Soluble Guanylyl Cyclase Inhibitor Research Articles

Nitric oxide attenuates overexpression of Giα proteins in vascular smooth muscle cells from SHR: Role of ROS and ROS-mediated signaling.

Vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) exhibit decreased levels of nitric oxide (NO) that may be responsible for the overexpression of Giα proteins that has been shown as a contributing factor for the pathogenesis of hypertension in SHR. The present study was undertaken to investigate if increasing the intracellular levels of NO by NO donor S-Nitroso-N-acetyl-DL-penicillamine (SNAP) could attenuate the enhanced expression of Giα proteins in VSMC from SHR and explore the underlying mechanisms responsible for this response. The expression of Giα proteins and phosphorylation of ERK1/2, growth factor receptors and c-Src was determined by Western blotting using specific antibodies. Treatment of VSMC from SHR with SNAP for 24 hrs decreased the enhanced expression of Giα-2 and Giα-3 proteins and hyperproliferation that was not reversed by 1H (1, 2, 4) oxadiazole (4, 3-a) quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, however, PD98059, a MEK inhibitor restored the SNAP-induced decreased expression of Giα proteins towards control levels. In addition, the increased production of superoxide anion, NAD(P)H oxidase activity, overexpression of AT1 receptor, Nox4, p22phox and p47phox proteins, enhanced levels of TBARS and protein carbonyl, increased phosphorylation of PDGF-R, EGF-R, c-Src and ERK1/2 in VSMC from SHR were all decreased to control levels by SNAP treatment. These results suggest that NO decreased the enhanced expression of Giα-2/3 proteins and hyperproliferation of VSMC from SHR by cGMP-independent mechanism and involves ROS and ROS-mediated transactivation of EGF-R/PDGF-R and MAP kinase signaling pathways.

Pancreatic-β-cell survival and proliferation are promoted by protein kinase G type Iα and downstream regulation of AKT/FOXO1.

Early studies showed nitric oxide as a pro-inflammatory-cytokine-induced toxin involved in pancreatic β-cell destruction during pathogenesis of type-1 diabetes. However, nitric oxide has both cytotoxic and cytoprotective effects on mammalian cells, depending on concentration and micro-environmental surroundings. Our studies have shown that low/physiological-level nitric oxide selectively activates protein kinase G type Iα isoform, promoting cytoprotective/pro-cell-survival effects in many cell types. In bone marrow-derived stromal/mesenchymal stem cells, protein kinase G type Iα mediates autocrine effects of nitric oxide and atrial natriuretic peptide, promoting DNA-synthesis/proliferation and cell survival. In this study, endothelial nitric oxide synthase/neuronal nitric oxide synthase inhibitor L-NIO (L-N(5)-(1-iminoethyl)ornithine), soluble guanylyl cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3,-a] quinoxalin-1-one), atrial natriuretic peptide-receptor inhibitor A71915 and protein kinase G type Iα kinase activity inhibitor DT-2 all increased apoptosis and decreased insulin secretion in RINm5F pancreatic β-cells, suggesting autocrine regulatory role for endogenous nitric oxide- and atrial natriuretic peptide-induced activation of protein kinase G type Iα. In four pancreatic β-cell lines, Beta-TC-6, RINm5F, INS-1 and 1.1B4, protein kinase G type Iα small-interfering RNA decreased phospho-serine-239-VASP (indicator of endogenous protein kinase G type Iα kinase activity), increased apoptosis and decreased proliferation. In protein kinase G type Iα-knockdown β-cell lines, expressions of phospho-protein kinase B (PKB/AKT) (AKT), phospho-Forkhead box protein O1 (FOXO1) (transcriptional repressor of pancreas duodenum homobox-1) and pancreas duodenum homobox-1 were decreased, suppressing proliferation and survival in pancreatic β-cells. The data suggest autocrine nitric oxide/atrial natriuretic peptide-induced activation of protein kinase G type Iα/p-AKT/p-FOXO1 promotes survival and proliferation in pancreatic β-cells, providing therapeutic implications for development of new therapeutic agents for diabetes.

Inhibitory action of oxytocin on spontaneous contraction of rat distal colon by nitrergic mechanism: involvement of cyclic GMP and apamin-sensitive K+ channels.

