Cross-activation: overriding cAMP/cGMP selectivities of protein kinases in tissues.

cAMP- and cGMP-dependent protein kinases are homologous proteins and are predicted to exhibit very similar three-dimensional structures. Their cyclic nucleotide binding domains share a high degree of amino acid sequence identity. cAMP- and cGMP-dependent protein kinases are activated relatively specifically by cAMP and cGMP, respectively; and a single alanine-threonine difference between cAMP- and cGMP-binding domains partially accounts for this specificity. Thus, it would be expected that cAMP and cGMP mediate separate physiological effects. However, owing in part to the lack of absolute specificity of either enzyme and to the relatively high level of cAMP or cGMP in certain tissues, it is also possible that either cyclic nucleotide could cross-activate the other kinase. Increases in either cAMP or cGMP cause pig coronary artery relaxation. However, only cGMP-dependent protein kinase specific cyclic nucleotide analogues are very effective in causing relaxation, and cAMP elevation in arteries treated with isoproterenol or forskolin activates cGMP-dependent protein kinase, in addition to cAMP-dependent protein kinase. Conversely, increases in either cAMP or cGMP cause Cl- secretion in T-84 colon carcinoma cells, and the cGMP level in T-84 cells can be elevated sufficiently by bacterial enterotoxin to activate cAMP-dependent protein kinase. These results imply specific regulation of cAMP- and cGMP-dependent protein kinases by the respective cyclic nucleotides, but either cyclic nucleotide is able to cross-activate the other kinase in certain tissues.     

One amino acid change produces a high affinity cGMP-binding site in cAMP-dependent protein kinase

Discrimination between cAMP and cGMP is a critical feature of cAMP- and cGMP-dependent protein kinases. An alanine/threonine difference in the cyclic nucleotide-binding sites has been proposed to provide a structural basis for this functional distinction. Site-directed mutagenesis of this alanine to a threonine in a cAMP-binding site of cAMP kinase produced a mutant with markedly increased cGMP affinity as determined by cGMP binding and protein kinase activation assays. Studies of other mutants at this position support the role of the threonine hydroxyl group as the component that enhances cGMP binding affinity.     

Role of neuronal nitric oxide synthase in hypoxia-induced anapyrexia in rats.

Anapyrexia (a regulated decrease in body temperature) is a response to hypoxia that occurs in organisms ranging from protozoans to mammals, but very little is known about the mechanisms involved. Recently, it has been shown that the NO pathway plays a major role in hypoxia-induced anapyrexia. However, very little is known about which of the three different nitric oxide synthase isoforms (neuronal, endothelial, or inducible) is involved. The present study was designed to test the hypothesis that neuronal nitric oxide synthase (nNOS) plays a role in hypoxia-induced anapyrexia. Body core temperature (T(c)) of awake, unrestrained rats was measured continuously using biotelemetry. Rats were submitted to hypoxia, 7-nitroindazole (7-NI; a selective nNOS inhibitor) injection, or both treatments together. Control animals received vehicle injections of the same volume. We observed a significant (P < 0.05) reduction in T(c) of approximately 2.8 degrees C after hypoxia (7% inspired O(2)), whereas intraperitoneal injection of 7-NI at 25 mg/kg caused no significant change in T(c). 7-NI at 30 mg/kg elicited a reduction in T(c) and was abandoned in further experiments. When the two treatments were combined (25 mg/kg of 7-NI and 7% inspired O(2)), we observed a significant attenuation of hypoxia-induced anapyrexia. The data indicate that nNOS plays a role in hypoxia-induced anapyrexia.     

Thermoregulatory response to hypoxia after inhibition of the central heme oxygenase–carbon monoxide pathway

(1) Centrally acting carbon monoxide (CO) seems to play thermoregulatory actions, but no report exists about its role in hypoxia-induced anapyrexia. (2) CO arises from the catabolism of heme by heme oxygenase (HO), an enzyme that is overexpressed during hypoxia. Thus, we tested the hypothesis that the central HO–CO pathway modulates hypoxia-induced anapyrexia by means of intracerebroventricular injection of the HO inhibitor ZnDPBG. (3) Core temperature (T_C) of awake rats was determined by biotelemetry. ZnDPBG did not alter basal T_c, but it exacerbated hypoxia-induced anapyrexia, indicating that the central HO–CO pathway is a modulator of hypoxia-induced anapyrexia, probably preventing excessive decreases in T_c.     

