NO-sensitive Guanylyl Cyclase Research Articles

Evaluation of ODQ as specific inhibitor of NO-sensitive guanylyl cyclase using mice deficient for the enzyme

The NO/cGMP signal transduction is involved in the regulation of a variety of physiological processes e.g. smooth muscle relaxation and platelet aggregation. As signalling molecule, NO has diverse effects with NO-sensitive guanylyl cyclase (NO-GC) being accepted as the most important NO receptor. To differentiate between cGMP-dependent and -independent effects of NO inhibitors for the NO-GC are broadly used. The commonly used inhibitor for NO-GC is ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one). The precise mechanism of NO-GC inhibition by ODQ remains unclear. Recently, we have generated mice deficient in NO-GC (GC-KO). GC-KO mice show a pronounced increase in blood pressure, underlining the importance of NO in the regulation of smooth muscle tone in vivo. We showed a total lack of NO affecting smooth muscle tone and platelet aggregation which confirms NO-GC as the only NO target regulating these two functions in mice. Using these KO mice we can evaluate the specificity of ODQ as inhibitor of NO-GC. In fact, ODQ used at low μM concentrations is a good inhibitor of cGMP signalling, allowing its use to investigate the cGMP dependence of NO effects. However, high NO concentrations elicited relaxation responses in ODQ-treated WT smooth muscle via NO-GC as they were absent in GC-KO tissue. This shows that NO-induced effects in the presence of ODQ do not necessarily indicate cGMP independence.

NO signalling in mice lacking the NO receptor guanylyl cyclase

NO-sensitive guanylyl cyclase (NO-GC) is accepted to be the major receptor for the signaling molecule NO. NO-GC catalyzes the production of the intracellular second messenger cGMP and thereby has a key regulatory function regarding various physiological processes. NO-GC is made up of one β subunit and one α subunit. On the protein level, there are two α subunits (α1 and α2) and one β subunit (β1) known, and thus two different GC isoforms exist (α1β1 and α2β1). In the enteric nervous system, non-adrenergic non-cholinergic (NANC) neurons control the motor function of the gastrointestinal tract. These NANC neurons express neuronal NO synthase. NO has been demonstrated to be an important physiological mediator of NANC relaxation of gastrointestinal smooth muscle, leading to relaxation via activation of NO-GC. In this study, we intended to clarify the role of NO-GC with regards to gastrointestinal (GI) motility. Immunohistochemical analysis revealed that GI smooth muscle expresses only minor amounts of NO-GC. Interestingly, strong expression was detected in the interstitial cells of Cajal (ICC). Using mice in which exon 10 of the β1 subunit is surrounded by loxP sites (floxed mice), we have generated mice lines that lack NO-GC either ubiquitously or specifically in smooth muscle. In isometric force studies we used endogenous NO production induced by electric field stimulation and pharmacological NO release using NO donors. Total gut transit time was measured to study the consequences of NO-GC deletion in vivo. General lack of NO-GC abolishes NO-dependent relaxation of GI smooth muscle. Neither physiological concentrations of NO using NO donors nor electric field stimulation at low frequencies led to relaxation of gastric fundus strips or sphincters. The whole gut transit time was increased approximately three times compared with WT. This dysfunction led to early mortality starting around day 18 after birth. Mortality was reduced by omission of dietary fiber. Surprisingly, in smooth muscle-specific knockout mice (SMKO) the NO-dependent relaxation was reduced, but still intact. Total gut transit time showed no difference and survival of SMKO animals was similar to that of WT mice. In conclusion, the NO receptor guanylyl cyclase in GI smooth muscle is dispensable for motility and survival.

Lack of correlation between sGC subunit expression and sGC activity in cerebral development is due to non-heterodimerizing subunits in vivo

Background A number of studies have shown that the nitric oxide (NO)/cGMP signalling pathway plays a major role in neuronal cell differentiation and in the central nervous system during development, but much less is known about the expression and regulation of the different subunits of NO sensitive guanylyl cyclase (sGC) in the developing brain. In the present study, we have analysed the regulation and expression of sGC in brain of rats during postnatal development using biochemical methods.

