Intracellular Cyclic GMP Content Research Articles

Essential role of the cGMP/PKG signaling pathway in regulating the proliferation and survival of human renal carcinoma cells

Phosphodiesterase type 5 (PDE5) plays a key role in regulating the intracellular cyclic GMP (cGMP) concentration, which influences anti-proliferative and pro-apoptotic mechanisms in multiple carcinomas. PDE5 inhibitors, such as exisulind and its analogs have anticancer activities. In this study, we found that suppressing PDE5 gene expression by PDE5 siRNA inhibited cell proliferation and induced apoptosis in OS-RC-2 human renal cell carcinoma cells. These effects were enhanced by 8-Br-cGMP, a cell membrane permeable cGMP derivative, and were inhibited by KT5823, a protein kinase G (PKG) inhibitor, indicating that PKG was activated by intracellular cyclic GMP. In addition, there was a reduction in both the mRNA and protein expression of cyclin D1, while p21 protein expression was increased; the reduction in cyclin D1 expression was blocked by the proteasome inhibitor, MG132, or c-Jun N-terminal kinase (JNK) inhibitor; both β-catenin and JNK were phosphorylated by activated PKG. Furthermore, p21 protein expression was decreased in Sp1 siRNA transfected-cells treated with 8-Br-cGMP, indicating that p21 may be partly controlled by the PKG activation through Sp1. Furthermore, we found that PKG Iβ was responsible for the anticancer activities. Our findings indicate that the downregulation of PKG-activated genes, such as cyclin D1 partly accounts for the pro-apoptotic effects in PDE5 siRNA-transfected OS-RC-2 cells.

A novel adipocytokine, chemerin exerts anti-inflammatory roles in human vascular endothelial cells

Chemerin is a recently identified adipocytokine which plays a role on inflammation and adipocytes metabolism. However, its function in vasculature is largely unknown. We examined the effects of chemerin on vascular endothelial inflammatory states. Treatment of human umbilical vein endothelial cells with chemerin (300ng/ml, 20min) induced phosphorylation of Akt (Ser473) and endothelial nitric oxide (NO) synthase (eNOS) (Ser1177). Consistently, chemerin increased intracellular cyclic GMP content. Pretreatment with chemerin (1–300ng/ml, 24h) significantly inhibited phosphorylation of nuclear factor (NF)-κB p65 (Ser536) and p38 as well as vascular cell adhesion molecule (VCAM)-1 expression induced by tumor necrosis factor (TNF)-α (5ng/ml, 20min–6h). Inhibitor of NF-κB or p38 significantly inhibited the TNF-α-induced VCAM-1 expression. Chemerin also inhibited TNF-α-induced VCAM-1 expression in rat isolated aorta. Moreover, chemerin significantly inhibited monocytes adhesion to TNF-α-stimulated endothelial cells. The inhibitory effect of chemerin on TNF-α-induced VCAM-1 was reversed by a NOS inhibitor. Conversely, an NO donor, sodium nitroprusside significantly inhibited TNF-α-induced VCAM-1. The present results for the first time demonstrate that chemerin plays anti-inflammatory roles by preventing TNF-α-induced VCAM-1 expression and monocytes adhesion in vascular endothelial cells. The effect is mediated via inhibiting activation of NF-κB and p38 through stimulation of Akt/eNOS signaling and NO production.

Inhibitory effect of NZ-105, a 1,4-dihydropyridine derivative, on cyclic nucleotide phosphodiesterase activity.

The effects of NZ-105, a 1,4-dihydropyridine calcium antagonist, on the intracellular cyclic nucleotide system were investigated in-vitro. In rabbit isolated aorta, both NZ-105 (1 and 10 microM) and nicardipine significantly and in a concentration-dependent manner increased intracellular cyclic AMP and cyclic GMP content. NZ-105 inhibited bovine cardiac phosphodiesterase activity (Ki 30 microM) by competitive antagonism. The concentration ranges for inhibition were consistent with the range of increases in cyclic nucleotides.