Dominant role of cAMP in regulation of microvessel permeability

We reported previously that increasing cAMP levels in endothelial cells attenuated ATP-induced increases in hydraulic conductivity (L(p)), and that the activation of cGMP-dependent pathways was a necessary step to increase L(p) in response to inflammatory mediators. The aim of the present study was to evaluate the role of basal levels of cAMP in microvessel permeability under resting conditions and to evaluate the cross talk between cAMP- and cGMP-dependent signaling mechanisms in regulation of microvessel permeability under stimulated conditions, using individually perfused microvessels from frog and rat mesenteries. We found that reducing cAMP levels by inhibition of adenylate cyclase or inhibiting cAMP-dependent protein kinase through the use of H-89 increased basal L(p) in both frog and rat mesenteric venular microvessels. We also found that 8-bromocAMP (8-BrcAMP, 0.2 and 2 mM) was sufficient to attenuate or abolish the increases in L(p) due to exposure of frog mesenteric venular microvessels to 8-BrcGMP (2 mM) and ATP (10 microM). Similarly, in rat mesenteric venular microvessels, application of 8-BrcAMP (2 mM) abolished the increases in L(p) due to exposure to 8-BrcGMP alone (2 mM) or with the combination of bradykinin (1 nM). In addition, application of erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of cGMP-stimulated phosphodiesterase, significantly attenuated both 8-BrcGMP- and bradykinin-induced increases in L(p). These results demonstrate that basal levels of cAMP are critical to maintaining normal permeability under resting conditions, and that increased levels of cAMP are capable of overcoming the activation of cGMP-dependent pathways, therefore preventing increases in microvessel permeability. The balance between endothelial concentrations of these two opposing cyclic nucleotides controls microvessel permeability, and cAMP levels play a dominant role.     

Effect of JBP485 on obstructive jaundice is related to regulation of renal Oat1, Oat3 and Mrp2 expression in ANIT-treated rats

Pulmonary vascular endothelial nitric oxide (NO) synthase (eNOS)-derived NO is the major stimulant of cyclic guanosine 5'-monophosphate (cGMP) production and NO/cGMP-dependent vasorelaxation in the pulmonary circulation. We recently synthesized multiple peptides and reported that an eleven amino acid (SSWRRKRKESS) peptide (P1) but not scrambled P1 stimulated the catalytic activity but not expression of eNOS and causes NO/cGMP-dependent sustained vasorelaxation in isolated pulmonary artery (PA) segments and in lung perfusion models. Since cGMP levels can also be elevated by inhibition of phosphodiesterase type 5 (PDE-5), this study was designed to test the hypothesis that P1-mediated vesorelaxation is due to its unique dual action as NO-releasing PDE-5 inhibitor in the pulmonary circulation. Treatment of porcine PA endothelial cells (PAEC) with P1 caused time-dependent increase in intracellular NO release and inhibition of the catalytic activity of cGMP-specific PDE-5 but not PDE-5 protein expression leading to increased levels of cGMP. Acute hypoxia-induced PA vasoconstriction ex vivo and continuous telemetry monitoring of hypoxia (10% oxygen)-induced elevated PA pressure in freely moving rats were significantly restored by administration of P1. Chronic hypoxia (10% oxygen for 4 weeks)-induced alterations in PA perfusion pressure, right ventricular hypertrophy, and vascular remodeling were attenuated by P1 treatment. These results demonstrate the potential therapeutic effects of P1 to prevent and/or arrest the progression of hypoxia-induced PAH via NO/cGMP-dependent modulation of hemodynamic and vascular remodeling in the pulmonary circulation.     