Gonadal hormones decrease temporomandibular joint κ-mediated antinociception through a down-regulation in the expression of kappa opioid receptors in the trigeminal ganglia

We have previously demonstrated that activation of κ-opioid receptor located in the temporomandibular joint (TMJ) of rats induces a significantly greater TMJ antinociception in diestrus females than in proestrus females (higher estradiol serum levels than diestrus) and males. These findings indicate that gonadal hormones decrease TMJ kappa-mediated antinociception. The aim of this study was to investigate some of the mechanisms by which gonadal hormones decrease TMJ kappa-mediated antinociception. Western blot analysis demonstrated a significantly lower κ-opioid receptor expression in the trigeminal ganglia of intact males than in intact and ovariechtomized (OVX) females and orchidectomized (ORX) males. In females, κ-opioid receptor expression in the trigeminal ganglia was significantly lower in proestrus than in diestrus and OVX females. Taken together these findings suggest that gonadal hormones, especially male gonadal hormones, down-regulate κ-opioid receptor expression. Co-application of the NOS inhibitor l-NMMA or the NO-sensitive guanylyl cyclase inhibitor ODQ with the κ-opioid receptor agonist U50,488 blocked TMJ kappa-mediated antinociception in males and females. These findings suggest that antinociception induced by activation of kappa opioid receptors in the TMJ region is mediated by the l-arginine/NO/cGMP pathway in both sexes. Despite the involvement of the l-arginine/NO/cGMP pathway in TMJ kappa-mediated antinociception in both sexes, gonadal hormones do not diminish the activity of this pathway to decrease TMJ kappa-mediated antinociception. Alternatively, they significantly reduce κ-opioid receptor expression in the trigeminal ganglia.

NO-sensitive guanylyl cyclase β1 subunit is peripherally associated to chromosomes during mitosis. Novel role in chromatin condensation and cell cycle progression

NO-sensitive guanylyl cyclase (GC NO), the major NO target, is involved in important regulatory functions in the cardiovascular, gastrointestinal and central nervous systems. GC NO exists as heterodimers of α(1/2) and β1 subunits. Deletion of the obligate β1 dimerizing partner abrogates NO/cGMP signaling and shortens the life span of KO mice. Localization studies in the CNS have shown that β1 is more widespread than α subunits and in some areas is the only GC NO subunit expressed, suggesting that β1 may have GC NO-independent functions. GC NO is predominantly cytosolic, but association to membranes and other intracellular structures has been described. Here, we show localization of β1 in cytoplasm and nucleus of cells expressing α subunits and GC NO activity (astrocytes, C6 cells), as well as in cells devoid of α subunits and GC NO activity (microglia). In both cell types β1 associates peripherally to chromosomes in all phases of mitosis. Immunodepletion of β1 in C6 cells enhances chromatin condensation in an in vitro assay. Moreover, silencing β1 by siRNA induces cell cycle re-entry as determined by flow cytometry, and increases proliferation rate in a MTT-assay, whereas infection with β1-containing adenovirus has the opposite effect. These actions are independent of cGMP formation. We postulate that β1 is a multifunctional protein that regulates chromatin condensation and cell cycle progression, in addition to being an obligate monomer in functional GC NO heterodimers.

Phenolphthalein as a Prototype Drug for a Group of Structurally Related Calcium Channel Blockers in Human Platelets

Thrombin increases intracellular free Ca ([Ca]i) in human platelets by 2 mechanisms: internal mobilization and the influx of Ca via store-operated Ca entry (SOCE). 2-Aminoethoxydiphenyl borate (2-APB) is an inhibitor of SOCE. In search for nonboron analogues of 2-APB, we identified a well-known compound, phenolphthalein, structurally related to 2-APB. Many phenolphthalein analogues inhibited the ability of thrombin and thapsigargin (a specific activator of SOCE) to increase [Ca]i. Phenolphthalein has an IC50 approximately 10 microM to inhibit thrombin-induced [Ca]i elevation, its active analogues have a similar potency. Several phenolphthalein analogues also inhibited thrombin-induced intracellular Ca mobilization, which indicates action on inositol 1,4,5-trisphosphate receptors. We identified structural features among active and inactive phenolphthalein analogues that are responsible for the activity. Opening of the 5-membered lactone ring of phenolphthalein resulted in a total loss of activity. If the diphenyl rings possessed primary amine, dimethyl amine, or a cyano group, there was no activity. Modifications to the diphenyl groups that were tolerated include phosphate, sulfate, iodine, bromine, methyl, nitrite, and methoxy. Inhibition of thrombin-induced [Ca]i increase by phenolphthalein was not mediated by an increase in cyclic adenosine monophosphate because the inhibitor of cyclic adenosine monophosphate-dependent protein kinase A, 4-cyano-3-methylisoquinoline, did not affect the inhibitory action of phenolphthalein. The inhibitory effect of phenolphthalein was not mediated by an increase in NO/cyclic guanosine monophosphate (cGMP) because the inhibitors of NO-sensitive soluble guanylyl cyclase, methylene blue, and ODQ did not affect the inhibition. Phytohemagglutinin and thapsigargin-induced SOCE in Jurkat cells was also inhibited by phenolphthalein and 2-APB to the same extent as seen in platelets. Therefore, phenolphthalein and its derivatives structurally similar to 2-APB inhibit SOCE in platelets and other cells.