The mechanisms underlying the inhibitory effects of oxytocin (OT) on colon tone are not totally understood. We explore the mechanisms of OT on spontaneous contractility in rat distal colon and identify the mediators involved in this action. In rat distal colon strips, mechanical activity was analysed and the production of nitric oxide (NO) in tissue loaded with the fluorochrome DAF-FM was visualized by confocal microscopy. OT receptor (OTR) expression was determined by Western blotting and immunofluorescence. In rat distal colon, OT produced a concentration-dependent reduction in the spontaneous contraction, which was abolished by the OTR antagonist atosiban, the neural blocker tetrodotoxin and the inhibitor of neuronal nitric oxide synthase (nNOS) NPLA. The inhibitory effects of OT were not affected by propranolol, atropine, the nicotinic cholinoceptor blocker hexamethonium, the vasoactive intestinal peptide receptor antagonist VIPHyb, the P2 purinoceptor antagonist PPADS, the adenosine A1 receptors antagonist DPCPX and the prostacyclin receptor antagonist Ro1138452. The soluble guanylyl cyclase (sGC) inhibitor ODQ and the small conductance Ca2+ -activated K+ (Ca K+ ) channels blocker apamin significantly reduced the relaxation induced by OT, nicotine, sodium nitroprusside and the sGC activator BAY 41-2272. The neural release of NO elicited by OT was prevented by NPLA, tetrodotoxin and atosiban. The presence of the OTR and its co-localization with nNOS was detected by immunohistochemistry and Western blotting experiments. These results demonstrate the NO release from enteric neurones induced by activation of OTR mediates distal colon relaxation. sGC and small conductance Ca K+ channels are involved in this relaxation.

The NO-cGMP-PKG signal transduction pathway is involved in the analgesic effect of early hyperbaric oxygen treatment of neuropathic pain

BackgroundHyperbaric oxygen (HBO) has the potential to relieve neuropathic pain. The purpose of this study was to determine whether the NO-cGMP-PKG signaling pathway is involved in the analgesic effects of early hyperbaric oxygen treatment of neuropathic pain in rats.MethodsRats were randomly grouped for establishment of chronic constriction injury (CCI) models. Intrathecal catheters were inserted and 2.5ATA HBO therapy was administered from day 1 post-surgery for 60 minutes daily, continuously for 5 days; menstruum NS, DMSO, NO synthase(NOS) nonspecific inhibitor (L-NAME), soluble guanylyl cyclase(sGC) inhibitor (ODQ) and protein kinase G(PKG) inhibitor (KT5823) were administered intrathecally 30 minutes prior to HBO therapy. Pain-related behaviors in rats were observed at specific time points. Western blot and real-time RT-PCR were used to observe the expressions of PKG1 mRNA and protein in the spinal dorsal horn.ResultsCompared with the CCI group, HBO could significantly relieve mechanical and thermal hyperalgesia in rats. After intrathecal administration of L-NAME, ODQ and KT5823, effects of HBO on relieving hyperalgesia in rats were reversed (P < 0.05 vs. HBO), and expression of PKG1 mRNA and protein decreased in the spinal dorsal horn of the animals (P < 0.05 vs. HBO).ConclusionsEarly HBO therapy could significantly improve symptoms of hyperalgesia of neuropathic pain in rats, possibly via activation of the NO-cGMP-PKG signaling transduction pathway.

NO production and potassium channels activation induced by Crotalus durissus cascavella underlie mesenteric artery relaxation

Animal toxins are natural resources for pharmacological studies. The venom of Crotalus durissus cascavella (C.d. cascavella) may be a source in the bio-prospecting of new anti-hypertensive agents. The aim of this study was to investigate vascular effects of the venom of C.d. cascavella in normotensive rats. Studies were performed using isolated mesenteric artery segments and aortic endothelial cells. The cumulative administration of the venom of C.d. cascavella (0.001–30 μg/mL) on phenylephrine (Phe; 10 μM) pre-contracted rings induced a concentration-dependent vasorelaxation in the presence of vascular endothelium (Emax = 47.9 ± 5.0% n = 8), and its effect was almost abolished in the absence of endothelium (Emax = 5.8± 2.4% n = 5 (∗∗∗p < 0.001)). Tissue viability was maintained as there was no difference in the contractile capacity of rings before and after the administration of venom. The vasorelaxant effect of the venom was also abolished when arteries were pre-contracted with potassium chloride (KCl; 80 mM) (Emax = 6.4± 0.9% n = 5, ∗∗∗p < 0.001). When assessing the participation of endothelium-derived relaxing factors, it was noted that non-selective COX inhibition with indomethacin (10 μM) caused a significant reduction in the vasorelaxant effect of C.d. cascavella (*p < 0.05). When investigating the participation of NO released by endothelium, there was a significant reduction of the vasorelaxant effect of venom in rings treated with L-NAME (100 μM; Emax = 17.5± 2.2% n = 6; **p < 0.01). Similar results were noted in the presence of ODQ (10 μM), an inhibitor of soluble guanylyl cyclase (Emax = 11.2± 3.5%, n = 6) and PTIO (100 μM), a stable radical scavenger for nitric oxide (Emax = 10.77± 3.6%, n = 6). Moreover, the venom induced the release of NO by isolated aortic endothelial cells through amperometric studies. When assessing the participation of K+ channels on the vasodilatory response of the venom, tyrode solution with 20 mM of KCl caused a significant reduction in the relaxation response (p < 0.001) (Emax = 21.3 ± 8%, n = 7), as did inhibitor of delayed rectifier K+ channels (4-amynopiridine 1 mM; Emax = 9.5 ± 1.3, %, n = 5, ***p < 0.001), and vasorelaxation was almost abolished in the presence of Iberiotoxin (IbTx 100 nM). Therefore, these results suggest that the venom of C.d. cascavella induces vasorelaxation in superior mesenteric artery rings of normotensive rats in an endothelium-dependent manner. Specifically, the venom stimulates the generation of endothelium-derived relaxing factors, especially NO, and activates vascular smooth muscle hyperpolarization through K+ channels. These data illustrate that C.d. cascavella is a source of bioactive molecules and therefore has therapeutic potential in the treatment of cardiovascular diseases such as hypertension.