Cell-cycle-specific activity of cyclic nucleotide phosphodiesterase in Physarum polycephalum

The cell-cycle-related activities of the cAMP- and cGMP-dependent phosphodiesterases of Physarum polycephalum were assayed. The activities of plasmodial homogenate and of selected subcellular fractions were measured. The results suggested the presence of both cAMP- and cGMP-dependent phosphodiesterase in the isolated nuclei of P. polycephalum. In addition, they reveal that the cAMP- and cGMP-dependent phosphodiesterase activities of the subcellular fractions fluctuate throughout the cell cycle. The whole-cell homogenates exhibit no cell-cycle-related changes in the presence of 5 X 10(-4) M cGMP. Kinetic data suggest the presence of multiple phosphodiesterase activities in the homogenate and its particulate fractions for the cGMP-dependent enzyme. Multiple cAMP activities are also suggested for the particulate fractions. The Km values indicate that the substrate affinities of the phosphodiesterases from P. polycephalum are similar to those found previously in mammalian systems.     

Regulatory subunit of the type I cAMP-dependent protein kinase as an inhibitor and substrate of the cGMP-dependent protein kinase

The regulatory subunit of the type I cAMP-dependent protein kinase (Rt) serves as a substrate for the phosphotransferase reaction catalyzed by cGMP-dependent protein kinase (Km = 2.2 microM). The reaction is stimulated by cGMP when RI . cAMP is the substrate, but not when nucleotide-free RI is used. The cGMP-dependent protein kinase catalyzes the incorporation of 2 mol of phosphate/mol of RI dimer in the presence of cAMP and a self-phosphorylation reaction to the extent of 4 mol of phosphate/mol of enzyme dimer. In the absence of cAMP, RI is a competitive inhibitor of the phosphorylation of histone H2B (Ki = 0.25 microM) and of the synthetic peptide substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly (Ki = 0.15 microM) by the cGMP-dependent enzyme. Nucleotide-free RI also inhibits the intramolecular self-phosphorylation of cGMP-dependent protein kinase. The inhibition of the phosphorylation reactions are reversed by cAMP. The catalytic subunit of cAMP-dependent protein kinase does not catalyze the phosphorylation of RIand does not significantly alter the ability of RI to serve as a substrate or an inhibitor of cGMP-dependent protein kinase. These observations are consistent with the concept that the cGMP- and cAMP-dependent protein kinases are closely related proteins whose functional domains may interact.     

cAMP inhibits IP(3)-dependent Ca(2+) release by preferential activation of cGMP-primed PKG

The singular effects and interplay of cAMP- and cGMP-dependent protein kinase (PKA and PKG) on Ca(2+) mobilization were examined in dispersed smooth muscle cells. In permeabilized muscle cells, exogenous cAMP and cGMP inhibited inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release and muscle contraction via PKA and PKG, respectively. A combination of cAMP and cGMP caused synergistic inhibition that was exclusively mediated by PKG and attenuated by PKA. In intact muscle cells, low concentrations (10 nM) of isoproterenol and sodium nitroprusside (SNP) inhibited agonist-induced, IP(3)-dependent Ca(2+) release and muscle contraction via PKA and PKG, respectively. A combination of isoproterenol and SNP increased PKA and PKG activities: the increase in PKA activity reflected inhibition of phosphodiesterase 3 activity by cGMP, whereas the increase in PKG activity reflected activation of cGMP-primed PKG by cAMP. Inhibition of Ca(2+) release and muscle contraction by the combination of isoproterenol and SNP was preferentially mediated by PKG. In light of studies showing that PKG phosphorylates the IP(3) receptor in intact and permeabilized muscle cells, whereas PKA phosphorylates the receptor in permeabilized cells only, the results imply that inhibition of IP(3)-induced Ca(2+) release is mediated exclusively by PKG. The effect of PKA on agonist-induced Ca(2+) release probably reflects inhibition of IP(3) formation.     