Pharmacological assessments of nitric oxide synthase isoforms and downstream diversity of NO signaling in the maintenance of thermal and mechanical hypersensitivity after peripheral nerve injury in mice

Nitric oxide synthase (NOS) isoforms and NO downstream signal pathways involved spinally in the maintenance of thermal and mechanical hypersensitivity were assessed in a mouse model of neuropathic pain developing after partial ligation of the sciatic nerve. Intrathecal injection of the NOS inhibitor N G-nitro- l-arginine methyl ester ( l-NAME), the highly selective neuronal NOS (nNOS) inhibitor N ω-propyl- l-arginine and the potent selective inducible NOS (iNOS) inhibitor 2-amino-5,6-dihydro-6-methyl-4 H-1,3-thiazine hydrochloride (AMT) exerted dose-dependent analgesic effects on thermal and mechanical hypersensitivity, which were assessed by the plantar and von Frey tests, respectively, suggesting that both nNOS and iNOS participate in producing NO to maintain neuropathic pain. Since the selective inhibitor of NO-sensitive guanylyl cyclase 1 H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one (ODQ) and the guanosine 3′,5′-cyclic monophosphate (cGMP)-dependent protein kinase (PKG) inhibitor Rp-8-pCPT-cGMPS intrathecally exerted dose-dependent analgesic effects on thermal and mechanical hypersensitivity, spinally released NO most likely stimulates the NO–cGMP–PKG pathway. Moreover, the superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), a potent superoxide scavenger, reduced thermal and mechanical hypersensitivity when administered intrathecally, suggesting that spinal release of superoxide, which can then react with NO to produce peroxynitrite, also appears to mediate neuropathic pain. Finally, intrathecal injection of phenyl- N- tert-butylnitrone (PBN), a reactive oxygen species (ROS) scavenger, ameliorated thermal and mechanical hypersensitivity, thus further confirming the importance of ROS including NO and superoxide in the maintenance of neuropathic pain. Together, the present results demonstrate that NO, produced presumably via nNOS and iNOS in the spinal cord, mediates the maintenance of neuropathic pain following peripheral nerve injury through both the NO–cGMP–PKG and the NO–peroxynitrite pathways.

Abstract 3894: Inhibition of iNOS Uncoupling by Tetrahydrobiopterin Confers Cardioprotection through Protein S- Nitrosylation in Streptozotocin-Induced Diabetic Rat

