction of NO release from GTN, which is caused through inhibition of a GTN-converting step due to preceding exposure to NO itself.     

Activation by Nitric Oxide of Guanylate Cyclase in Endothelial Cells from Brain Capillaries

Endothelial cells (ECs) from brain microvessels respond to exogenous nitric oxide (NO) donor molecules (N-ethoxycarbonyl-3-morpholinosydnonimine and sodium nitroprusside) with large (greater than 15-fold) increases in cyclic GMP (cGMP) levels. Comparable actions of sodium nitroprusside were observed in vascular smooth muscle cells and in neuroblastoma cells. Coculturing brain capillary ECs in the presence of N1E-115 neuroblastoma cells increased their cGMP levels fourfold. A further increase was observed in the presence of 50 nM neurotensin, although brain capillary ECs lack receptor sites for neurotensin. The neuroblastoma cell-dependent formation of cGMP was suppressed by 0.1 mM L-NG-monomethylarginine, indicating that NO, produced by N1E-115 cells in response to neurotensin, activated guanylate cyclase in brain capillary ECs. Similarly, culturing brain capillary ECs in the presence of aortic ECs increased their cGMP content in a manner that was amplified by bradykinin and that was inhibited by L-NG-monomethylarginine. Bradykinin had no action in pure cultures of brain capillary ECs. It is concluded that brain capillary ECs express high levels of guanylate cyclase activity that could be activated by exogenous NO donor molecules and by NO produced by neuroblastoma cells and by aortic ECs in response to specific agonists. Brain capillary ECs are thus potential target cells for brain-derived NO.     

Nitric oxide-epoxygenase interactions and arachidonate-induced dilation of rat renal microvessels

Nitric oxide (NO) is an inhibitor of hemoproteins including cytochrome P-450 enzymes. This study tested the hypothesis that NO inhibits cytochrome P-450 epoxygenase-dependent vascular responses in kidneys. In rat renal pressurized microvessels, arachidonic acid (AA, 0.03-1 microM) or bradykinin (BK, 0.1-3 microM) elicited NO- and prostanoid-independent vasodilation. Miconazole (1.5 microM) or 6-(2-propargyloxyphenyl)hexanoic acid (30 microM), both of which are inhibitors of epoxygenase enzymes, or the fixing of epoxide levels with 11,12-epoxyeicosatrienoic acid (11,12-EET; 1 and 3 microM) inhibited these responses. Apamin (1 microM), which is a large-conductance Ca2+-activated K+ (BKCa) channel inhibitor, or 18alpha-glycyrrhetinic acid (30 microM), which is an inhibitor of myoendothelial gap junctional electromechanical coupling, also inhibited these responses. NO donors spermine NONOate (1 and 3 microM) or sodium nitroprusside (0.3 and 3 microM) but not 8-bromo-cGMP (100 microM), which is an analog of cGMP (the second messenger of NO), blunted the dilation produced by AA or BK in a reversible manner without affecting that produced by hydralazine. However, the non-NO donor hydralazine did not affect the dilatory effect of AA or BK. Spermine NONOate did not affect the dilation produced by 11,12-EET, NS-1619 (a BKCa channel opener), or cromakalim (an ATP-sensitive K+ channel opener). AA and BK stimulated EET production, whereas hydralazine had no effect. On the other hand, spermine NONOate (3 microM) attenuated basal (19 +/- 7%; P < 0.05) and AA stimulation (1 microM, 29 +/- 9%; P < 0.05) of renal preglomerular vascular production of all regioisomeric EETs: 5,6-; 8,9-; 11,12-; and 14,15-EET. These results suggest that NO directly and reversibly inhibits epoxygenase-dependent dilation of rat renal microvessels without affecting the actions of epoxides on K+ channels.     