Salvia fruticosa Induces Vasorelaxation In Rat Isolated Thoracic Aorta: Role of the PI3K/Akt/eNOS/NO/cGMP Signaling Pathway

Salvia fruticosa (SF) Mill. is traditionally used for its antihypertensive actions. However, little is known about its pharmacologic and molecular mechanisms of action. Here we determined the effects of an ethanolic extract of SF leaves on rings of isolated thoracic aorta from Sprague-Dawley rats. Our results show that SF extract increased nitric oxide production and relaxed endothelium-intact rings in a dose-dependent (0.3 µg/ml–1 mg/ml) manner, and the maximum arterial relaxation (Rmax) was significantly reduced with endothelium denudation. Pretreatment of endothelium-intact rings with L-NAME (a non-selective inhibitor of nitric oxide synthase, 100 µM), or ODQ (an inhibitor of soluble guanylyl cyclase, 10 µM) significantly diminished SF-mediated vasorelaxation. Furthermore, SF induced Akt phosphorylation as well as increased cGMP levels in rings treated with increasing doses of SF. Prior exposure to PI3K inhibitors, wortmannin (0.1 µM) or LY294002 (10 µM), decreased cGMP accumulation and attenuated the SF-induced vasorelaxation by approximately 50% (Rmax). SF-evoked relaxation was not affected by indomethacin, verapamil, glibenclamide, tetraethylammonium, pyrilamine or atropine. Taken together, our results indicate that SF induces endothelium-dependent vasorelaxation through the PI3K/Akt/eNOS/NO/sGC/cGMP signaling pathway. Our data illustrate the health-orientated benefits of consuming SF which may act as an antihypertensive agent to reduce the burden of cardiovascular complications.

Platelet inhibition and increased phosphorylated vasodilator-stimulated phosphoprotein following sodium nitrite inhalation

In the presence of red blood cells (RBCs), nitrite inhibits platelets through its conversion to nitric oxide (NO) by the reductase activity of partially deoxygenated hemoglobin. Inhaled sodium nitrite is being investigated as a therapy for pulmonary hypertension. Here, we measured platelet aggregation, P-selectin expression, platelet-leukocyte aggregates and phosphorylated vasodilator-stimulated phosphoprotein (P-VASPSer239) following sodium nitrite inhalation in healthy subjects. In vitro incubation of nitrite with deoxygenated whole blood showed an increase in P-VASPSer239, which was inhibited by ODQ, a soluble guanylyl cyclase (sGC) inhibitor. Immediately and 60 min after nitrite inhalation, P-VASPSer239 increased in platelets. Platelet aggregation, P-selectin expression, platelet-monocyte and platelet-lymphocyte aggregates decreased after inhalation. In conclusion, sodium nitrite administered to healthy subjects by inhalation can inhibit platelet activation and increase P-VASPSer239 in platelets. Platelet inhibition by nitrite administration may be useful in disorders associated with platelet hyperactivity.