Although expression of inducible nitric oxide synthase (iNOS) and oxidative stress are increased in diabetic (DM) hearts, the role of iNOS uncoupling in ischemia/reperfusion (IR) injury remains unknown. Because iNOS-derived NO is known to play a crucial role in cardioprotection against IR injury in non-DM hearts, we hypothesized that iNOS uncoupling may compromise tolerance to IR injury in the DM heart by decreasing the bioavailability of NO. The expression and activity of iNOS but not n/eNOS were increased in the streptozotocin-induced DM rat heart. Under Langendorff perfusion, superoxide generation as evaluated by dihydroethidium accumulation in the nucleus was significantly increased in cardiomyocytes of the DM heart, but it was inhibited by treatment with the NOS co-factor tetrahydrobiopterin (BH4; 10 μM) or an iNOS selective inhibitor 1400W (10 μM). BH4 increased NOx, a marker of NO bioavailability, and cGMP in the DM heart. The increase in cGMP by BH4 was abrogated by co-treatment with 1400W or a NO-sensitive guanylyl cyclase inhibitor ODQ (10 μM). BH4 significantly decreased nitrotyrosin formation but increased protein S -nitrosylation in the DM heart. The increase in protein S -nitrosylation by BH4 was abolished by co-treatment with a thiol reducing agent dithiothreitol (DTT; 5 mM). The isolated rat heart was subjected to 30 min global ischemia followed by 120 min reperfusion. Post-ischemic recovery of left ventricular (LV) function and infarct size was comparable between the non-DM and the DM hearts. Pre-ischemic treatment with BH4 significantly improved post-ischemic LV function and reduced infarct size only in the DM heart. Co-treatment with BH4 and 1400W, ODQ, or DTT had no significant effect on post-ischemic LV function and infarct size in the non-DM heart. However, co-treatment with BH4 and 1400W or DTT but not ODQ abolished BH4-induced improvement of post-ischemic LV function and reduction of infarct size in the DM heart. These results suggest that inhibition of iNOS uncoupling by BH4 confers cardioprotection against IR injury in the streptozotocin-induced DM rat heart by increasing the bioavailability of NO and this cardioprotective effect is mediated by protein S -nitrosylation but not cGMP.

Olfactory bulb interneurons releasing NO exhibit the Reelin receptor ApoEr2 and part of those targeted by NO express Reelin

Nitric oxide (NO) and Reelin both modulate neuronal plasticity in developing and mature synaptic networks. We recently showed a loss of neuronal nitric oxide synthase (nNOS) protein in the olfactory bulb of reeler mutants and advanced the hypothesis that the Reelin and NO signalling pathways may influence each other. We now studied the distribution of NO sensitive guanylyl cyclase (NOsGC), Reelin and its receptor Apolipoprotein E2 (ApoEr2) in the olfactory bulb by multiple fluorescence labelling and tested whether nNOS and ApoEr2 colocalize in this area. We also essayed the protein content of NOsGC in the reeler olfactory bulb and tested whether there are any changes in nNOS and NOsGC protein in other reeler brain areas. Olfactory bulb interneurons expressing ApoEr2 and nNOS are only few in the glomerular layer but represent the large majority of granule cell layer interneurons. Conversely, NOsGC interneurons are rare in the granule cell layer and abundant as periglomerular cells. Reelin containing periglomerular cells almost entirely belong to the NOsGC subset. These data further support the hypothesis of a reciprocal signalling between Reelin/NOsGC and ApoEr2/nNOS expressing neurons to affect olfactory bulb activity. We also show that a significant rise in NOsGC content accompanies the decrease of nNOS protein in the reeler olfactory bulb. The same reciprocal changes present in the cortex/striatum and the hippocampus of reeler mice. Thus, the influence that the deficit of extracellular Reelin seems to exert on nNOS and its receptor is not limited to the olfactory bulb but is a general feature of the reeler brain.

CGMP Produced by NO-Sensitive Guanylyl Cyclase Essentially Contributes to Inflammatory and Neuropathic Pain by Using Targets Different from cGMP-Dependent Protein Kinase I

A large body of evidence indicates that the release of nitric oxide (NO) is crucial for the central sensitization of pain pathways during both inflammatory and neuropathic pain. Here, we investigated the distribution of NO-sensitive guanylyl cyclase (NO-GC) in the spinal cord and in dorsal root ganglia, and we characterized the nociceptive behavior of mice deficient in NO-GC (GC-KO mice). We show that NO-GC is distinctly expressed in neurons of the mouse dorsal horn, whereas its distribution in dorsal root ganglia is restricted to non-neuronal cells. GC-KO mice exhibited a considerably reduced nociceptive behavior in models of inflammatory or neuropathic pain, but their responses to acute pain were not impaired. Moreover, GC-KO mice failed to develop pain sensitization induced by intrathecal administration of drugs releasing NO or carbon monoxide. Surprisingly, during spinal nociceptive processing, cGMP produced by NO-GC may activate signaling pathways different from cGMP-dependent protein kinase I (cGKI), whereas cGKI can be activated by natriuretic peptide receptor-B dependent cGMP production. Together, our results provide evidence that NO-GC is crucially involved in the central sensitization of pain pathways during inflammatory and neuropathic pain.

