cGMP stimulation of cystic fibrosis transmembrane conductance regulator Cl- channels co-expressed with cGMP-dependent protein kinase type II but not type Ibeta

In order to investigate the involvement of cGMP-dependent protein kinase (cGK) type II in cGMP-provoked intestinal Cl- secretion, cGMP-dependent activation and phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels was analyzed after expression of cGK II or cGK Ibeta in intact cells. An intestinal cell line which stably expresses CFTR (IEC-CF7) but contains no detectable endogenous cGK II was infected with a recombinant adenoviral vector containing the cGK II coding region (Ad-cGK II) resulting in co-expression of active cGK II. In these cells, CFTR was activated by membrane-permeant analogs of cGMP or by the cGMP-elevating hormone atrial natriuretic peptide as measured by 125I- efflux assays and whole-cell patch clamp analysis. In contrast, infection with recombinant adenoviruses expressing cGK Ibeta or luciferase did not convey cGMP sensitivity to CFTR in IEC-CF7 cells. Concordant with the activation of CFTR by only cGK II, infection with Ad-cGK II but not Ad-cGK Ibeta enabled cGMP analogs to increase CFTR phosphorylation in intact cells. These and other data provide evidence that endogenous cGK II is a key mediator of cGMP-provoked activation of CFTR in cells where both proteins are co-localized, e. g. intestinal epithelial cells. Furthermore, they demonstrate that neither the soluble cGK Ibeta nor cAMP-dependent protein kinase are able to substitute for cGK II in this cGMP-regulated function.     

Protoporphyrin IX accumulation in cells treated with 5-aminolevulinic acid: dependence on cell density, cell size and cell cycle

NO and cGMP have emerged as important signal transduction mediators of the effects of certain hormones, inter-/intracellular signals, toxins and drugs. However, a major challenge is to define relevant criteria for determining which of the many NO and/or cGMP effects are dependent on cGMP-dependent protein kinases (cGKs). Important criteria include that: (1) the cell types/tissues investigated contain at least one form of cGK which is activated by the cGMP-elevating agent in the intact cell system; (2) specific activators/inhibitors of cGKs mimic/block the effects of cGMP-elevating agents in the intact cell system; and (3) the cGMP effect is absent or blunted in cGK-deficient systems, or can be reconstituted by the introduction of active cGKs. Previously, analysis of cGK activity in intact cells has been very difficult. However, the analysis of vasodilator-stimulated phosphoprotein (VASP) phosphorylation by polyclonal antibodies and newly developed monoclonal antibodies, each of which specifically recognize different phosphorylation sites, allows the quantitative measurement of cGK activity in intact cells. With the use of these methods, the properties of certain cGK mutants, cGK activators (cGMP, 8-Br-cGMP, 8-pCPT-cGMP) as well as various "specific cGK inhibitors" (KT 5823, Rp-8Br-PET-cGMPS, Rp-8-pCPT-cGMPS, H8 and H89) were investigated. Although these "specific cGK inhibitors" have been widely used to establish or rule out functional roles of cGKs, very few studies have actually addressed the efficiency/specificity of such compounds in intact cells. Our results demonstrate that these inhibitors are useful tools only when used in combination with other experimental approaches and biochemical evidence.     

N-terminal myristoylation is required for membrane localization of cGMP-dependent protein kinase type II