Mitogen-activated Protein Kinase p38 Regulates the Wnt/Cyclic GMP/Ca2+ Non-canonical Pathway

The non-canonical Wnt/cyclic GMP/Ca(2+)/NF-AT pathway operates via Frizzled-2, a member of the superfamily of G protein-coupled receptors. In scanning for signaling events downstream of the Frizzled-2/G alpha t2/PDE6 triad activated in response to Wnt5a, we observed a strong activation of the mitogen-activated protein kinase p38 in mouse F9 teratocarcinoma embryonal cells. The activation of p38 is essential for NF-AT transcriptional activation mediated via Frizzled2. Wnt5a-stimulated p38 activation was rapid, sensitive to pertussis toxin, to siRNA against either G alpha t2 or p38 alpha, and to the p38 inhibitor SB203580. Real-time analysis of intracellular cyclic GMP using the Cygnet2 biosensor revealed p38 to act at the level of cyclic GMP, upstream of the mobilization of intracellular Ca(2+). Fluorescence resonance energy transfer (FRET) imaging reveals the changes in cyclic GMP in response to Wnt5a predominate about the cell membrane, and likewise sensitive to either siRNA targeting p38 or to treatment with SB203580. Dishevelled is not required for Wnt5a activation of p38; siRNAs targeting Dishevelleds and expression of the Dishevelled antagonist Dapper-1 do not suppress the p38 response to Wnt5a stimulation. These novel results are the first to detail a Dishevelled-independent Wnt response, demonstrating a critical role of the mitogen-activated protein kinase p38 in regulating the Wnt non-canonical pathway.

Spare guanylyl cyclase NO receptors ensure high NO sensitivity in the vascular system

In the vascular system, the receptor for the signaling molecule NO, guanylyl cyclase (GC), mediates smooth muscle relaxation and inhibition of platelet aggregation by increasing intracellular cyclic GMP (cGMP) concentration. The heterodimeric GC exists in 2 isoforms (alpha1-GC, alpha2-GC) with indistinguishable regulatory properties. Here, we used mice deficient in either alpha1- or alpha2-GC to dissect their biological functions. In platelets, alpha1-GC, the only isoform present, was responsible for NO-induced inhibition of aggregation. In aortic tissue, alpha1-GC, as the major isoform (94%), mediated vasodilation. Unexpectedly, alpha2-GC, representing only 6% of the total GC content in WT, also completely relaxed alpha1-deficient vessels albeit higher NO concentrations were needed. The functional impact of the low cGMP levels produced by alpha2-GC in vivo was underlined by pronounced blood pressure increases upon NO synthase inhibition. As a fractional amount of GC was sufficient to mediate vasorelaxation at higher NO concentrations, we conclude that the majority of NO-sensitive GC is not required for cGMP-forming activity but as NO receptor reserve to increase sensitivity toward the labile messenger NO in vivo.

Nitric oxide synthase and soluble guanylyl cyclase underlying the modulation of electrical oscillations in a central olfactory organ.

We have isolated and characterized the cDNAs for nitric oxide synthase (NOS) and soluble guanylyl cyclase (sGC) from the terrestrial slug Limax marginatus, and examined the presence and distribution of their mRNAs in the central nervous system using histological techniques and a reverse transcription-polymerase chain reaction method. Our results showed that both bursting and nonbursting neurons in the procerebral lobes contain the mRNAs for both NOS and sGC. We further found that the oscillation frequency of electrical activity in the procerebral lobes increases with increasing intracellular concentrations of cyclic GMP (cGMP). Taken together with previous data on the NO-induced cGMP-like immunoreactivity and on the anatomical distribution of neurites and the localization of synapses of bursting and nonbursting neurons, our present results suggest that NO-induced changes in cGMP concentration modulate the oscillation frequency in the procerebral lobes by acting on the olfactory input pathways, but possibly not on the output pathways, in slugs. .

WNT-frizzled signaling via cyclic GMP.

Wnt-Frizzled signaling is an essential aspect of early development, regulating cell fate, polarity, differentiation, and migration. In addition to the well-known Wnt/beta-catenin pathway characterized for Frizzled-1, there are other pathways regulated by Wnts that are not mediated by Frizzled-1 and do not lead to stabilization of beta-catenin and activation of the Lef/Tcf-sensitive transcription of genes. The first of these non-canonical pathways to be identified is the Wnt/Ca++ pathway in which Frizzled-2 activation leads to release of beta/gamma subunit complexes from heterotrimeric G-proteins (presumably Go and Gt) to activate phospholipase C and other effectors to stimulate a mobilization of intracellular Ca++. More recently a second, related pathway of Wnt-Frizzled signaling has been discovered that regulates the intracellular levels of cyclic GMP. Frizzled-2, established as a member of the family of 7TMS receptors that couple by heterotrimeric G-proteins to effectors, can signal via the G-protein Gt2, transducin, a G-protein prominent in phototransduction in the eye, to cyclic GMP phosphodiesterase. The discovery of the expression of Gt2 in embryonic cells was co-incident with the demonstration that inhibitors of cyclic GMP phosphodiesterase potently blocked various features of Frizzled-2 signaling in mouse embryonic F9 cells and in zebrafish embryos. The signal linkage map from Wnt to changes in intracellular cyclic GMP and development is the focus of this review. The molecular features of how changes in intracellular cyclic GMP concentrations control development remain to be elucidated.