L-Citrulline ameliorates cerebral blood flow during cortical spreading depression in rats: Involvement of nitric oxide- and prostanoids-mediated pathway

l-Citrulline is a potent precursor of l-arginine, and exerts beneficial effect on cardiovascular system via nitric oxide (NO) production. Migraine is one of the most popular neurovascular disorder, and imbalance of cerebral blood flow (CBF) observed in cortical spreading depression (CSD) contributes to the mechanism of migraine aura. Here, we investigated the effect of l-citrulline on cardiovascular changes to KCl-induced CSD. in rats. Intravenous injection of l-citrulline prevented the decrease in CBF, monitored by laser Doppler flowmetry, without affecting mean arterial pressure and heart rate during CSD. Moreover, l-citrulline attenuated propagation velocity of CSD induced by KCl. The effect of l-citrulline on CBF change was prevented by l-NAME, an inhibitor of NO synthase, but not by indomethacin, an inhibitor of cyclooxygenase. On the other hand, attenuation effect of l-citrulline on CSD propagation velocity was prevented not only by l-NAME but also by indomethacin. In addition, propagation velocity of CSD was attenuated by intravenous injection of NOR3, a NO donor, which was diminished by ODQ, an inhibitor of soluble guanylyl cyclase. These results suggest that NO/cyclic GMP- and prostanoids-mediated pathway differently contribute to the effect of l-citrulline on the maintenance of CBF.

Fermented garlic extract ameliorates monocrotaline-induced pulmonary hypertension in rats

This study investigated the effect of fermented garlic extract (FGE) on pulmonary hypertension in monocrotaline (MCT)-treated rats. Sprague-Dawley rats were subcutaneously injected with either 50mg/kg MCT or a vehicle 3days after the FGE feeding (0.97mg nitrite/ml/day) was initiated. MCT treatment increased the weight, systolic pressure, and atrial natriuretic peptide concentration in the right ventricle but not in the left ventricle. FGE feeding attenuated these effects as well as the endothelial damage and medial hypertrophy of the pulmonary arterioles and the pulmonary fibrosis induced by MCT. These FGE effects were blocked by a soluble guanylyl cyclase (sGC) inhibitor. Increases in VCAM-1 and MMP-9 protein expressions, and decreases in PKG and eNOS protein expressions in the lung of MCT rats were attenuated by FGE feeding. These findings suggest that FGE ameliorates MCT-induced pulmonary hypertension by decreasing the inflammatory reaction via the NO–sGC–PKG pathway.

11,12-Epoxyeicosatrienoic acid induces vasodilator response in the rat perfused mesenteric vasculature.

Summary Epoxyeicosatrienoic acids (EETs) are endogenous ligands that undergo hydrolysis by soluble epoxide hydrolase (sEH). The responses of 11, 12‐EET in comparison with other vasodilator agonists including carbachol and sodium nitroprusside (SNP) were investigated. The effect of 1‐cyclohexyl‐3‐dodecyl urea (CDU), a sEH, was tested on the vasodilator effect induced by 11, 12‐EET in the perfused mesenteric beds isolated from normo‐glycaemic and type‐1 STZ‐diabetic rats.In the perfused mesenteric beds of control and diabetic animals, 11, 12‐EET produced vasodilation in a dose‐dependent manner. The vasodilator response induced by 11, 12‐EET was significantly decreased in tissues obtained from diabetic animals, but this was significantly corrected through inhibition of sEH.The effects of nitric oxide synthase inhibitor, cyclo‐oxygenase inhibitor, specific potassium channel inhibitors, soluble guanylyl cyclase inhibitor and transient receptor potential channel V4 inhibitor, on vasodilator response to 11, 12‐EET were investigated.In tissues isolated from control animals, vasodilator responses to 11, 12‐EET were not inhibited by acute incubation with l‐NAME, l‐NAME with indomethacin, glibenclamide, iberiotoxin, charybdotoxin, apamin or ODQ.Incubation with the transient receptor potential channel V4 inhibitor ruthenium red caused significantly reduced vasodilator responses induced by 11, 12‐EET.In conclusion, results from this study indicate that 11, 12‐EET has a vasodilator effect in the perfused mesenteric bed, partly through activation of vanilloid receptor. A strategy to elevate the levels of EETs may have a significant impact in correcting microvascular abnormality associated with diabetes.