The apical membrane of intestinal epithelial cells harbors a unique isozyme of cGMP-dependent protein kinase (cGK type II) which acts as a key regulator of ion transport systems, including the cystic fibrosis transmembrane conductance regulator (CFTR)-chloride channel. To explore the mechanism of cGK II membrane-anchoring, recombinant cGK II was expressed stably in HEK 293 cells or transiently in COS-1 cells. In both cell lines, cGK II was found predominantly in the particulate fraction. Immunoprecipitation of solubilized cGK II did not reveal any other tightly associated proteins, suggesting a membrane binding motif within cGK II itself. The primary structure of cGK II is devoid of hydrophobic transmembrane domains; cGK II does, however, contain a penultimate glycine, a potential acceptor for a myristoyl moiety. Metabolic labeling showed that cGK II was indeed able to incorporate [3H]myristate. Moreover, incubation of cGK II-expressing 293 cells with the myristoylation inhibitor 2-hydroxymyristic acid (1 mM) significantly increased the proportion of cGK II in the cytosol from 10 +/- 5 to 35 +/- 4%. Furthermore, a nonmyristoylated cGK II Gly2 --> Ala mutant was localized predominantly in the cytosol after transient expression in COS-1 cells. The absence of the myristoyl group did not affect the specific enzyme activity or the Ka for cGMP and only slightly enhanced the thermal stability of cGK II. These results indicate that N-terminal myristoylation fulfills a crucial role in directing cGK II to the membrane.     

Arginine 75 in the pseudosubstrate sequence of type Ibeta cGMP-dependent protein kinase is critical for autoinhibition, although autophosphorylated serine 63 is outside this sequence

Autoinhibitory domains in many protein kinases include either a phosphorylatable substrate-like sequence or a pseudosubstrate sequence. This study shows that Ibeta cGMP-dependent protein kinase (cGK) autophosphorylates Ser-63, which is in an atypical cGK substrate sequence (-59AQKQAS-) that is amino-terminal to the pseudosubstrate motif (-74KRQAI-). cGMP increases the rate of autophosphorylation (approximately 0.8 phosphate/cGK monomer) approximately 3-fold. Autophosphorylation is an intramolecular process since it is independent of cGK concentration. cGMP activation of cGK enhances proteolysis within and near the pseudosubstrate site; treatment of dimeric cGK with three proteases produces three cGK monomers (approximately 67-70 kDa each). Their amino-terminal sequences are 75RQAISAEPT-, 76QAISAEPTAF-, and 86DIQDLSXV-, respectively. cGMP stimulates these kinases by 10-, 2.5-, and 1.4-fold, respectively, compared with a 10-fold effect on intact cGK. Increased basal activity accounts for the diminished stimulation. Thus, the primary autophosphorylation site of Ibeta cGK is well outside the pseudosubstrate site, but Arg-75 in the pseudosubstrate site is critical for autoinhibition. Autoinhibition also involves elements that are carboxyl-terminal to Arg-75.     

YC-1 potentiates nitric oxide- and carbon monoxide-induced cyclic GMP effects in human platelets

Nitric oxide (NO), the physiological activator of soluble guanylyl cyclase (sGC), induces inhibitory effects on platelet activation via elevation of cGMP levels and stimulation of the cGMP-dependent protein kinase. YC-1, a benzylindazole derivative, was shown to activate sGC in intact platelets, resulting in inhibition of platelet aggregation. In a previous study, we demonstrated that YC-1 not only stimulates purified sGC but also potentiates the stimulatory action of submaximally effective NO and carbon monoxide (CO) concentrations. Here, we investigated the potentiating effect of YC-1 in intact platelets. YC-1 together with NO or CO led to complete inhibition of platelet aggregation at concentrations that were ineffective by themselves. Maximally effective 2, 2-diethyl-1-nitroso-oxyhydrazine (3 microM) and YC-1 (100 microM) concentrations each elevated the cGMP levels in intact platelets approximately 13-fold, and administration of the two drugs together resulted in enormous potentiation of cGMP formation, which greatly exceeded the effect on the purified enzyme and yielded a >1300-fold increase in cGMP levels. Similar results were obtained using CO instead of NO. Furthermore, YC-1 not only stimulated sGC but also inhibited cGMP-hydrolyzing phosphodiesterases in platelets. The enormous elevation of cGMP levels led to enhanced phosphorylation of the cGMP-dependent protein kinase substrate vasodilator-stimulated phosphoprotein. Thus, by the combination of two effects (i.e., potentiation of NO-induced sGC stimulation and phosphodiesterase inhibition), YC-1-like substances are potent activators of the sGC/cGMP pathways and are therefore interesting candidates to act as modulators of cGMP-mediated effects, especially within the cardiovascular system.     