Demonstration of cGMP-dependent protein kinase and cGMP-dependent phosphorylation in cell-free extracts of platelets

Homogenates, membranes and cytosol of rat and human platelets were found to contain cGMP-dependent protein kinase immunoreactivity. Specific cGMP-dependent protein kinase immunoreactivity was about 1.7 pmol protein kinase/mg protein for homogenates of human platelets and 0.7 pmol/mg for homogenates of rat platelets; the majority appeared to be associated with the membrane fraction. In membranes of platelets low concentrations of cAMP (0.5-2 microM) stimulated the phosphorylation of five major proteins with apparent relative molecular masses, Mr, of 240 000, 130 000, 50 000, 42 000 and 22 000 while low concentrations of cGMP (0.5-2 microM) stimulated the phosphorylation of three major proteins with apparent Mr of 130 000, 50 000 and 46 000. An affinity-purified antibody against the cGMP-dependent protein kinase was prepared which specifically inhibited the activity of cGMP-dependent protein kinase. In membranes of human platelets this affinity-purified antibody inhibited the cGMP-stimulated phosphorylation of the three proteins with Mr of 130 000, 50 000 and 46 000 while it had no effect on the cAMP-dependent and cyclic-nucleotide-independent protein phosphorylation. The results demonstrate that platelets contain a cGMP-dependent protein kinase and at least three specific substrates for this enzyme. Two of these substrates, the proteins with apparent molecular Mr of 130 000 and 50 000, are substrates for both cAMP- and cGMP-dependent protein kinase. The protein with apparent Mr of 130 000 appears to be closely related to an intrinsic plasma membrane protein of vascular smooth muscle cells which is a substrate for a membrane-associated cGMP-dependent protein kinase. Therefore, cGMP-dependent protein kinase and cGMP-regulated phosphoproteins may mediate in platelets the intracellular effects of those hormones, vasodilators and drugs which elevate the level of cGMP and inhibit platelet aggregation.     

STa and cGMP stimulate CFTR translocation to the surface of villus enterocytes in rat jejunum and is regulated by protein kinase G

The cystic fibrosis transmembrane conductance regulator (CFTR) is critical to cAMP- and cGMP-activated intestinal anion secretion and the pathogenesis of secretory diarrhea. Enterotoxins released by Vibrio cholerae (cholera toxin) and Escherichia coli (heat stable enterotoxin, or STa) activate intracellular cAMP and cGMP and signal CFTR on the apical plasma membrane of small intestinal enterocytes to elicit chloride and fluid secretion. cAMP activates PKA, whereas cGMP signals a cGMP-dependent protein kinase (cGKII) to phosphorylate CFTR in the intestine. In the jejunum, cAMP also regulates CFTR and fluid secretion by insertion of CFTR from subapical vesicles to the surface of enterocytes. It is unknown whether cGMP signaling or phosphorylation regulates the insertion of CFTR associated vesicles from the cytoplasm to the surface of enterocytes. We used STa, cell-permeant cGMP, and cAMP agonists in conjunction with PKG and PKA inhibitors, respectively, in rat jejunum to examine whether 1) cGMP and cGK II regulate the translocation of CFTR to the apical membrane and its relevance to fluid secretion, and 2) PKA regulates cAMP-dependent translocation of CFTR because this intestinal segment is a primary target for toxigenic diarrhea. STa and cGMP induced a greater than fourfold increase in surface CFTR in enterocytes in association with fluid secretion that was inhibited by PKG inhibitors. cAMP agonists induced a translocation of CFTR to the cell surface of enterocytes that was prevented by PKA inhibitors. We conclude that cAMP and cGMP-dependent phosphorylation regulates fluid secretion and CFTR trafficking to the surface of enterocytes in rat jejunum. small intestine; cystic fibrosis transmembrane conductance regulator; membrane traffic; phosphorylation     