The action of nitric oxide to enhance cell survival in chick cardiomyocytes is mediated through a cGMP and ERK1/2 pathway while p38 mitogen‐activated protein kinase‐dependent pathways do not alter cell death

The objective of this study was to determine whether the dual action of nitric oxide (NO) on cardiomyocyte cell viability is mediated through p38 mitogen-activated protein kinase (MAPK)-induced cell death and extracellular signal-regulated kinase (ERK1/2)-mediated cell survival pathways, and whether either of these is mediated through a cGMP-protein kinase G (PKG) pathway. Cell viability of embryonic chick cardiomyocytes was assessed by the MTT assay, which is based on the ability of viable cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. The NO donor sodium nitroprusside (SNP) produced a significant (P < 0.01) concentration-dependent reduction in cell viability or increase in cell death. Sodium nitroprusside induced ERK1/2 phosphorylation, and the mitogen-activated protein kinase (MEK1/2) inhibitor PD 98059 significantly increased cell death. In contrast, SB202190, a relatively selective inhibitor of p38 MAPK, did not affect SNP-induced cell death. The cardioprotective effect of NO was prbably mediated in part via cGMP because 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a selective inhibitor of NO-sensitive guanylyl cyclase, produced a significant enhancement of SNP-induced cell death. In contrast, the PKG inhibitor KT5823 did not affect cell viability. In summary, these data suggest that NO, via stimulation of soluble guanylyl cyclase, activates MEK1/2 whose product, ERK1/2, protects against cell death. In contrast, SNP-induced p38 MAPK activation does not modulate NO-induced cardiomyocyte cell death. Not all cGMP targets affect NO-induced cell death, since the PKG pathway does not enhance or suppress NO-induced cardiomyocyte cell death. Enhancement of the ERK1/2 responses to NO may permit the beneficial effects of NO to predominate.

Tanshinone IIA: A new activator of human cardiac KCNQ1/KCNE1 ( IKs) potassium channels

Tanshinone IIA, one of the main active components from Chinese herb Danshen, is widely used to treat cardiovascular diseases including arrhythmia in Asian countries especially in China. However, the mechanisms underlying its anti-arrythmia effects are not clear. In this study we investigate the effects of tanshinone IIA on human KCNQ1/KCNE1 potassium channels ( I Ks), human ether-a-go-go-related gene potassium channels (hERG), Kv1.5 potassium channels, inward rectifier potassium channels ( I K1) expressed in HEK 293 cells using patch clamp technique. Tanshinone IIA potently and reversibly enhanced the amplitude of I Ks in a concentration dependent manner with an EC 50 of 64.5 μM, accelerated the activation rate of I Ks channels, decelerated their deactivation and shifted the voltage dependence of I Ks activation to negative direction. Isoproteronol, a stimulator of β-adrenergic receptor, at 1 μM and sodium nitroprusside (SNP), a NO donor, at 1 mM, had no significant effects on the enhancement of I Ks by 30 μM tanshinone IIA. N-[2-( p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89), a selective protein kinase A inhibitor, at 0.1 μM and 1 H-(1,2,4)oxadiazolo(4,3- a)quinoxalin-1-one (ODQ), a selective nitric oxide-sensitive guanylyl cyclase inhibitor, at 10 μM, also had no significant effects on the enhancement of I Ks by 30 μM tanshinone IIA. Tanshinone IIA did not affect expressed hERG channels, Kv1.5 channels and I K1 channels. These results indicate that tanshinone IIA directly and specifically activate human cardiac KCNQ1/KCNE1 potassium channels ( I Ks) in HEK 293 cell through affecting the channels' kinetics.

Enhanced nitric oxide and cyclic GMP formation plays a role in the anti‐platelet activity of simvastatin