Cyclic GMP and cGMP-binding phosphodiesterase are required for interleukin-1-induced nitric oxide synthesis in human articular chondrocytes

This study addressed the role of guanylyl cyclase (GC) and phosphodiesterase (PDE) in interleukin (IL)-1 activation of human articular chondrocytes. The GC inhibitors LY83583 and methylene blue dose-dependently inhibited IL-1-induced nitric oxide (NO) production, inducible NO synthase (iNOS) protein, and mRNA expression. These effects of GC inhibition were consistent with the rapid induction of cGMP by IL-1, which reached maximal levels after 5 min. The effects of GC inhibitors were selective as they did not reduce IL-1-induced cyclooxygenase II protein and mRNA. An inhibitor specific for soluble GC did not affect IL-1-induced NO production, and activators of soluble GC did not induce NO. However, the expression of iNOS mRNA was induced by atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP), activators of particulate GC, indicating that particulate rather than soluble guanylyl cyclases were involved in iNOS induction. The expression of iNOS mRNA and the production of NO were induced by a slowly hydrolyzable analog of cGMP, 8-bromo-cGMP, but not by nonhydrolyzable analog, dibutyryl cGMP, suggesting that PDE rather than cGMP-dependent protein kinase mediates the cGMP effects. Chondrocytes contained extensive cGMP PDE activity. This had PDE5 biochemical features and an inhibitor profile consistent with PDE5. Furthermore, the nonisoformspecific PDE inhibitor IBMX and PDE5-specific inhibitors suppressed IL-1-induced NO release and iNOS mRNA expression. PDE5 mRNA was constitutively expressed in chondrocytes. In addition to increasing PDE5 activities, IL-1 treatment reduced the sensitivity of PDE5 to several pharmacological inhibitors by up to 50-fold. In summary, inhibitors of either GC or PDE5 prevented IL-1 induction of iNOS; IL-1 increased the rates of both cGMP generation and hydrolysis; and exogenous PDE hydrolyzable cGMP analog induced iNOS and NO. These results suggest that increased cGMP metabolic flux is sufficient to induce iNOS, and GC and PDE5 activities are required for IL-1 induction of iNOS expression via increases in coupled cGMP synthesis and hydrolysis.     

Cyclic GMP-dependent protein kinase and cellular signaling in the nervous system

Nitric oxide (NO) and natriuretic peptide hormones play key roles in a surprising number of neuronal functions, including learning and memory. Most data suggest that they exert converging actions by elevation of intracellular cyclic GMP (cGMP) levels through activation of soluble and particulate guanylyl cyclases. However, cGMP is only the starting point for multiple signaling cascades, which are now beginning to be defined. A primary action of elevated cGMP levels is the stimulation of cGMP-dependent protein kinase (PKG), the major intracellular receptor protein for cGMP, which phosphorylates substrate proteins to exert its actions. It has become increasingly clear that PKG mediates some of the neuronal effects of cGMP, but how is not yet clear. One clear illustration of this pathway has been reported in striatonigral nerve terminals, where NO mediates phosphorylation of the protein phosphatase regulator dopamine- and cyclic AMP-regulated phosphoprotein having a molecular mass of 32,000 (DARPP-32) by PKG. There are remarkably few PKG substrates in brain whose identities are known. A survey of these proteins and those known from other tissues that might also be found in the nervous system reveals the key molecular sites where cGMP and PKG signaling is likely to be regulating neural function. These potential substrates are critically placed to have profound effects on the protein phosphorylation network through regulation of protein phosphatases, intracellular calcium levels, and the function of many ion channels and neurotransmitter receptors. The brain also contains a rich diversity of specific PKG substrates whose identities are not yet known. Their future identification will provide exciting new leads that will permit better understanding of the role of PKG signaling in both basic and higher orders of brain function.     