Endogenous vasopressin does not mediate hypoxia-induced anapyrexia in rats

The present study was designed to test the hypothesis thatarginine vasopressin (AVP) mediates hypoxia-induced anapyrexia. Therectal temperature of awake, unrestrained rats was measured before andafter hypoxic hypoxia, AVP-blocker injection, or a combination of thetwo. Control animals received saline injections of the same volume.Basal body temperature was 36.52 ± 0.29°C. We observed asignificant (P < 0.05) reduction inbody temperature of 1.45 ± 0.33°C after hypoxia (7% inspiredO₂), whereas systemic andcentral injections of AVP V₁- andAVP V₂-receptor blockers caused nochange in body temperature. When intravenous injection of AVP blockerswas combined with hypoxia, we observed a reduction in body temperatureof 1.49 ± 0.41°C(V₁-receptor blocker) and of 1.30 ± 0.13°C (V₂-receptorblocker), similar to that obtained by application of hypoxia only.Similar results were observed when the blockers were injectedintracerebroventricularly. The data indicate that endogenous AVP doesnot mediate hypoxia-induced anapyrexia in rats.     

Chemoattractant-mediated increases in cGMP induce changes in Dictyostelium myosin II heavy chain-specific protein kinase C activities

Myosin II heavy chain (MHC)-specific protein kinase C (MHC-PKC) isolated from the ameba, Dictyostelium discoideum, regulates myosin II assembly and localization in response to the chemoattractant cAMP (Abu- Elneel et al. 1996. J. Biol. Chem. 271:977- 984). Recent studies have indicated that cAMP-induced cGMP accumulation plays a role in the regulation of myosin II phosphorylation and localization (Liu, G., and P. Newell. 1991. J. Cell. Sci. 98: 483-490). This report describes the roles of cAMP and cGMP in the regulation of MHC-PKC membrane association, phosphorylation, and activity (hereafter termed MHC-PKC activities). cAMP stimulation of Dictyostelium cells resulted in translocation of MHC-PKC from the cytosol to the membrane fraction, as well as increasing in MHC-PKC phosphorylation and in its kinase activity. We present evidence that MHC is phosphorylated by MHC-PKC in the cell cortex which leads to myosin II dissociation from the cytoskeleton. Use of Dictyostelium mutants that exhibit aberrant cAMP- induced increases in cGMP accumulation revealed that MHC-PKC activities are regulated by cGMP. Dictyostelium streamer F mutant (stmF), which produces a prolonged peak of cGMP accumulation upon cAMP stimulation, exhibits prolonged increases in MHC-PKC activities. In contrast, Dictyostelium KI-10 mutant that lacks the normal cAMP-induced cGMP response, or KI-4 mutant that shows nearly normal cAMP-induced cGMP response but has aberrant cGMP binding activity, show no changes in MHC- PKC activities. We provide evidence that cGMP may affect MHC-PKC activities via the activation of cGMP-dependent protein kinase which, in turn, phosphorylates MHC-PKC. The results presented here indicate that cAMP-induced cGMP accumulation regulates myosin II phosphorylation and localization via the regulation of MHC-PKC.     

A study of signal transduction for the two diuretic peptides of Diploptera punctata

We investigated second messengers involved in the action of the CRF-related peptide Dippu-DH46 and the calcitonin-like peptide Dippu-DH31 in Diploptera punctata. Dippu-DH46 causes a dose-dependent increase in intracellular cAMP levels, its diuretic activity is mimicked by cAMP agonists, but is attenuated by Rp-cAMPS. Dippu-DH46 acts synergistically with kinins and thapsigargin; both mobilize intracellular Ca2+. Dippu-DH46 also acts synergistically with cAMP agonists, and its effect is inhibited by a PKC inhibitor, suggesting it also activates intracellular Ca2+. Dippu-DH31 has no effect on cAMP levels and its activity is not blocked by cAMP agonists. Neither peptide stimulated cGMP levels in a dose-dependent manner, nor does cGMP have any effect on fluid secretion.     

Phosphorylation of tyrosine hydroxylase by cGMP-dependent protein kinase in intact bovine chromaffin cells.