Expression of Constitutively Active Guanylate Cyclase in Cardiomyocytes Inhibits the Hypertrophic Effects of Isoproterenol and Aortic Constriction on Mouse Hearts

Evidence from several rodent models has suggested that a reduction of either atrial natriuretic peptide or its receptor in the heart affects cardiac remodeling by promoting the onset of cardiac hypertrophy. The atrial natriuretic peptide receptor mediates signaling at least in part via the generation of intracellular cyclic GMP. To directly test whether accumulation of intracellular cyclic GMP conveys protection against cardiac hypertrophy, we engineered transgenic mice that overexpress a catalytic fragment of constitutively active guanylate cyclase domain of the atrial natriuretic peptide receptor in a cardiomyocyte-specific manner. Expression of the transgene increased the intracellular concentration of cyclic GMP specifically within cardiomyocytes and had no detectable effect on cardiac performance under basal conditions. However, expression of the transgene attenuated the effects of the pharmacologic hypertrophic agent isoproterenol on cardiac wall thickness and prevented the onset of the fetal gene expression program normally associated with cardiac hypertrophy. Likewise, expression of the transgene inhibited the hypertrophic effects of abdominal aortic constriction, since it abolished its effects on ventricular wall thickness and greatly attenuated its effects on cardiomyocyte size. Altogether, our results suggest that cyclic GMP is a cardioprotective agent against hypertrophy that acts via a direct local effect on cardiomyocytes.

Gene expression profiling of rat uterus at different stages of parturition.

A fuller understanding of the process of parturition is needed in view of the current lack of efficient treatment for preterm labor. Using DNA microarrays, we have analyzed patterns of uterine gene expression at d 0 and 20 of pregnancy, at term in labor or not in labor, and at 1 d post partum. Of the 8740 genes analyzed, 562 genes undergoing significant changes were grouped into 5 distinct clusters, each containing many genes not previously known to be involved with uterine functions. Cluster 1 genes were up-regulated at labor and encompassed immune defense and immediate early response genes, including transcription factors NGFI-B/nurr77 and estrogen-responsive gene 1. Cluster 3 genes were acutely suppressed at labor and included extracellular matrix products and genes related to hormonal signaling, implying novel intrauterine mechanisms regulating intracellular cyclic GMP and local steroid hormone concentrations. At labor, more genes were suppressed than activated, indicating that, for the process of labor induction, gene suppression is at least equally important as the more extensively studied processes of gene activation. The study also points to the existence of novel uterine signaling pathways, including Wnt/frizzled and receptor activator of nuclear factor-kappaB (RANK) and its ligand, as well as the involvement of novel signaling molecules such as estrogen-responsive gene 1, decay-accelerating factor 1, and ebnerin. The present results provide the basis for further studies that will enlarge our knowledge of the mechanisms underlying labor and parturition under physiological and pathophysiological conditions.

Contribution of bradykinin and nitric oxide to AT2 receptor-mediated differentiation in PC12 W cells.

We investigated the effect of angiotensin II on intracellular cyclic GMP content and neurite outgrowth as an indicator of cell differentiation in PC12 W cells. Neurite outgrowth was examined by phase-contrast microscopy. Outgrown neurites were classified as small, medium and large, and were expressed as neurites per 100 cells. Angiotensin II (10-7 m) increased the outgrowth of medium and large neurites by mean +/- SEM 20.2 +/- 2.3 and 6.6 +/- 1.4 compared with 1.66 +/- 0.5 and 0.1 +/- 0.06 neurites per 100 cells in control. Cellular cyclic GMP content increased by 50-250% with angiotensin II at concentrations of 10-6-10-4 m. Both blockade of AT2 receptors and of nitric oxide synthase markedly reduced angiotensin II-induced neurite outgrowth and cyclic GMP production. In contrast, B2 receptor blockade had no effect or even increased these angiotensin II effects. Sodium nitroprusside and 8-bromo-cyclic GMP both mimicked the effects of angiotensin II on cell differentiation. The protein kinase G inhibitor KT-5823 inhibited the neurite outgrowth induced by both angiotensin II and 8-bromo-cyclic GMP. Our results demonstrate that angiotensin II can stimulate cell differentiation in PC12 W cells by nitric oxide-related and cyclic GMP-dependent mechanisms. The effects of angiotensin II on cell differentiation and cyclic GMP production were mediated via the AT2 receptor and further enhanced by bradykinin B2 receptor blockade.