cAMP- and cGMP-dependent protein kinase phosphorylation sites of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) in vitro and in intact human platelets

The vasodilator-stimulated phosphoprotein (VASP) is a major substrate for cAMP-dependent- (cAK) and cGMP-dependent protein kinase (cGK) in human platelets and other cardiovascular cells. To identify the VASP phosphorylation sites, purified VASP was phosphorylated by either protein kinase and subjected to trypsin, V8 and Lys-C proteolysis. The phosphorylated proteolytic fragments obtained were separated by reversed phase high performance liquid chromatography. Sequence analysis of the phosphorylated peptides and 32P measurement of the released 32P-labeled amino acids revealed three phosphorylation sites: a serine 1-containing site (LRKVSKQEEA), a serine 2-containing site (HIERRVSNAG), and a threonine-containing site (MNAVLARRRKATQVGE). Additional experiments with purified VASP demonstrated that both cAK and cGK phosphorylated serine 2 rapidly and the threonine residue slowly, whereas cGK phosphorylated the serine 1 residue more rapidly than the cAK. These differences in the phosphorylation rates of VASP by the two protein kinases were also observed with synthetic peptides corresponding to the sequences of the three identified phosphorylation sites. These experiments also established the synthetic peptide serine 1 as one of the best in vitro cGK substrates and the serine 2-containing site as the site responsible for the phosphorylation-induced mobility shift of VASP in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Experiments with 32P-labeled platelets provided evidence that VASP is phosphorylated at the same three identified sites also in intact cells and that selective activation of cAK or cGK primarily increased the phosphorylation of both serine 2 and serine 1 but not threonine. Our results demonstrated overlapping substrate specificities of cAK and cGK in vitro and in intact cells. However, important quantitative and qualitative differences between cAK- and cGK-mediated phosphorylation of the focal adhesion protein VASP in human platelets were also observed, suggesting distinct functions of the two types of cyclic nucleotide-mediated VASP phosphorylation.     

Activation of p38 mitogen-activated protein kinase by lipopolysaccharide in human neutrophils requires nitric oxide-dependent cGMP accumulation

This study examined the signal transduction pathway(s) leading to phosphorylation of p38 in human neutrophils stimulated with lipopolysaccharide and formyl peptides. Blockade of the nitric oxide (NO) pathway in neutrophils with the NO synthase inhibitor N-nitro-L-arginine methyl ester or by treatment with the NO scavenger 2-phenyl-tetramethylimidazoline-1-oxyl-3-oxide attenuated phosphorylation of the mitogen-activated protein kinase p38 in response to lipopolysaccharide but not fMet-Leu-Phe. Using the NO releasing agents S-nitroso-N-acetylpenicillamine and sodium nitroprusside it was determined that nitric oxide is sufficient to cause an increase in phosphorylation of p38. Increasing cellular cGMP with phosphodiesterase inhibitors, by stimulation of soluble guanylyl cyclase with YC-1 or with exogenous dibutyryl cGMP resulted in mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 3,6 (MEK3,6) activation and phosphorylation of p38. This phenomenon was specific for MEK3,6, because these agents had no effect on the phosphorylation state of MEK1,2. A role for protein kinase G but not protein kinase A downstream of lipopolysaccharide but not formylmethionylleucylphenylalanine was shown using the specific inhibitors KT5823 and H89, respectively. These data indicate that activation of p38 by fMet-Leu-Phe and lipopolysaccharide involve different mechanisms, and that activation of protein kinase G by NO-dependent stimulation of guanylyl cyclase is necessary and sufficient for phosphorylation of p38 downstream of lipopolysaccharide.     