Regulation of Intracellular Calcium by Carbon Monoxide in Human Bronchial Epithelial Cells

Background/Aims: Carbon monoxide (CO) is an important autocrine/paracrine messenger involved in a variety of physiological and pathological processes. This study aimed to investigate the regulatory role of CO released by CO-releasing molecule-2 (CORM-2) in a P2Y receptor-mediated calcium-signaling pathway in the human bronchial epithelial cell line, 16HBE14o-. Methods: Intracellular calcium ([Ca²⁺]_i) was measured by fura-2 microspectrofluorimetry. D-myo-inositol-1-phosphate (IP₁) levels and cGMP-dependent protein kinase activity (PKG) were also quantified. Results: The exogenous application of CORM-2 increased both intracellular Ca²⁺ and IP₁, which are inhibited by U73122, a phospholipase C (PLC) inhibitor. In contrast, the P2Y₂/P2Y₄ receptor-mediated intracellular Ca²⁺ release and influx induced by UTP were inhibited in the presence of CORM-2. However, CORM-2 did not affect the store-operated Ca²⁺ entry (SOCE) induced by thapsigargin (Tg). Moreover, the inhibitory effect of CORM-2 on UTP-induced calcium increase could be attenuated by a soluble guanylyl cyclase (sGC) inhibitor, 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), or a Protein Kinase G (PKG) inhibitor, KT5823, suggesting the involvement of sGC/PKG signaling in this process. Conclusion: CORM-2 serves a dual role in modulating [Ca²⁺]_i in 16HBE14o- cells. Thus, CO released by CORM-2 may act as a regulator of calcium homeostasis in human airway epithelia. These findings help further elucidate the function of CO in many physiological and pathological conditions.

Cross-talking between lymphocytes and platelets and its regulation by nitric oxide and peroxynitrite in physiological condition and endotoxemia

AimsCross-talk between platelets and lymphocytes may play a role in different pathological conditions like sepsis. This study aimed to investigate the effect of lymphocytes on platelet aggregation in lipopolysaccharide (LPS)-stimulated and non-stimulated cells. Main methodsLymphocytes and platelet-rich plasma (PRP) were obtained from rat arterial blood. Platelets (1.2×108platelets/ml) were incubated with lymphocytes (0.8×106cells/ml) in the presence or not of LPS (100μg/ml), after which ADP (5μM)-induced platelet aggregation was carried out. Key findingsLymphocytes inhibited by 51% the platelet aggregation, which was significantly prevented by the non-selective NO inhibitor l-NAME (300μM) or the selective iNOS inhibitor 1400W (100μM), as well as by the soluble guanylyl cyclase (sGC) inhibitor ODQ (10μM). The platelet inhibition by lymphocytes was accompanied by 2-fold increase of intraplatelet cGMP levels. Next, lymphocytes and platelets were co-incubated with LPS for 6h. In LPS-treated cells, lymphocytes produced a larger inhibition of platelet aggregation (62%), despite the same elevation of cGMP levels (2.2-fold increase). This inhibitory effect was prevented by l-NAME and 1400W, but rather unaffected by ODQ. The peroxynitrite (ONOO−) scavenger −(−)epigallocatechin gallate (ECG, 100μM) abolished the inhibition by lymphocytes on platelet aggregation in LPS-treated cells, but not in non-treated cells. SignificanceOur results show that lymphocytes act to inhibit platelet aggregation via iNOS-derived NO release and cGMP generation. In presence of LPS, ONOO− production accounts for the platelet inhibition.

Vascular Tone Regulation Induced by C-Type Natriuretic Peptide: Differences in Endothelium-Dependent and -Independent Mechanisms Involved in Normotensive and Spontaneously Hypertensive Rats.

Given that the role of C-type natriuretic peptide (CNP) in the regulation of vascular tone in hypertensive states is unclear, we hypothesized that impaired response of the nitric oxide system to CNP in spontaneously hypertensive rats (SHR) could affect vascular relaxation induced by the peptide in this model of hypertension, and that other endothelial systems or potassium channels opening could also be involved. We examined the effect of CNP on isolated SHR aortas, and the hindlimb vascular resistance (HVR) in response to CNP administration compared to normotensive rats. Aortas were mounted in an isometric organ bath and contracted with phenylephrine. CNP relaxed arteries in a concentration-dependent manner but was less potent in inducing relaxation in SHR. The action of CNP was diminished by removal of the endothelium, inhibition of nitric oxide synthase by Nω-nitro-L-arginine methyl ester, and inhibition of soluble guanylyl cyclase by 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one in both groups. In contrast, blockade of cyclooxygenase or subtype 2 bradykinin receptor increased CNP potency only in SHR. In both Wistar and SHR, CNP relaxation was blunted by tetraethylammonium and partially inhibited by BaCl2 and iberiotoxin, indicating that it was due to opening of the Kir and BKCa channels. However, SHR seem to be more sensitive to Kir channel blockade and less sensitive to BKCa channel blockade than normotensive rats. In addition, CNP decreases HVR in Wistar and SHR, but the effect of CNP increasing blood flow was more marked in SHR. We conclude that CNP induces aorta relaxation by activation of the nitric oxide system and opening of potassium channels, but the response to the peptide is impaired in conductance vessel of hypertensive rats.