The phosphorylation of the enzyme tyrosine hydroxylase by the cGMP pathway was investigated in chromaffin cells from the bovine adrenal medulla. The nitric oxide donor, sodium nitroprusside, and the natriuretic peptide, C-type natriuretic peptide, which are able to increase cGMP levels and cGMP-dependent protein kinase activity, produced significant increases in the phosphorylation level of tyrosine hydroxylase in a time- and concentration-dependent manner. The pretreatment of the cells with the soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one blocked the effect of sodium nitroprusside. This result indicates that cGMP production by this enzyme mediated this effect. Experiments performed with a cGMP-dependent protein kinase inhibitor, the Rp-isomer of 8-(4-chlorophenylthio)-cyclic guanosine monophosphorothioate, which blocked the effects of both sodium nitroprusside and C-type natriuretic peptide, demonstrated that the phosphorylation increases evoked by both compounds were mediated by the activation of cGMP-dependent protein kinase. In cells incubated with the adenylyl cyclase activator, forskolin, an increase in the phosphorylation level of the tyrosine hydroxylase was also found. When cells were treated simultaneously with forskolin and sodium nitroprusside or C-type natriuretic peptide, an additive effect on tyrosine hydroxylase phosphorylation was not observed. This suggests that cAMP- and cGMP-dependent protein kinases may phosphorylate the same amino acid residues in the enzyme. Western blot analysis of soluble extracts from chromaffin cells detected specific immunoreactivity for two different commercial antibodies raised against cGMP-dependent protein kinase (both Ialpha and Ibeta isoforms). Electrophoretic mobility correlates with that of purified PKG Ialpha. Because the phosphorylation of the tyrosine hydroxylase correlates with increases in its enzymatic activity and thus with augmentation in the cell capacity to synthesize catecholamines, our results indicate that a cGMP-based second messenger pathway participates in catecholamine biosynthesis regulation in chromaffin cells, a mechanism which may be widespread in other catecholamine-synthesizing cells.     

Cell-Cycle-Specific Activity of Cyclic Nucleotide Phosphodiesterase In Physarum polycephalum

The cell-cycle-related activities of the cAMP- and cGMP-dependent phosphodiesterases of Physarum polycephalum were assayed. the activities of plasmodial homogenate and of selected subcellular fractions were measured. the results suggested the presence of both cAMP- and cGMP-dependent phosphodiesterase in the isolated nuclei of P. polycephalum. In addition, they reveal that the cAMP- and cGMP-dependent phosphodiesterase activities of the subcellular fractions fluctuate throughout the cell cycle. the whole-cell homogenates exhibit no cell-cycle-related changes in the presence of 5 × 10^-4 m cGMP. Kinetic data suggest the presence of multiple phosphodiesterase activities in the homogenate and its particulate fractions for the cGMP-dependent enzyme. Multiple cAMP activities are also suggested for the particulate fractions. the K_m values indicate that the substrate affinities of the phosphodiesterases from P. polycephalum are similar to those found previously in mammalian systems.     

Immunofluorescent localization of cGMP,cGMP-dependent protein kinase,calmodulin and cAMP in the rat uterus

Cyclic guanosine 3',5' monophosphate (cGMP), cGMP-dependent protein kinase, calmodulin and cyclic adenosine 3',5' monophosphate (cAMP) were localized in the uterus of the immature rat by an indirect immunofluorescence technique. cGMP, cGMP-dependent protein kinase and calmodulin were detected predominantly along epithelial and myometrial plasma membranes and in the adjacent cytoplasm. In contrast, cAMP immunoreactive material was found principally in the cytoplasm of connective tissue. After administration of 17 beta-estradiol, similar time-dependent changes were observed in the localization of cGMP, cGMP-dependent protein kinase and calmodulin in all uterine cell types. For the three compounds, nucleolar-like distribution of the immunofluorescence appeared approximately 12 h after treatment. A more dispersed, reticular distribution of the nuclear fluorescent staining was observed 20-24 h after hormonal treatment. Estrogen did not affect the localization of cAMP. The simultaneous mobilization of cGMP, cGMP-dependent protein kinase and calmodulin towards the same nuclear loci suggests concerted roles for these three molecules in nuclear metabolic processes during the development of the uterotrophic action of estrogens.     