It has been found that 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) exert various vascular protective effects, beyond their cholesterol-lowering property, including inhibition of platelet-dependent thrombus formation. The objective of the present study was to determine whether the nitric oxide (NO)/cyclic GMP-mediated processes in platelets contribute to the anti-aggregatory activity of simvastatin. After rabbit platelets were incubated with simvastatin for 5 min, aggregation was induced and the platelet aggregation, nitric oxide synthase activity, guanylyl cyclase activity, NO and cyclic GMP formation were measured appropriately. Treatment with simvastatin concentration-dependently inhibited platelet aggregation induced by collagen or arachidonic acid with an IC(50) range of 52-158 microM. We also demonstrated that simvastatin (20-80 microM) concentration-dependently further enhanced collagen-induced NO and cyclic GMP formation through increasing NOS activity (from 2.64+/-0.12 to 3.52+/-0.21-5.10+/-0.14 micromol min(-1) mg protein(-1)) and guanylyl cyclase activity (from 142.9+/-7.2 to 163.5+/-17.5-283.8+/-19.5 pmol min(-1) mg protein(-1)) in the platelets. On the contrary, inhibition of platelet aggregation by simvastatin was markedly attenuated (by about 50%) by addition of a nitric oxide synthase inhibitor, a NO scavenger or a NO-sensitive guanylyl cyclase inhibitor. The anti-aggregatory effects of simvastatin were significantly increased by addition of a selective inhibitor of cyclic GMP phosphodiesterase. Our findings indicate that enhancement of a NO/cyclic GMP-mediated process plays an important role in the anti-aggregatory activity of simvastatin.

LPS-induced down-regulation of NO-sensitive guanylyl cyclase in astrocytes occurs by proteasomal degradation in clastosomes

We previously showed that treatment with bacterial lipopolysaccharide (LPS) or pro-inflammatory cytokines decreases NO-sensitive guanylyl cyclase (GC NO) activity in astrocytes by decreasing the half-life of the obligate GC NO β1 subunit in a NO-independent but transcription- and translation-dependent process. Here we show that LPS-induced β1 degradation requires proteasome activity and is independent of NFκB activation or β1 interaction with HSP90. Immunocytochemistry and confocal microscopy analysis revealed that LPS promotes colocalization of the predominantly soluble β1 protein with ubiquitin and the 20S proteasome in nuclear aggregates that present characteristics of clastosomes, nuclear bodies involved in proteolysis via the ubiquitin–proteasome system. Proteasome and protein synthesis inhibitors prevented LPS-induced clastosome assembly and nuclear colocalization of β1 with ubiquitin and 20S proteasome, strongly supporting a role for these transient nuclear structures in GC NO down-regulation during neuroinflammation.

Involvement of Nitric Oxide in Depolarization-Induced Suppression of Inhibition in Hippocampal Pyramidal Cells during Activation of Cholinergic Receptors

Several types of neurons are able to regulate their synaptic inputs via releasing retrograde signal molecules, such as endocannabinoids or nitric oxide (NO). Here we show that, during activation of cholinergic receptors, retrograde signaling by NO controls CB1 cannabinoid receptor (CB1R)-dependent depolarization-induced suppression of inhibition (DSI). Spontaneously occurring IPSCs were recorded in CA1 pyramidal neurons in the presence of carbachol, and DSI was induced by a 1-s-long depolarization step. We found that, in addition to the inhibition of CB1Rs, blocking the NO signaling pathway at various points also disrupted DSI. Inhibitors of NO synthase (NOS) or NO-sensitive guanylyl cyclase (NO-sGC) diminished DSI, whereas a cGMP analog or an NO donor inhibited IPSCs and partially occluded DSI in a CB1R-dependent manner. Furthermore, an NO scavenger applied extracellularly or postsynaptically also decreased DSI, whereas L-arginine, the precursor for NO, prolonged it. DSI of electrically evoked IPSCs was also blocked by an inhibitor of NOS in the presence, but not in the absence, of carbachol. In line with our electrophysiological data, double immunohistochemical staining revealed an NO-donor-induced cGMP accumulation in CB1R-positive axon terminals. Using electron microscopy, we demonstrated the postsynaptic localization of neuronal NOS at symmetrical synapses formed by CB1R-positive axon terminals on pyramidal cell bodies, whereas NO-sGC was found in the presynaptic terminals. These electrophysiological and anatomical results in the hippocampus suggest that NO is involved in depolarization-induced CB1R-mediated suppression of IPSCs as a retrograde signal molecule and that operation of this cascade is conditional on cholinergic receptor activation.