Defective smooth muscle regulation in cGMP kinase I-deficient mice

Regulation of smooth muscle contractility is essential for many important biological processes such as tissue perfusion, cardiovascular haemostasis and gastrointestinal motility. While an increase in calcium initiates smooth muscle contraction, relaxation can be induced by cGMP or cAMP. cGMP-dependent protein kinase I (cGKI) has been suggested as a major mediator of the relaxant effects of both nucleotides. To study the biological role of cGKI and its postulated cross-activation by cAMP, we inactivated the gene coding for cGKI in mice. Loss of cGKI abolishes nitric oxide (NO)/cGMP-dependent relaxation of smooth muscle, resulting in severe vascular and intestinal dysfunctions. However, cGKI-deficient smooth muscle responded normally to cAMP, indicating that cAMP and cGMP signal via independent pathways, with cGKI being the specific mediator of the NO/cGMP effects in murine smooth muscle.     

Growth factor-induced Ca2+ responses are differentially modulated by nitric oxide via activation of a cyclic GMP-dependent pathway

Nitric oxide (NO) plays a modulatory role on cell growth and differentiation, biological processes that occur under the control of various signal transduction mechanisms, including those triggered by activation of membrane receptors for polypeptide growth factors. The increases in intracellular Ca2+ concentration elicited by the activation of these receptors are sustained by release of the cation from intracellular stores and by stimulation of this influx from the extracellular medium. Using NIH 3T3 cells overexpressing the human epidermal growth factor receptor, we investigated both of these processes stimulated by the administration of epidermal and platelet-derived growth factors as the receptor agonists. Pharmacological and functional analyses carried out on Fura-2-loaded cells showed that Ca2+ influx elicited by both growth factors is the summation of two distinct pathways, with the major pathway dependent on and the minor pathway independent of store depletion. Exposure of the cells to either No donors or NO synthase inhibitors induced increase and inhibition, respectively, of the two components of Ca2+ influx. When Ca2+ release was investigated, the above drugs were also active but in the opposite direction. The effects of NO were mimicked by the cGMP analogue 8-Br-cGMP and abolished by two cGMP-dependent protein kinase I inhibitors, whereas the cAMP analogue 8-Br-cAMP and two protein kinase A inhibitors had no appreciable effects. In addition, growth factors induced an increase in cGMP formation, an effect that was prevented by NO synthase inhibitors. In conclusion, NO appears to exert a feedback modulatory control on CA2+ responses to growth factor administration. Such a control might contribute to the inhibitory effect of NO on growth previously reported with various cell types.     

Reversal of radiation-induced cisplatin resistance in murine fibrosarcoma cells by selective modulation of the cyclic GMP-dependent transduction pathway

Cisplatin resistance, induced in murine fibrosarcoma cells (SSK) in vitro or in vivo by low-dose irradiation, can be overcome by activation of the cyclic GMP(cGMP)-dependent transduction pathway. This is mediated either by stimulating cGMP formation with sodium nitroprusside or by replacing cGMP with a selective activator of the cGMP-dependent protein kinase, 8-bromo-cGMP. The cyclic AMP-dependent transduction pathway is not involved in cisplatin resistance. Instead, activation of cAMP sensitises both parental and resistant SSK cells equally to the action of cisplatin. There is a 1.8 to 2.5-fold increase in drug toxicity, depending on the activating agent. Enhancement of cisplatin sensitivity is induced by specific inhibition of cAMP hydrolysis, increase in cAMP formation or by increasing the activation potential to cAMP-dependent protein kinase by specific cAMP analogues. Cells that have lost cisplatin resistance respond to cGMP- or cAMP-elevating agents in the same way as the parental SSK cells. The radiation sensitivity is unchanged in all cell lines, even after activation of cAMP or cGMP. These results suggest that specific DNA repair pathways are altered by radiation but affected only in cisplatin damage repair, which is regulated by cGMP. Although there is ample cooperativity and interaction between the cAMP- and the cGMP-dependent transduction pathways, specific substrate binding by cGMP appears to play an important role in radiation-induced cisplatin resistance.     