Cyclic nucleotide crosstalk in salivary glands from partially fed Dermacentor variabilis (Say)

Enzyme immunosorbent assays were used to measure cyclic nucleotide concentrations in homogenates of salivary glands from partially fed female Dermacentor variabilis. The adenylyl cyclase activator forskolin (100 μM) increased homogenate cGMP concentrations greater than three-fold over controls. Competitive inhibition of nitric oxide synthase with 1 mM l-NMMA, an l-arginine analog, demonstrated that crosstalk occurs downstream of nitric oxide synthesis. Forskolin-stimulated synthesis of cGMP was diminished 58% by the soluble guanylyl cyclase inhibitor ODQ (2 μM). The protein kinase A selective inhibitor Rp-cAMPS (50 μM) inhibited forskolin-stimulated cGMP by 49%. Whole glands treated with 10 μM dopamine increased cGMP levels two-fold in the presence of 1 mM IBMX. Treatment of whole salivary glands with equimolar concentrations of 8-Br-cAMP and 8-Br-cGMP produced no greater fluid uptake than in glands treated with 8-Br-cGMP alone, suggesting that cAMP and cGMP share a downstream target. The protein kinase G-selective inhibitor Rp-8-pCPT-cGMPS (100 μM) impeded 10 mM 8-Bromo-cGMP-stimulated gland weight increases. Pretreatment with verapamil, a Ca²⁺ channel blocker, attenuated cyclic nucleotide-stimulated fluid uptake indicating that whole gland fluid changes are dependent on extracellular Ca²⁺. Together, our data suggest that cGMP production is mediated in part by cAMP-dependent activation of soluble guanylyl cyclase. Experiments measuring changes in whole salivary gland weight support the hypothesis that cAMP and cGMP signaling cascades have a common target and that cyclic nucleotide-stimulated fluid movement is dependent on Ca²⁺ influx.     

Effect of sex hormones on the content of cyclic nucleotides in the rat denervated uterus

Estradiol dipropionate increased the amount of cGMP and to less extent that of cAMP in the uterine tissues of ovariectomized rats. The combined effect of estradiol and progesterone promoted rise only in the cAMP level. Surgical and pharmacological denervation of both sympathetic and parasympathetic nerve influences in uterus suppressed the effect of the above hormones. Thus, neuromediator processes play an essential role in realization of cAMP- and cGMP-dependent effects in the general program of the sex hormone action in the above organ.     

Inhibition of hypoxia-induced proliferation and collagen synthesis by vasonatrin peptide in cultured rat pulmonary artery smooth muscle cells

The aim of the present research is to investigate the effects of vasonatrin peptide (VNP) on hypoxia-induced proliferation and collagen synthesis in pulmonary artery smooth muscle cells (PASMCs). Smooth muscle cells isolated from rat pulmonary artery were cultured and used at passages 3-5. Cell proliferation and collagen synthesis were evaluated by cell counts, [(3)H] thymidine and [(3)H] proline incorporation. The results showed that cells exposed to hypoxia for 24 h exhibited a significant increase in [(3)H] thymidine (93%) and [(3)H] proline (52%) incorporation followed by a significant increase in cell number (47%) at 48 h in comparison with the respective normoxic controls. VNP reduced hypoxia-stimulated increase in cell proliferation in a concentration-dependent manner from 10(-8) to 10(-6) mol/L and attenuated hypoxia-induced collagen synthesis ranging from 10(-6) to 10(-5) mol/L, which is similar to but more potent than both ANP and CNP. The action of VNP on PASMCs was mimicked by 8-bromo-cGMP (10(-4) mol/L, the membrane-permeable cGMP analog), and blocked by HS-142-1 (2 x 10(-5) mol/L), the particulate guanylyl cyclase-coupled natriuretic peptide receptor antagonist, or KT-5823 (10(-6) mol/L), the cGMP-dependent protein kinase (PKG) inhibitor. The results suggest that VNP inhibits hypoxia-stimulated proliferation and collagen synthesis in cultured rat PASMCs via particulate guanylyl cyclase-coupled receptors through cGMP/PKG dependent mechanisms.