Effects of elevated ecdysteroid on tissue expression of three guanylyl cyclases in the tropical land crab Gecarcinus lateralis: possible roles of neuropeptide signaling in the molting gland

Two eyestalk (ES) neuropeptides, molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH), increase intracellular cGMP levels in target tissues. Both MIH and CHH inhibit ecdysteroid secretion by the molting gland or Y-organ (YO), but apparently through different guanylyl cyclase (GC)-dependent pathways. MIH signaling may be mediated by nitric oxide synthase (NOS) and NO-sensitive GC. CHH binds to a membrane receptor GC. As molting affects neuropeptide signaling, the effects of ecdysteroid on the expression of the land crab Gecarcinus lateralis beta subunit of a NO-sensitive GC (Gl-GC-Ibeta), a membrane receptor GC (Gl-GC-II) and a NO-insensitive soluble GC (Gl-GC-III) were determined. Gl-GC-Ibeta isoforms differing in the absence or presence of an N-terminal 32-amino acid sequence and Gl-GC-III were expressed at higher mRNA levels in ES ganglia, gill, hepatopancreas, ovary and testis, and at lower levels in YO, heart and skeletal muscle. Three Gl-GC-II isoforms, which vary in the length of insertions (+18, +9 and +0 amino acids) within the N-terminal ligand-binding domain, differed in tissue distribution. Gl-GC-II(+18) was expressed highly in striated muscle (skeletal and cardiac muscles); Gl-GC-II(+9) was expressed in all tissues examined (ES ganglia, YO, gill, hepatopancreas, striated muscles and gonads); and Gl-GC-II(+0) was expressed in most tissues and was the dominant isoform in ES and thoracic ganglia. ES ablation, which increased hemolymph ecdysteroid, increased Gl-GC-II(+18) mRNA level in claw muscle. Using real-time RT-PCR, ES ablation increased Gl-GC-Ibeta, Gl-GC-III and ecdysone receptor mRNA levels in the YOs approximately ten-, approximately four- and approximately twofold, respectively, whereas Gl-GC-II mRNA level was unchanged. A single injection of 20-hydroxyecdysone into intact animals transiently lowered Gl-GC-Ibeta in hepatopancreas, testis and skeletal muscle, and certain Gl-GC-II isoforms in some of the tissues. These data suggest that YO and other tissues can modulate responses to neuropeptides by altering GC expression.

Aldosterone-induced increases in superoxide production counters nitric oxide inhibition of epithelial Na channel activity in A6 distal nephron cells

Oxygen radicals play an important role in signal transduction and have been shown to influence epithelial sodium channel (ENaC) activity. We show that aldosterone, the principal hormone regulating renal ENaC activity, increases superoxide (O2*) production in A6 distal nephron cells. Aldosterone (50 nM to 1.5 microM) induced increases in dihydroethidium fluorescence in a dose-dependent manner in confluent A6 epithelial cells. Using single-channel measurements, we showed that sequestering endogenous O2* (with the O2* scavenger 2,2,6,6-tetramethylpiperidine 1-oxyl) significantly decreased ENaC open probability from 0.10 +/- 0.03 to 0.03 +/- 0.01. We also found that increasing endogenous O2* in A6 cells, by applying a superoxide dismutase inhibitor, prevented nitric oxide (NO) inhibition of ENaC activity. ENaC open probability values did not significantly change from control values (0.23 +/- 0.05) after superoxide dismutase and 1.5 microM NO coincubation (0.21 +/- 0.04). We report that xanthine oxidase and hypoxanthine compounds increase local concentrations of O2* by approximately 30%; with this mix, an increase in ENaC number of channels times the open probability (from 0.1 to 0.3) can be achieved in a cell-attached patch. Our data also suggest that O2* alters NO activity in a cGMP-independent mechanism, since pretreating A6 cells with ODQ compound (a selective inhibitor of NO-sensitive guanylyl cyclase) failed to block 2,2,6,6-tetramethylpiperidine 1-oxyl inhibition of ENaC activity.

Guanylyl cyclases in the tropical land crab, Gecarcinus lateralis: Cloning of soluble (NO-sensitive and -insensitive) and membrane receptor forms