Dual effect of nitric oxide on the hyperpolarization-activated inward current (I(f)) in sino-atrial node cells of the rabbit

Using the whole-cell voltage-clamp technique, we have investigated the effect of nitric oxide (NO) donor (sodium nitroprusside, SNP) on hyperpolarization-activated inward current, I(f), in isolated rabbit sinoatrial node (SAN) cells. I(f) in the basal state increased when NO was applied but decreased when I(f) was pre-stimulated by isoproterenol (ISO) or by adding cAMP to the pipette solution. Both the stimulatory and the inhibitory effects of NO were abolished by guanylyl cyclase inhibitor, methylene blue (MB), suggesting that the effect of NO is mediated by cGMP. The inhibitory effect of NO was abolished when I(f) was pre-stimulated by 3-isobutyl-1-methylxanthine (IBMX), which is a phosphodiesterase (PDE) inhibitor, or by adding 8Br-cAMP (which is resistant to PDE) to the pipette solution. An analogue of cGMP, 8Br-cGMP, which is a potent stimulator of cGMP-dependent protein kinase (PKG) but has little effect on PDE, did not inhibit I(f) when I(f) was pre-stimulated by ISO. In its basal state, I(f) was still increased by 8Br-cGMP, and this effect was not prevented by the pretreatment with H-7, PKG inhibitor. The effect of acetylcholine (ACh) was not identical to that of NO: I(f) decreased when pre-stimulated not only by ISO, but also by IBMX. The above results suggest that via cGMP, NO exerts a dual effect on I(f): the inhibitory effect is mediated by cGMP-stimulated PDE, and the stimulatory effect may be attributable to direct binding of cGMP to I(f) channels.     

Cloning and expression of a neuronal rat brain glutamate transporter

Recent evidence suggests that nitric oxide (NO) may function as a second messenger in the intracellular signal transduction pathways. We explored the possibility that NO was involved in the signal for triggering apoptosis in smooth muscle cells (SMCs). Chemical NO donors induced SMCs apoptosis in a concentration- and time-dependent manner. The membrane-permeable cGMP analogue, dibutyryl-cGMP, did not induce SMCs apoptosis, and the highly selective inhibitor of cGMP-dependent protein kinase, KT5823, was unable to inhibit the induction of NO-induced SMCs apoptosis. Inhibitor of ADP-ribosyltransferase slightly attenuated the induction of SMCs apoptosis by S-nitroso-N-acetyl penicillamine (SNAP). The inhibitor of Na+-H+ antiporter, amiloride, completely inhibited the induction of SMCs apoptosis by SNAP. These results demonstrate for the first time that NO can induce apoptosis in SMCs, suggesting that NO acts as a mediator in the development of atherosclerosis lesion via alterations in the number of SMCs. In addition, the results suggest that NO exert these effects through a pathway that does not involve guanylate cyclase and cGMP-dependent protein kinase.     

Messenger RNA and protein expression of basic fibroblast growth factor receptor after cortical ablation

In a previous report we demonstrated that basic fibroblast growth factor (bFGF), as a multipotent neurotrophic factor, could prevent retrograde degeneration of the thalamic neurons after ablation of the somatosensory cortex. To elucidate the mechanism of this bFGF action, we examined changes in FGF receptor (FGFR) mRNA (flg) expression with in situ hybridization. The FGF receptor protein was detected with the immunoblotting method. The FGFR mRNA expression was found to be diffusely increased in the affected cortex. Microscopic observation indicated that FGFR mRNA was expressed in several types of cortical cells including neurons and non-neuronal cells. This increase could be observed as early as 6 hours after surgery and lasted for 48 hours. In the thalamus, however no change in FGFR mRNA signals was observed. Western blotting detected a protein immunoreactive to anti-FGFR antibody. Samples from the periablated cortex showed an increase in FGFR protein. Samples from the thalamus, however, showed no difference in FGFR protein level between the lesion side and the contralateral side. Application of exogenous bFGF in Gelfoam to the cortical ablation cavity did not show any effect on the gene expression or protein level of FGFR. These results suggest that FGFR is diffusely induced throughout the injured cortex in the early phase after injury and that bFGF may play an important role after injury. Topically applied bFGF might thus modulate cellular responses in the cortex and have a neurotrophic effect on the affected thalamic neurons.     