Cyclic GMP-dependent protein kinase signaling pathway inhibits RhoA-induced Ca2+ sensitization of contraction in vascular smooth muscle

The potent vasodilator action of cyclic GMP-dependent protein kinase (cGK) involves decreasing the Ca(2+) sensitivity of contraction of smooth muscle via stimulation of myosin light chain phosphatase through unknown mechanisms (Wu, X., Somlyo, A. V., and Somlyo, A. P. (1996) Biochem. Biophys. Res. Commun. 220, 658-663). Myosin light chain phosphatase activity is controlled by the small GTPase RhoA and its target Rho kinase. Here we demonstrate cGMP effects mediated by cGK that inhibit RhoA-dependent Ca(2+) sensitization of contraction of blood vessels and actin cytoskeleton organization in cultured vascular myocytes. Ca(2+) sensitization and actin organization were inhibited by both 8-bromo-cGMP and sodium nitroprusside (SNP). SNP also caused translocation of activated RhoA from the membrane to the cytosol. SNP-induced actin disassembly was lost in vascular myocytes in culture after successive passages but was restored by transfection of cells with cGK I. Furthermore, cGK phosphorylated RhoA in vitro, and addition of cGK I inhibited RhoA-induced Ca(2+) sensitization in permeabilized smooth muscle. 8-Bromo-cGMP-induced actin disassembly was inhibited in vascular myocytes expressing RhoA(Ala-188), a mutant that could not be phosphorylated. Collectively, these results indicate that cGK phosphorylates and inhibits RhoA and suggest that the consequent inhibition of RhoA-induced Ca(2+) sensitization and actin cytoskeleton organization contributes to the vasodilator action of nitric oxide.     

Dual Effect of Diazepam on cGMP Levels in Rat Brain Slices

The effect of diazepam on NO-mediated cGMP synthesis was studied in rat brain slices. It was found that diazepam dose-dependently decreased cGMP synthesis in cerebellar slices, with an inhibition of 90% at 1 mM diazepam. cGMP levels in the presence of diazepam were not restored to control levels by the addition of 0.1 mM sodium nitroprusside, whereas the decrease in cerebellar cGMP levels induced by 0.1 mM L-NAME was restored by the simultaneous application of NO-donors. In addition to the decrease of cGMP levels in neuronal structures induced by 1 mM diazepam, we observed increased cGMP immunoreactivity in glial cells in the cerebellum, the hippocampus, and the cerebral cortex. The significance of this observation is discussed.     

Stimulation of aortic tissue calcium uptake by an extract of spontaneously hypertensive rat erythrocytes possessing hypertensive properties

In earlier reports we have described the isolation of a fraction from the erythrocytes of spontaneously hypertensive rats that produced hypertension when administered to normotensive rats. In addition, it was found that the fraction stimulated the uptake of "lanthanum-resistant" calcium by aortic rings excised from normotensive rats. In these studies we have found that the fraction causes a greater increase in the in vitro uptake of calcium by aortic tissue than that produced by depolarization of the tissue with high K+ or the receptor-mediated influx of calcium induced with norepinephrine. The hypertensive fraction appeared to be more effective in promoting increased calcium uptake in rabbit than in rat aortic tissue, suggesting that significant differences in tissue sensitivity to the active compound(s) may exist between species. In addition, we obtained evidence indicating that the tissue sensitivity to the action of the hypertensive fraction was greater in aortae from spontaneously hypertensive rats than from those of normotensive animals. Attempts to block the action of the hypertensive fraction with verapamil, nifedipine, and sodium nitroprusside had no significant effect on the elevation in tissue calcium. It was found, however, that the action of the hypertensive fraction was temperature dependent with reduced activity at lower temperatures. The data suggest that a compound(s) is present in the erythrocytes of rats that may have a marked effect on vascular tissue metabolism of calcium.     