355 - Approaches for Overcoming Diabetes-Induced Cardiovascular Nitric Oxide Resistance

In patients with cardiovascular disease, impaired nitric oxide (NO●) signalling is an independent predictor of poor outcomes, including mortality. This loss of NO●-responsiveness (termed ‘NO●-resistance’) is particularly debilitating in diabetes, where cardiovascular emergencies (acute myocardial infarction [MI], transient ischaemia, cardiogenic shock) occur more frequently, but NO●-based pharmacotherapies are unable to effectively counteract platelet aggregation and vasoconstriction. Further, the extent of NO●-resistance appears critically dependent on concomitant circulating levels of blood glucose. We have now examined whether the myocardium, like platelets and vessels, is also susceptible to NO●-resistance such that NO● can no longer enhance left ventricular (LV) relaxation. Hearts isolated from anaesthetised adult male streptozotocin (STZ) diabetic rats with severe (~30mM) or moderate hyperglycaemia(titrated to ~22mM with insulin), or non-diabetic shams were Langendorff-perfused. Following U46619 preconstriction of the coronary vasculature to 50%, dose-response curves to acute bolus doses of the NO● donor diethylamine NONOate (DEA-NO 10pmol-10μmol) were performed. Results were compared to those obtained in response to a donor of the NO● redox sibling nitroxyl, isopropylamine NONOate (IPA-NO). Using the selective inhibitor of soluble guanylyl cyclase, ODQ (10µM), the relative contribution of cGMP to these haemodynamic responses under normoglycaemia, moderate and severe hyperglycaemia were also elucidated. Our results demonstrated that diabetes impairs NO●-mediated coronary vasodilatation, LV relaxation and LV contractile function. The impaired NO vasodilatation persisted in hearts from moderately hyperglycaemic rats. In contrast, nitroxyl-mediated vasodilatation was preserved, and the positive inotropic and lusitropic effects were further enhanced, in diabetic myocardium. In conclusion, we have demonstrated for the first time that the haemodynamic responses to nitroxyl are preserved or enhanced in diabetes, whereas those of NO● are attenuated. We have hence identified in nitroxyl an exciting potential strategy for circumventing impaired NO● signalling.

Nicorandil inhibits osteoclast differentiation in vitro

Nicorandil is a hybrid angina therapeutic agent that has nitric oxide (NO) action and the ability to open ATP-sensitive K+ channels (KATP channels). A transient increase in NO and intracellular Ca2+ has been demonstrated to be highly involved in the differentiation and activation of osteoclasts. The objective of this study was to verify that the pharmacological effect of nicorandil suppresses the differentiation process of osteoclasts in vitro.Although little authentic NO production was detected in the culture medium in osteoclast formation assays, NO production increased only in the presence of nicorandil. The number of osteoclasts decreased markedly at late time-points after nicorandil addition compared with the number at early time-points. Both the number of TRAP-positive multinucleated cells and the number of cells that obtained F-actin rings decreased in the presence of nicorandil in a concentration-dependent manner. The osteo assay showed that the bone resorption area was also reduced with nicorandil in a concentration-dependent manner. An inhibition recovery experiment was conducted by adding a soluble guanylyl cyclase (sGC) inhibitor (ODQ) and a KATP channel-opening inhibitor (glibenclamide) during the osteoclast formation process. In the inhibition recovery experiment, the inhibitory effect of nicorandil on osteoclastogenesis was blocked by the addition of ODQ and glibenclamide. These results suggest that both the NO and KATP channel-opening activity of nicorandil inhibit osteoclast differentiation. Further study of nicorandil may lead to the development of drugs for osteoporosis treatment.

Hydrogen sulfide donors alleviate itch secondary to the activation of type-2 protease activated receptors (PAR-2) in mice