Defense gene induction in tobacco by nitric oxide, cyclic GMP, and cyclic ADP-ribose

Reactive oxygen species are believed to perform multiple roles during plant defense responses to microbial attack, acting in the initial defense and possibly as cellular signaling molecules. In animals, nitric oxide (NO) is an important redox-active signaling molecule. Here we show that infection of resistant, but not susceptible, tobacco with tobacco mosaic virus resulted in enhanced NO synthase (NOS) activity. Furthermore, administration of NO donors or recombinant mammalian NOS to tobacco plants or tobacco suspension cells triggered expression of the defense-related genes encoding pathogenesis-related 1 protein and phenylalanine ammonia lyase (PAL). These genes were also induced by cyclic GMP (cGMP) and cyclic ADP-ribose, two molecules that can serve as second messengers for NO signaling in mammals. Consistent with cGMP acting as a second messenger in tobacco, NO treatment induced dramatic and transient increases in endogenous cGMP levels. Furthermore, NO-induced activation of PAL was blocked by 6-anilino-5,8-quinolinedione and 1H-(1,2,4)-oxadiazole[4,3-a]quinoxalin-1-one, two inhibitors of guanylate cyclase. Although 6-anilino-5,8-quinolinedione fully blocked PAL activation, inhibition by 1H-(1,2,4)-oxadiazole[4, 3-a]quinoxalin-1-one was not entirely complete, suggesting the existence of cGMP-independent, as well as cGMP-dependent, NO signaling. We conclude that several critical players of animal NO signaling are also operative in plants.     

Effects of the spin trap-alpha-phenyl-N-tert-butyl nitrone (PBN) in transient forebrain ischaemia in the rat

In crustaceans, ecdysteroid production by the molting glands (Y-organs) is negatively regulated by a neuropeptide, molt-inhibiting hormone (MIH). The involvement of cyclic nucleotide-dependent kinases in the mechanism of action of this neuropeptide was investigated with regard to the steroidogenic activity of Carcinus maenas Y-organs. Regardless of the activity level, the major phosphotransferase activity measured in cytosolic fraction was cGMP-dependent, indicating a relatively high cytosolic concentration of cGMP-kinase in these cells. Phosphotransferase activity was nearly twofold higher in the intermolt (low steroidogenic activity) than in premolt (high steroidogenic activity) animals. In vitro incubation of premolt Y-organs with MIH for 1 hr increased by 3.7-fold the cGMP-kinase activity ratio (-cGMP/ +cGMP). Numerous endogenous protein substrates were predominantly phosphorylated in a cGMP-dependent manner in cytosolic, particulate, and membrane fractions. Similar phosphoprotein patterns were observed in both molting stages. By contrast, cAMP-kinase activity, which was low in intermolt Y-organs, increased significantly in the active steroidogenic premolt Y-organs. The increase in cAMP-kinase activity was accompanied by a cAMP-dependent phosphorylation of several specific endogenous proteins. Taken together these results strongly suggest that activation of cGMP-kinase and subsequent phosphorylation of an endogenous protein(s) may be responsible, at least in part, for the MIH-induced inhibition of steroidogenesis. By contrast, it is most unlikely that cAMP-kinase is involved in these processes.     

Redox modulation of L-type calcium channels in ferret ventricular myocytes. Dual mechanism regulation by nitric oxide and S-nitrosothiols