Inhibition of excitatory neurotransmitter–nitric oxide signaling pathway by inhalational anesthetics

Primary cultures of cerebral neurons of Sprague–Dawley rats increased cyclic GMP production in response to the stimulation of excitatory amino acids, including N-methyl- d-aspartate, quisqualate, kainate and (±)-1-aminocylopentane- trans-1,3-dicarboxylic acid. This increased cyclic GMP production was significantly inhibited by halothane or isoflurane at clinically relevant concentrations (0.5–2%). This inhibition was reversible by treatment with l-arginine, the substrate of nitric oxide synthase. However, the increase of cyclic GMP production stimulated by sodium nitroprusside, an activator of soluble guanylate cyclase, was not inhibited by halothane or isoflurane. Neither halothane nor isoflurane affected the basal cyclic GMP production. Activation of the excitatory amino acid neurotransmitter-stimulated nitric oxide–guanylate cyclase signaling pathway increases intracellular cyclic GMP content in neurons. Our results suggest that halothane or isoflurane inhibited this signaling pathway stimulated by selective agonists of each subtype of receptors for excitatory amino acid neurotransmitters. This inhibition may be involved in mechanisms of anesthesia and analgesia. The site(s) of the inhibition is (are) proximal to the activation of neuronal nitric oxide synthase.

Effects of the soluble guanylyl cyclase activator, YC-1, on vascular tone, cyclic GMP levels and phosphodiesterase activity.

The vasomotor and cyclic GMP-elevating activity of YC-1, a novel NO-independent activator of soluble guanylyl cyclase (sGC), was studied in isolated rabbit aortic rings and compared to that of the NO donor compounds sodium nitroprusside (SNP) and NOC 18. Similarly to SNP and NOC 18, YC-1 (0.3-300 microM) caused a concentration-dependent, endothelium-independent relaxation that was greatly reduced by the sGC inhibitor 1-H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ 10 microM; 59% inhibition of dilation induced by 100 microM YC-1) suggesting the activation of sGC as one mechanism of action. Preincubation with YC-1 (3 and 30 microM) significantly increased the maximal dilator responses mediated by endogenous NO in aortic rings that was released upon exposure to acetylcholine, and enhanced the dilator response to the exogenous NO-donors, SNP and NOC 18, by almost two orders of magnitude. Vasoactivity induced by SNP and YC-1 displayed different kinetics as evidenced by a longlasting inhibition by YC-1 (300 microM) on the phenylephrine (PE)-induced contractile response, which was not fully reversible even after extensive washout (150 min) of YC-1, and was accompanied by a long-lasting elevation of intracellular cyclic GMP content. In contrast, SNP (30 microM) had no effect on the vasoconstrictor potency of PE, and increases in intravascular cyclic GMP levels were readily reversed after washout of this NO donor compound. Surprisingly, YC-1 not only activated sGC, but also affected cyclic GMP metabolism, as it inhibited both cyclic GMP break down in aortic extracts and the activity of phosphodiesterase isoforms 1-5 in vitro. In conclusion, YC-1 caused persistent elevation of intravascular cyclic GMP levels in vivo by activating sGC and inhibiting cyclic GMP break down. Thus, YC-1 is a highly effective vasodilator compound with a prolonged duration of action, and mechanisms that are unprecedented for any previously known sGC activator.

Cyclic-GMP-dependent protein kinase inhibits the Ras/Mitogen-activated protein kinase pathway.