Hydrogen sulfide (H2S) has been highlighted as an endogenous signaling molecule and we have previously found that it can inhibit histamine-mediated itching. Pruritus is the most common symptom of cutaneous diseases and anti-histamines are the usual treatment; however, anti-histamine-resistant pruritus is common in some clinical settings. In this way, the involvement of mediators other than histamine in the context of pruritus requires new therapeutic targets. Considering that the activation of proteinase-activated receptor 2 (PAR-2) is involved in pruritus both in rodents and humans, in this study we investigated the effect of H2S donors on the acute scratching behavior mediated by PAR-2 activation in mice, as well as some of the possible pharmacological mechanisms involved. The intradermal injection of the PAR-2 peptide agonist SLIGRL-NH2 (8–80nmol) caused a dose-dependent scratching that was unaffected by intraperitoneal pre-treatment with the histamine H1 antagonist pyrilamine (30mg/kg). Co-injection of SLIGRL-NH2 (40nmol) with either the slow-release H2S donor GYY4137 (1 and 3nmol) or the spontaneous donor NaHS (1 and 0.3nmol) significantly reduced pruritus. Co-treatment with the KATP channel blocker glibenclamide (200nmol) or the nitric oxide (NO) donor sodium nitroprusside (10nmol) abolished the antipruritic effects of NaHS; however, the specific soluble guanylyl cyclase inhibitor ODQ (30μg) had no significant effects. The transient receptor potential ankyrin type 1 (TRPA1) antagonist HC-030031 (20μg) significantly reduced SLIGRL-NH2-induced pruritus; however pruritus induced by the TRPA1 agonist AITC (1000nmol) was unaffected by NaHS. Based on these data, we conclude that pruritus secondary to PAR-2 activation can be reduced by H2S, which acts through KATP channel opening and involves NO in a cyclic guanosine monophosphate (cGMP)-independent manner. Furthermore, TRPA1 receptors mediate the pruritus induced by activation of PAR-2, but H2S does not interfere with this pathway. These results provide additional support for the development of new therapeutical alternatives, mainly intended for treatment of pruritus in patients unresponsive to anti-histamines.

Inhibition of soluble guanylyl cyclase by small molecules targeting the catalytic domain

Soluble guanylyl cyclase (sGC) plays a crucial role in cyclic nucleotide signaling that regulates numerous important physiological processes. To identify new sGC inhibitors that may prevent the formation of the active catalytic domain conformation, we carried out an in silico docking screen targeting a 'backside pocket' of the inactive sGC catalytic domain structure. Compounds 1 and 2 were discovered to inhibit sGC even at high/saturating nitric oxide concentrations. Both compounds also inhibit the BAY 58-2667-activated sGC as well as BAY 41-2272-stimulated sGC activity. Additional biochemical analyses showed that compound 2 also inhibits the isolated catalytic domain, thus demonstrating functional binding to this domain. Both compounds have micromolar affinity for sGC and are potential leads to develop more potent sGC inhibitors.

Paracrine nitric oxide induces expression of cardiac sarcomeric proteins in adult progenitor cells through soluble guanylyl cyclase/cyclic-guanosine monophosphate and Wnt/β-catenin inhibition

Cardiac progenitor cells (CPC) from adult hearts can differentiate to several cell types composing the myocardium but the underlying molecular pathways are poorly characterized. We examined the role of paracrine nitric oxide (NO) in the specification of CPC to the cardiac lineage, particularly through its inhibition of the canonical Wnt/β-catenin pathway, a critical step preceding cardiac differentiation. Sca1 + CPC from adult mouse hearts were isolated by magnetic-activated cell sorting and clonally expanded. Pharmacologic NO donors increased their expression of cardiac myocyte-specific sarcomeric proteins in a concentration and time-dependent manner. The optimal time window for NO efficacy coincided with up-regulation of CPC expression of Gucy1a3 (coding the alpha1 subunit of guanylyl cyclase). The effect of paracrine NO was reproduced in vitro upon co-culture of CPC with cardiac myocytes expressing a transgenic NOS3 (endothelial nitric oxide synthase) and in vivo upon injection of CPC in infarcted hearts from cardiac-specific NOS3 transgenic mice. In mono- and co-cultures, this effect was abrogated upon inhibition of soluble guanylyl cyclase or nitric oxide synthase, and was lost in CPC genetically deficient in Gucy1a3. Mechanistically, NO inhibits the constitutive activity of the canonical Wnt/β-catenin in CPC and in cell reporter assays in a guanylyl cyclase-dependent fashion. This was paralleled with decreased expression of β-catenin and down-regulation of Wnt target genes in CPC and abrogated in CPC with a stabilized, non-inhibitable β-catenin. Exogenous or paracrine sources of NO promote the specification towards the myocyte lineage and expression of cardiac sarcomeric proteins of adult CPC. This is contingent upon the expression and activity of the alpha1 subunit of guanylyl cyclase in CPC that is necessary for NO-mediated inhibition of the canonical Wnt/β-catenin pathway.

Endothelium- and smooth muscle-dependent vasodilator effects of Citrus aurantium L. var. amara: Focus on Ca(2+) modulation.