The effects of NO-related activity and cellular thiol redox state on basal L-type calcium current, ICa,L, in ferret right ventricular myocytes were studied using the patch clamp technique. SIN-1, which generates both NO. and O2-, either inhibited or stimulated ICa,L. In the presence of superoxide dismutase only inhibition was seen. 8-Br-cGMP also inhibited ICa,L, suggesting that the NO inhibition is cGMP-dependent. On the other hand, S-nitrosothiols (RSNOs), which donate NO+, stimulated ICa,L. RSNO effects were not dependent upon cell permeability, modulation of SR Ca2+ release, activation of kinases, inhibition of phosphatases, or alterations in cGMP levels. Similar activation of ICa,L by thiol oxidants, and reversal by thiol reductants, identifies an allosteric thiol-containing "redox switch" on the L-type calcium channel subunit complex by which NO/O2- and NO+ transfer can exert effects opposite to those produced by NO. In sum, our results suggest that: (a) both indirect (cGMP-dependent) and direct (S-nitrosylation/oxidation) regulation of ventricular ICa,L, and (b) sarcolemma thiol redox state may be an important determinant of ICa,L activity.     

Twelve-month outcome of patients with DSM-III-R schizoaffective disorder: comparisons to matched patients with bipolar disorder

We examined potential mechanisms by which angiotensin subtype-2 (AT2) receptor stimulation induces net fluid absorption and serosal guanosine cyclic 3',5'-monophosphate (cGMP) formation in the rat jejunum. L-arginine (L-ARG) given intravenously or interstitially enhanced net fluid absorption and cGMP formation, which were completely blocked by the nitric oxide (NO) synthase inhibitor, N-nitro-L-arginine methylester (L-NAME), but not by the specific AT2 receptor antagonist, PD-123319 (PD). Dietary sodium restriction also increased jejunal interstitial fluid cGMP and fluid absorption. Both could be blocked by PD or L-NAME, suggesting that the effects of sodium restriction occur via ANG II at the AT2 receptor. L-ARG-stimulated fluid absorption was blocked by the soluble guanylyl cyclase inhibitor 1-H-[1,2,4]oxadiazolo[4, 2-alpha]quinoxalin-1-one (ODQ). Cyclic GMP-specific phosphodiesterase in the interstitial space decreased extracellular cGMP content and prevented the absorptive effects of L-ARG. Angiotensin II (ANG II) caused an increase in net Na+ and Cl- ion absorption and 22Na+ unidirectional efflux (absorption) from the jejunal loop. In contrast, intraluminal heat-stable enterotoxin of Escherichia coli (STa) increased loop cGMP and fluid secretion that were not blocked by either L-NAME or ODQ. These findings suggest that ANG II acts at the serosal side via AT2 receptors to stimulate cGMP production via soluble guanylyl cyclase activation and absorption through the generation of NO, but that mucosal STa activation of particulate guanylyl cyclase causes secretion independently of NO, thus demonstrating the opposite effects of cGMP in the mucosal and serosal compartments of the jejunum.     

Modulation by protein kinase C of nitric oxide and cyclic GMP poffation in cultured cerebellar granule cells

The possible modulation of nitric oxide (NO) synthase (NOS) activity by protein kinase C (PKC) was investigated in primary cultures of rat cerebellar neurons. Incubation of the cells with L-arginine and nicotinamide-adenine dinucleotide phosphate (NADPH) produced detectable levels of NO, as quantified by photometric assay [0.14 +/- 0.03 nmol/h/dish (2.5 x 10(6) cells)]. The NO producing activity was paralleled by concomitant accumulation of cyclic GMP (cGMP) (0.12 +/- 0.02 pmol/dish). Downregulation of PKC by prolonged treatment with phorbol esters or inhibition of the kinase by treatment with 4taurosporine raised the basal levels of NO and cGMP five fold. When granule cells were incubated in the absence of extracellular Mg2+, N-methyl-D-aspartate and to a lesser extent, glutamate became effective in enhancing NO formation and cGMP accumulation with respect to the control. The NO and cGMP increases induced by the two agonists were almost doubled by treatment of the cells with staurosporine or depletion of PKC. Calphostin C. an inhibitor of the regulatory domain of PKC, was as effective as staurosporine in increasing the formation of NO in both resting and excited cells. These results indicate that downregulation or inhibition of PKC increase NOS activity in cerebellar neurons, and suggest that phosphorylation of NOS by PKC negatively modulates the catalytic activity of the enzyme in these cells.