cGMP-dependent and -independent angiogenesis-related properties of nitric oxide

Nitric oxide exerts a stimulatory role during postnatal angiogenesis. Although soluble guanylyl cyclase (sGC) mediates many of the effects of nitric oxide (NO) in the vascular system, the contribution of cGMP-dependent vs cGMP-independent pathways in NO-induced angiogenesis remains unclear. Herein, we determined the effects of a NO donor (sodium nitroprusside; SNP) and a NO-independent sGC activator (BAY 41-2272) in the growth and migration of rat aortic endothelial cells (RAEC). RAEC lack enzymatically active sGC as suggested by their inability to accumulate cGMP upon exposure to SNP. However, treatment of RAEC with SNP promoted a modest increase in their proliferation and migration that was dependent on extracellular signal regulated kinase1/2 activation. Moreover, when RAEC were exposed to vascular endothelial growth factor we observed an increase in migration that was inhibited by NO synthase, but not sGC, inhibition. Infection of cells with adenoviruses containing sGC greatly increased the efficacy of SNP as a mitogenic and migratory stimulus. We conclude that NO is capable of stimulating EC proliferation and mobility in the absence of sGC; however, increased intracellular levels of cGMP following sGC activation greatly amplify the angiogenic potential of NO.     

Effects of hsp90 binding inhibitors on sGC-mediated vascular relaxation

Vascular soluble guanylate cyclase (sGC) exists in multimeric complexes with endothelial nitric oxide (NO) synthase (eNOS) and heat shock protein 90 (hsp90). Whereas disruption of hsp90-eNOS complexes clearly attenuates eNOS-dependent vascular relaxation, the contribution of sGC-hsp90 complexes to eNOS- or NO donor-dependent relaxations remains unclear. Isolated rat thoracic aortic rings were preincubated with structurally diverse hsp90 binding inhibitors, radicicol (RA) or geldanamycin (GA), or vehicle for 0.5, 1, or 15 h. Preconstricted vessels were exposed to ACh, 8-bromo-cGMP (8-BrcGMP), forskolin, or one of three NO donors: nitroglycerin (NTG), sodium nitroprusside, or spermine NONOate (SNN). Both RA and GA inhibited endothelium-dependent relaxations dose dependently. Indomethacin or the antioxidant tiron did not affect the inhibition of ACh-induced relaxations by GA. Long-term (15 h) exposure to RA inhibited all NO donor-induced relaxations; however, GA inhibited SNN-induced relaxation only. The effects of GA and RA appeared to be selective because 15-h treatment with either agent did not affect forskolin-induced relaxations and only slightly decreased 8-BrcGMP-induced relaxations. Similarly to their effects on NO-donor-induced relaxation, 15-h exposure to RA, but not to GA, decreased hsp90-bound sGC protein expression and NTG-stimulated cGMP formation in aortic rings, whereas RA more than GA reduced SNN-stimulated cGMP formation. We conclude that RA, much more so than GA, selectively inhibits sGC-dependent relaxations of aortic rings by reducing sGC expression, disrupting sGC-hsp90 complex formation and decreasing cGMP formation. These studies suggest that hsp90 regulates both eNOS- and sGC-dependent relaxations.     

A growth factor- and hormone-stimulated NADH oxidase from rat liver plasma membrane.

NADH oxidase activity (electron transfer from NADH to molecular oxygen) of plasma membranes purified from rat liver was characterized by a cyanide-insensitive rate of 1 to 5 nmol/min per mg protein. The activity was stimulated by growth factors (diferric transferrin and epidermal growth factor) and hormones (insulin and pituitary extract) 2- to 3-fold. In contrast, NADH oxidase was inhibited up to 80% by several agents known to inhibit growth or induce differentiation (retinoic acid, calcitriol, and the monosialoganglioside, GM3). The growth factor-responsive NADH oxidase of isolated plasma membranes was not inhibited by common inhibitors of oxidoreductases of endoplasmic reticulum or mitochondria. As well, NADH oxidase of the plasma membrane was stimulated by concentrations of detergents which strongly inhibited mitochondrial NADH oxidases and by lysolipids or fatty acids. Growth factor-responsive NADH oxidase, however, was inhibited greater than 90% by chloroquine and quinone analogues. Addition of coenzyme Q10 stimulated the activity and partially reversed the analogue inhibition. The pH optimum for NADH oxidase was 7.0 both in the absence and presence of growth factors. The Km for NADH was 5 microM and was increased in the presence of growth factors. The stoichiometry of the electron transfer reaction from NADH to oxygen was 2 to 1, indicating a 2 electron transfer. NADH oxidase was separated from NADH-ferricyanide reductase, also present at the plasma membrane, by ion exchange chromatography. Taken together, the evidence suggests that NADH oxidase of the plasma membrane is a unique oxidoreductase and may be important to the regulation of cell growth.     