Agents which increase the intracellular cyclic GMP (cGMP) concentration and cGMP analogs inhibit cell growth in several different cell types, but it is not known which of the intracellular target proteins of cGMP is (are) responsible for the growth-suppressive effects of cGMP. Using baby hamster kidney (BHK) cells, which are deficient in cGMP-dependent protein kinase (G-kinase), we show that 8-(4-chlorophenylthio)guanosine-3', 5'-cyclic monophosphate and 8-bromoguanosine-3',5'-cyclic monophosphate inhibit cell growth in cells stably transfected with a G-kinase Ibeta expression vector but not in untransfected cells or in cells transfected with a catalytically inactive G-kinase. We found that the cGMP analogs inhibited epidermal growth factor (EGF)-induced activation of mitogen-activated protein (MAP) kinase and nuclear translocation of MAP kinase in G-kinase-expressing cells but not in G-kinase-deficient cells. Ras activation by EGF was not impaired in G-kinase-expressing cells treated with cGMP analogs. We show that activation of G-kinase inhibited c-Raf kinase activation and that G-kinase phosphorylated c-Raf kinase on Ser43, both in vitro and in vivo; phosphorylation of c-Raf kinase on Ser43 uncouples the Ras-Raf kinase interaction. A mutant c-Raf kinase with an Ala substitution for Ser43 was insensitive to inhibition by cGMP and G-kinase, and expression of this mutant kinase protected cells from inhibition of EGF-induced MAP kinase activity by cGMP and G-kinase, suggesting that Ser43 in c-Raf is the major target for regulation by G-kinase. Similarly, B-Raf kinase was not inhibited by G-kinase; the Ser43 phosphorylation site of c-Raf is not conserved in B-Raf. Activation of G-kinase induced MAP kinase phosphatase 1 expression, but this occurred later than the inhibition of MAP kinase activation. Thus, in BHK cells, inhibition of cell growth by cGMP analogs is strictly dependent on G-kinase and G-kinase activation inhibits the Ras/MAP kinase pathway (i) by phosphorylating c-Raf kinase on Ser43 and thereby inhibiting its activation and (ii) by inducing MAP kinase phosphatase 1 expression.

Organization of the canine gene encoding the E isoform of retinal guanylate cyclase (cGC-E) and exclusion of its involvement in the inherited retinal dystrophy of the Swedish Briard and Briard–Beagle dogs

Intracellular cyclic GMP concentration is known to change in response to a wide variety of agents, including hormones, neurotransmitters or light. In vertebrate photoreceptors, different membrane-bound guanylate cyclase isoforms are responsible for cGMP synthesis and thus directly involved in termination of light signalling via the phototransduction cascade and recovery of the dark state. We have characterized a 4.7 kb long cDNA for the canine retinal guanylate cyclase isoform E (cGC-E) predicting a polypeptide of 1109 amino acids. The genomic structure and the complete sequence of the canine GC-E gene, which consists of 20 exons and spans about 14.5 kb, has also been determined. Northern blot analysis showed that GC-E was expressed in the canine retina as a 4.7 and 6.1 kb large transcript. RT-PCR analysis also detected low expression in cerebrum (occipital lobe). We performed a sequence analysis of the cGC-E gene in animals of a Swedish Briard and Briard–Beagle dog kinship in which an inherited retinal dystrophy is segregating. Several intragenic DNA polymorphisms were identified and used for segregation analysis which excluded cGC-E as a candidate gene for this type of canine retinal dystrophy.

Effect of the phosphodiesterase III inhibitor amrinone on cytokine and nitric oxide production in immunostimulated J774.1 macrophages

The level of intracellular cyclic nucleotides is a regulatory factor in a variety of immune processes. Increases in intracellular cyclic AMP (cAMP) and/or cyclic GMP (cGMP) concentration by the inhibition of phosphodiesterase have been shown to modulate the inflammatory response. Amrinone is a clinically used positive inotropic agent which elevates intracellular cAMP and cGMP levels by selective inhibition of the phosphodiesterase III isoenzyme. In the current study, we investigated the effect of various concentrations (1–300 μM) of amrinone on lipopolysaccharide-induced production of pro- and anti-inflammatory cytokines and of nitric oxide (NO) in vitro. In cultured murine J774.1 macrophages, 1 ng/ml–10 μg/ml of lipopolysaccharide from Escherichia coli O55:B5 induced production of tumor necrosis factor- α (TNF- α), interleukin-10, and nitrite (breakdown product of NO). Pretreatment of cells with amrinone caused a dose-dependent suppression of TNF- α production in the concentration range of 1–100 μM. Furthermore, this drug suppressed NO production in the range of 30–300 μM. Similarly to the results in the J774.1 cells, amrinone also inhibited TNF- α and NO production in the range of 10–100 μM in primary rat peritoneal macrophages. At 300 μM, but not at lower concentrations, amrinone inhibited interleukin-10 production in lipopolysaccharide-treated J774.1 macrophages. Pretreatment of the macrophages with 100 and 300 μM amrinone increased the lipopolysaccharide-elicited translocation of nuclear factor- κB. Taken together, our results indicate that the phosphodiesterase III inhibitor amrinone modulates the activation/production of many pro- and anti-inflammatory factors in endotoxin-stimulated cells. It remains to be further investigated how such immunomodulatory effects contribute to the clinical profile of the agent.