Neroli, the essential oil of Citrus aurantium L. var. amara, is a well-characterized alleviative agent used to treat cardiovascular symptoms. However, because it has been found to have multiple effects, its mechanism of action requires further exploration. We sought to clarify the mechanism underlying the actions of neroli in mouse aorta. In aortic rings from mice precontracted with prostaglandin F2 alpha, neroli induced vasodilation. However, relaxation effect of neroli was decreased in endothelium-denuded ring or pre-incubation with the nitric oxide synthase inhibitor NG-Nitro-l-arginine-methyl ester (L-NAME). And also, neroli-induced relaxation was also partially reversed by 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ), a soluble guanylyl cyclase (sGC) inhibitor. In addition, neroli inhibited extracellular Ca(2+)-dependent, depolarization-induced contraction, an effect that was concentration dependent. Pretreatment with the non-selective cation channel blocker, Ni(2+), attenuated neroli-induced relaxation, whereas the K(+) channel blocker, tetraethylammonium chloride, had no effect. In the presence of verapamil, added to prevent Ca(2+) influx via smooth muscle voltage-gated Ca(2+) channels, neroli-induced relaxation was reduced by the ryanodine receptor (RyR) inhibitor ruthenium red. Our findings further indicate that the endothelial component of neroli-induced vasodilation is partly mediated by the NO-sGC pathway, whereas the smooth muscle component involves modulation of intracellular Ca(2+) concentration through inhibition of cation channel-mediated extracellular Ca(2+) influx and store-operated Ca(2+) release mediated by the RyR signaling pathway.

Mirabegron causes relaxation of human and rat corpus cavernosum: could it be a potential therapy for erectile dysfunction?

To examine the effects of mirabegron, a selective β3 -adrenoceptor agonist that has recently been approved for the treatment of overactive bladder (OAB), on erectile function. Stimulation of β3 -adrenoceptors localised in cavernosal smooth muscle cells may play a physiological role in mediating penile erection, and offer a beneficial pharmacological action for patients who have OAB and erectile dysfunction (ED). Corpus cavernosal (CC) specimens were obtained from patients with ED and Peyronie's disease undergoing penile prosthesis implantation. Erectile responses were also evaluated in vivo after intracavernosal injection (ICI) of mirabegron in anaesthetised rats. Mirabegron-elicited relaxation responses (10(-8) -10(-3) m) on phenylephrine-induced contraction were seen in human CC (HCC) and rat CC strips in isolated organ-bath studies. The effects of inhibitors, namely L-NAME [N(G) -nitro-L-arginine methyl ester, a competitive inhibitor of nitric oxide synthase (NOS), 100 μm], ODQ [1H-(1,2,4) oxadiazolo(4,3-α) quinoxalin-1-one, a soluble guanylyl cyclase (sGC) inhibitor, 30μm], methylene blue (a NOS and sGC inhibitor, 20μm), SR59230A (β3 -adrenoceptor blocker, 1 μm), and fasudil [Rho-associated protein kinase (ROCK) inhibitor, 0.1 μm], on mirabegron-induced relaxation responses were evaluated. Responses to mirabegron were compared with responses to isoprenaline and nebivolol. Immunohistochemistry was used to localise β3 -adrenoceptors and ROCK in CC smooth muscle cells. In vivo rat data were expressed as intracavernosal pressure (ICP)/mean arterial pressure, and total ICP. Mirabegron resulted in a relaxation of phenylephrine-evoked CC contractions in a concentration-dependent manner and SR59230A antagonised the mirabegron-induced relaxations in HCC and rat CC. Other inhibitors, L-NAME, ODQ, and methylene blue, did not affect the mirabegron-induced relaxation responses. Mirabegron relaxation responses at concentrations (0.1-10 μm) were enhanced by fasudil (ROCK inhibitor) in rat but not in HCC strips. KCl-induced contractions in HCC and rat CC were partially inhibited by mirabegron. In vivo, ICI of mirabegron (doses of 0.1-1 mg/kg) had a minor effect on ICP when compared with vehicle administration. Immunohistochemistry data showed β3 -adrenoceptors localised in the smooth muscle cells of the HCC and rat CC. Mirabegron markedly relaxed isolated CC strips by activating β3 -adrenoceptors independently of the NO-cGMP pathway. There is also evidence of the existence of a close functional link between β3 -adrenoceptors and the RhoA/ROCK pathway. These results may support further clinical studies using combinations of mirabegron with ROCK and phosphodiesterase type 5 inhibitors (PDE5i) for the treatment of ED, especially in patients who do not respond to PDE5i therapy.