Dietary L-arginine restores aspirin-induced endothelial dysfunction in rat aorta

L-Arginine induced elevation of the vascular prostanoid led us to think that the risk of coronary spasm may increase in L-arginine consumers when they are subjected to cyclooxygenase inhibitors and this limits the therapeutic value of aspirin. So the aim was to investigate the interaction of aspirin and dietary L-arginine in male rats. Animals were divided into four groups and fed with normal food. The first group received tap water while the second, third and fourth groups were subjected daily to aspirin (8.6 mg/kg), L-arginine (143 mg/kg) and aspirin + L-arginine combination in their drinking water respectively for 7 days. Vasomotor responses were recorded in the aortic rings suspended for isometric-force recordings. Aspirin treatment significantly reduced the dilation to acetylcholine and sodium nitroprusside. Attenuated phenylephrine contractility was associated with normal acetylcholine response in L-arginine group. Addition of L-arginine to aspirin treatment completely prevented aspirin-induced endothelial dysfunction but defective response to sodium nitroprusside persisted. Dietary L-arginine without affecting maximal dilation to acetylcholine significantly increased the share of dilator prostanoid which appears to resist aspirin. These results demonstrated that dietary L-arginine increases dilator prostaoid in rat aortic rings. Contrary to our expectation, co-administered L-arginine protected aspirin induced endothelial dysfunction and ruled out the limitation of aspirin use in L-arginine consumers.     

Immunocytochemical localization of cAMP and cGMP in cells of the rat carotid body following natural and pharmacological stimulation

Summary Although the chemoreceptive function of the carotid body has been known for many decades, the cellular mechanisms of sensory transduction in this organ remain obscure. Common elements in the transductive processes of many cells are the cyclic nucleotide second messengers, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Studies from our laboratory have revealed stimulus-induced changes in cyclic nucleotide levels in the carotid body as measured by RIA, but such changes in second messenger levels have not been localized to specific cellular elements in the organ. The present immunocytochemical study utilized the avidin-biotin-peroxidase method to investigate the distribution of cAMP and cGMP in the rat carotid body and to assess changes in the intensity of immunostaining following in vitro stimulation by hypoxia, forskolin, sodium nitroprusside, high potassium, and atrial natriuretic peptide. Both cAMP and cGMP immunoreactivity were localized to type I cells of organs maintained in vivo and fixed by perfusion. Organs exposed to 100% O₂-equilibrated media in vitro produced low but visible levels of cAMP immunoreactivity in a majority of type I cells; hypoxia (5% O₂-equilibrated media) for 10 min moderately increased the level of immunoreactivity; forskolin (10^–5 M), or forskolin combined with hypoxia, dramatically increased cAMP levels in virtually all cells. Moderate levels of cGMP immunoreactivity in control carotid bodies in vitro were strikingly reduced by hypoxia; a significant increase in cGMP levels occurred following incubation in high potassium (100 mM), and under these conditions, the decrease in cGMP immunoreactivity with hypoxia was much more pronounced. The synthetic analog of atrial natriuretic peptide, atriopeptin III (10^–7 M), greatly elevated cGMP immunoreactivity in the type I cells. On the other hand, sodium nitroprusside (1 mM) elevated cGMP staining mostly in vascular elements of the carotid body in vitro. The data implicate the involvement of cyclic nucleotides in transduction of natural chemosensory stimuli by the type I cells in rat carotid body.