Neuronal nitric oxide synthase activation by vasoactive intestinal peptide in bovine cerebral arteries.

The participation of nitric oxide and vasoactive intestinal peptide (VIP) in the neurogenic regulation of bovine cerebral arteries was investigated. Nitrergic nerve fibers and ganglion-like groups of neurons were revealed by NADPH-diaphorase staining in the adventitial layer of bovine cerebral arteries. NADPH diaphorase also was present in endothelial cells but not in the smooth muscle layer. Double immunolabeling for neuronal nitric oxide synthase and VIP indicated that both molecules co-localized in the same nerve fibers in these vessels. Transmural nerve stimulation (200 mA, 0.2 milliseconds, 1 to 8 Hz) of endothelium-denuded bovine cerebral artery rings precontracted with prostaglandin F2 alpha, produced tetrodotoxin-sensitive relaxations that were completely suppressed by NG-nitro-L-arginine methyl ester (L-NAME) and by the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline (ODQ), but were not affected by the adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536), nor by VIP tachyphylaxis induced by pretreatment with 1 mumol/L VIP. Transmural nerve stimulation also elicited increases in intracellular cyclic GMP concentration, which were prevented by L-NAME, and small decreases in intracellular cyclic AMP concentration. Addition of VIP to bovine cerebral artery rings without endothelium produced a concentration-dependent relaxation that was partially inhibited by L-NAME, ODQ, and SQ 22,536. The effects of L-NAME and SQ 22,536 were additive. VIP induced a transient increase in intracellular cyclic GMP concentration, which was maximal 1 minute after VIP addition, when the highest relaxation rate was observed, and which was blocked by L-NAME. It is concluded that nitric oxide produced by perivascular neurons and nerve fibers fully accounts for the experimental neurogenic relaxation of bovine cerebral arteries and that VIP, which also is present in the same perivascular fibers, acts as a neuromodulator by activating neuronal nitric oxide synthase.

Cyclic GMP-independent effects of nitric oxide on guinea-pig uterine contractility.

1. The role of nitric oxide (NO) in the regulation of uterine contractility has yet to be clearly defined. We evaluated the effect of NO (in the form of S-nitroso-cysteine, CysNO) upon uterine contractility and guanosine 3',5'-cyclic monophosphate (cyclic GMP) accumulation in pregnant and nonpregnant guinea-pig myometrium. 2. While CysNO had no effect upon spontaneous contractile activity in either pregnant or nonpregnant uterine tissues, addition of CysNO resulted in an immediate and reversible relaxation of oxytocin- or acetylcholine (ACh)-evoked contractions. 3. Relaxation of agonist-evoked contractions in response to CysNO was associated with significant elevations in intracellular cyclic GMP concentrations ([cyclic GMP]i). 4. Elevations in [cyclic GMP]i were not required for relaxation, as inhibition of guanylyl cyclase by methylene blue prevented [cyclic GMP]i accumulation while having no effect upon the ability of CysNO to relax agonist-evoked contractions. 5. Addition of the cyclic GMP-analogues, 8-Br-cyclic GMP and PET-cyclic GMP, only at high concentrations, produced partial relaxation of agonist-contracted tissues, suggesting the possibility that cyclic GMP may be sufficient but not necessary for myometrial relaxation. 6. Our studies not only provide evidence for a functional role for NO-modulation of agonist-evoked contractions in the pregnant and nonpregnant guinea-pig uterus, but also that these occur by a mechanism which is not dependent upon guanylyl cyclase activity.