cDNAs encoding three guanylyl cyclases (GCs), which catalyze the production cGMP from GTP, were cloned from the blackback land crab, Gecarcinus lateralis: the β subunit of a NO-sensitive soluble GC (Gl-GC-Iβ; 2380 bp; 603 amino acids; 68,435 Da), a membrane receptor GC (Gl-GC-II; 4609 bp; 1349 amino acids; 150,999 Da), and a NO-insensitive soluble GC (Gl-GC-III; 1416 bp; 285 amino acids; 32,487 Da). All three have a highly-conserved catalytic domain located in the C-terminal region and are therefore likely to be enzymatically active. Gl-GCIβ contains heme/NO-binding (HNOB) and HNOB-associated domains characteristic of the catalytic subunit of NO-sensitive GCs. Gl-GC-II encodes a single-pass membrane protein containing ligand-binding (LB), transmembrane (TM), kinase homology (KH), and dimerization domains. Gl-GC-III is similar to Gl-GC-II but lacks the LB, TM, and most of the KH domains. Isoforms of Gl-GC-Iβ and Gl-GC-II appear to be generated by alternative splicing. Two Gl-GC-Iβ isoforms differed in the absence (Δ32N) or presence (Δ0N) of a 32-amino acid sequence at the N-terminus. The truncated form may not bind a heme group, but still would be able to dimerize with the alpha subunit to form a NO-insensitive enzyme. Three Gl-GC-II isoforms varied in length within the N-terminal ligand-binding domain (+ 18, + 9, and + 0 amino acid insertions). As GC-II is the putative receptor for crustacean hyperglycemic hormone (CHH), these data suggest that the isoforms differ in binding to CHH and related neuropeptides.

Hippocampal GABAergic Synapses Possess the Molecular Machinery for Retrograde Nitric Oxide Signaling

Nitric oxide (NO) plays an important role in synaptic plasticity as a retrograde messenger at glutamatergic synapses. Here we describe that, in hippocampal pyramidal cells, neuronal nitric oxide synthase (nNOS) is also associated with the postsynaptic active zones of GABAergic symmetrical synapses terminating on their somata, dendrites, and axon initial segments in both mice and rats. The NO receptor nitric oxide-sensitive guanylyl cyclase (NOsGC) is present in the brain in two functional subunit compositions: alpha1beta1 and alpha2beta1. The beta1 subunit is expressed in both pyramidal cells and interneurons in the hippocampus. Using immunohistochemistry and in situ hybridization methods, we describe that the alpha1 subunit is detectable only in interneurons, which are always positive for beta1 subunit as well; however, pyramidal cells are labeled only for beta1 and alpha2 subunits. With double-immunofluorescent staining, we also found that most cholecystokinin- and parvalbumin-positive and smaller proportion of the somatostatin- and nNOS-positive interneurons are alpha1 subunit positive. We also found that the alpha1 subunit is present in parvalbumin- and cholecystokinin-positive interneuron terminals that establish synapses on somata, dendrites, or axon initial segments. Our results demonstrate that NOsGC, composed of alpha1beta1 subunits, is selectively expressed in different types of interneurons and is present in their presynaptic GABAergic terminals, in which it may serve as a receptor for NO produced postsynaptically by nNOS in the very same synapse.

Cannabinoid receptor-mediated translocation of NO-sensitive guanylyl cyclase and production of cyclic GMP in neuronal cells

Cannabinoid agonists regulate NO and cyclic AMP production in N18TG2 neuroblastoma cells, leading to the hypothesis that neuronal cyclic GMP production could be regulated by CB1 cannabinoid receptors. NO (nitric oxide)-sensitive guanylyl cyclase (GC) is a heterodimeric cytosolic protein that mediates the down-stream effects of NO. Genes of proteins in the cyclic GMP pathway (α1, α2, and β1 subunits of NO-sensitive GC and PKG1, but not PKG2) were expressed in N18TG2 cells, as was the CB1 but not the CB2 cannabinoid receptor. Stimulation of N18TG2 cells by cannabinoid agonists CP55940 and WIN55212-2 increased cyclic GMP levels in an ODQ-sensitive manner. GC-β1 in membrane fractions was increased after 5 or 20min stimulation, and was significantly depleted in the cytosol by 1h. The cytosolic pool of GC-β1 was replenished after 48h of continued cannabinoid drug treatment. Translocation of GC-β1 from the cytosol was blocked by the CB1 antagonist rimonabant (SR141716) and by the Gi/o inactivator pertussis toxin, indicating that the CB1 receptor and Gi/o proteins are required for translocation. Long-term treatment with rimonabant or pertussis toxin reduced the amount of GC-β1 in the cytosolic pool. We conclude that CB1 receptors stimulate cyclic GMP production and that intracellular translocation of GC from cytosol to the membranes is intrinsic to the mechanism and may be a tonically active or endocannabinoid-regulated process.