Tricyclic Antidepressants Inhibit Human Natural Killer Cells

The commonly used antidepressants imipramine, amitriptyline, and nortriptyline were found to significantly inhibit human natural killer (NK) cell-mediated cytolysisin vitroand suppress the stimulation of NK cells by IFN-γ. This is a previously unrecognized biologic property of these drugs with psychotropic activity. Tricyclic antidepressants did not decrease effector–target cell conjugation formation, nor did they induce target cell resistance to NK lysis, indicating that the drugs might interfere with the killing mechanism of the effector cells. Kinetic data reveal that the drug interference is related to an early postbinding event in the activation of NK cells. Results also showed that the inhibitory effect of tricyclic antidepressants on human NK cell activity occurred in parallel to an increase in intracellular cyclic GMP concentration. However, the attenuation in the cyclic GMP formation by methylene blue, a selective inhibitor of soluble guanylate cyclase, was not accompanied by a corresponding increase in NK cell cytolytic activity. It is suggested that the stimulation of cyclic GMP was not directly involved in the inhibitory effect of antidepressants on NK cells and perhaps was a secondary phenomenon. This immune cell modulatory property of tricyclic antidepressants seems to indirectly provide evidence for the concept that human brain neurons and NK cells might share regulatory system(s).

Rapid regulation of PDE-2 and PDE-4 cyclic AMP phosphodiesterase activity following ligation of the T cell antigen receptor on thymocytes: Analysis using the selective inhibitors erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) and rolipram

The PDE2, cyclic GMP-stimulated, and the PDE4, cyclic AMP-specific enzymes provide the major, detectable cyclic AMP phosphodiesterase activities in murine thymocytes. In the absence of cyclic GMP, PDE4 activity predominated (∼80% total) but in the presence of low (10 μM) cyclic GMP concentrations, PDE2 activity constituted the major PDE activity in thymocytes (∼80% total). The PDE4 selective inhibitor rolipram dose-dependently inhibited thymocyte PDE4 activity ( ic 50 ∼ 65 nM). PDE2 was dose-dependently activated ( ec 50 ∼ 1 μM) by cyclic GMP and inhibited by erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) ( ic 50 ∼ 4 μM). EHNA was shown to serve as a selective inhibitor of PDE-2 activity as assessed from studies using separated PDE1, PDE2, PDE3 and PDE4 species from hepatocytes as well as human PDE2 and PDE4 enzymes. EHNA completely ablated the ability of cyclic GMP to activate PDE2 activity, whilst having a much smaller inhibitory effect on the unstimulated PDE2 activity. EHNA exhibited normal Michaelian kinetics of inhibition for the cyclic GMP-stimulated PDE2 activity with Hill plots near unity. Apparent negative co-operative effects were seen in the absence of cyclic GMP with Hill coefficients of ∼0.3 for inhibition of PDE2 activity. Within 5 min of challenge of thymocytes with the lectin phytohaemagglutinin (PHA) there was a transient decrease (∼83%) in PDE-4 activity and in PDE2 activity (∼40%). Both anti-CD3 and anti-TCR antibodies also caused an initial reduction in the PDE4 activity which was followed by a sustained and profound increase in activity. In contrast to that observed with PHA, anti-TCR/CD3 antisera had little effect on PDE2 activity. It is suggested that, dependent upon the intracellular concentrations of cyclic GMP, thymocyte cyclic AMP metabolism can be expected to switch from being under the predominant control of PDE4 activity to that determined predominantly by PDE2 activity. These activities may be rapidly and differentially regulated following ligation of different cell surface receptors.

The reaction of Helix photosensitive neurons to light and cyclic GMP.

The effect of light and cyclic nucleotides (cGMP and cAMP) on a group of identified photosensitive neurons in Helix pomatia left parietal ganglion was examined. The onset of light induces in these cells a slow inward current shift associated with decrement of slope conductance. The reversal potential (Erev) of the light induced current (IL) and its changes due to altered extracellular concentration of potassium support the assumption that (IL) is due to suppression of K+ conductance. Elevation of intracellular concentration of cyclic GMP by adding 8-bromo-cGMP into the bathing solution or by injecting the nucleotide into the cell mimics, in all cases, the effect of light: it produces an inward current accompanied by decrement of slope conductance. The effect of elevation of cAMP was inconsistent. The I-V relations of IL and IcGMP follow a similar course and have a common Erev. The assumption that 3',5'-cyclic GMP is increased by light is also supported by comparing the effect of light and the cyclic nucleotide on the action potential. Both light and 8-bromo-cGMP broaden the action potential by increasing the voltage-dependent Ca2+ current.