Generation Of Cyclic Guanosine Monophosphate Research Articles

Expression of nitric oxide synthases in rat odontoblasts and the role of nitric oxide in odontoblastic differentiation of rat dental papilla cells.

As precursor cells of odontoblasts, dental papilla cells (DPCs) form the dentin-pulp complex during tooth development. Nitric oxide (NO) regulates the functions of multiple cells and organ tissues, including stem cell differentiation and bone formation. In this paper, we explored the involvement of NO in odontoblastic differentiation. We verified the expression of NO synthase (NOS) in rat odontoblasts by nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) staining and immunohistochemistry in vivo. The expression of all three NOS isoforms in rat DPCs was confirmed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), immunofluorescence, and western blotting in vitro. The expression of neuronal NOS and endothelial NOS was upregulated during the odontoblastic differentiation of DPCs. Inhibition of NOS function by NOS inhibitor l-NG -monomethyl arginine (L-NMMA) resulted in reduced formation of mineralized nodules and expression of dentin sialophosphoprotein (DSPP) and dentin matrix protein (DMP1) during DPC differentiation. The NO donor S-nitroso-N-acetylpenicillamine (SNAP, 0.1, 1, 10, and 100μM) promoted the viability of DPCs. Extracellular matrix mineralization and odontogenic markers expression were elevated by SNAP at low concentrations (0.1, 1, and 10μM) and suppressed at high concentration (100μM). Blocking the generation of cyclic guanosine monophosphate (cGMP) with 1H-(1,2,4)oxadiazolo-(4,3-a)quinoxalin-1-one (ODQ) abolished the positive influence of SNAP on the odontoblastic differentiation of DPCs. These findings demonstrate that NO regulates the odontoblastic differentiation of DPCs, thereby influencing dentin formation and tooth development.

The effect of long-acting PDE-5 inhibitor on renal ischemia/ reperfusion injury in Wistar rats; a study on serum cystatin C and renal histopathological findings

Background: Ischemia/reperfusion (I/R) is often a complication of bleeding shock, renal dysfunction and renal vessel operation. Nitric oxide (NO) as an important vasodilator is produced by endothelial cells. NO stimulates the generation of cyclic guanosine monophosphate (cGMP). Phosphodiesterase (PDE) is an intracellular enzyme which hydrolyzes cGMP into an inactive metabolite. It effectively decreases cGMP level. PDE is an intracellular enzyme which hydrolyzes cGMP into an inactive metabolite. It effectively decreases cGMP level. Therefore, an inhibition of PDE can increase cGMP level. PDE5 inhibitor is a compound which inhibits or acts antagonistically against biosynthesis or act of PDE. PDE5 inhibitor is now commonly used for the treatment of pulmonary artery hypertension and erectile dysfunction. According to some latest researches, long-acting PDE5 inhibitor (Tadalafil) reduces renal I/R injury in experiments with Wistar rats. Objectives: The purpose of this study was to determine the effect of long-acting PDE5 inhibitor on renal I/R injury in Wistar rats. Materials and Methods: Rats were divided into three groups; sham group, a right nephrectomy was performed. Control group, a right nephrectomy was performed followed by an occlusion on left renal pedicle for 60 minutes and a perfusion was performed for 60 minutes. Tadalafil group; the same treatment was performed as to group control, plus administering tadalafil as a PDE5 inhibitor (10 mg/kg), given by a nasogastric tube 60 minutes before the operation. A left nephrectomy was performed on the mice to determine the value of cystatin C level and histopathology. Results: The mean necrosis of tubular renal cells indicates that highest mean necrosis of tubular renal cells was at group control (mean score, 8.6±0.84), and the lowest mean necrosis of tubular renal cells was at sham group (mean score, 4.4±0.52) which indicates a significant difference between the sham and control groups (P<0.05). For the tadalafil group mean score of renal tubular necrosis cell was 6.9±1.45, which also indicates a significant difference between this group with sham group and control (P<0.05). Highest mean cystatin C levels related to group control, mean score was 1.51 ± 0.13 mg/dL, which indicates a significant difference with the sham group (P<0.05), but there is no significant difference with the tadalafil group. Conclusion: The results of this study showed that the administration of PDE5 inhibitor (tadalafil) improves reperfusion ischemic injury. Although it did not decrease the level of cystatin C, it significantly reduced tubular necrosis.

Riociguat: First Global Approval

Riociguat (Adempas(®)), an oral first-in-class soluble guanylate cyclase (sGC) stimulator, is under global development by Bayer Healthcare Pharmaceuticals Inc. for the treatment of adult patients with inoperable or chronic/persistent chronic thromboembolic pulmonary hypertension (CTEPH) and for the treatment of adult patients with pulmonary arterial hypertension (PAH). The drug directly stimulates sGC in a nitric oxide independent manner, thereby increasing the sensitivity of sGC to nitric oxide, leading to increased cyclic guanosine monophosphate generation (a key signalling molecule involved in regulating vascular tone, proliferation, fibrosis and inflammation). Riociguat is the world's first approved pharmacotherapy for CTEPH, with its first global approval in this indication occurring in Canada. It has subsequently been approved in the USA for the treatment of patients with CTEPH and also received its first global approval in patients with PAH in the USA. It is undergoing regulatory review for these indications in Europe and for use in patients with CTEPH in Japan. This article summarizes the milestones in the development of riociguat, leading to its first global approvals in patients with CTEPH and PAH.

Nitric Oxide Inhibits IP3R‐mediated Activation of TRPM4 in Cerebral Artery Smooth Muscle Cells

The melastatin transient receptor potential channel TRPM4 is essential for pressure‐induced smooth muscle cell contraction and is activated by Ca2+ released from the SR via inositol trisphosphate receptors (IP3R). Endothelium‐derived nitric oxide (NO) activates soluble guanylyl cyclase (sGC) leading to the generation of cyclic guanosine monophosphate (cGMP), activation of protein kinase G (PKG), and smooth muscle relaxation. We hypothesized that NO blocks IP3R‐dependent activation of TRPM4 channels in cerebral artery myocytes. Using perforated whole‐cell patch clamp electrophysiology, we observe a dose‐dependent reduction in TRPM4 channel activity in response to the NO donor S‐Nitroso‐N‐Acetyl‐D,L‐Penicillamine (SNAP). TRPM4 channel activity is also diminished following exposure to the cell‐permeable cGMP analog dibutyryl‐cGMP. TRPM4 currents inhibited by NO or cGMP did not return following administration of the cell‐permeable IP3 analog, Bt‐IP3. In addition, the sGC inhibitor NS‐2028 prevented NO‐mediated inhibition of TRPM4 channel activity. These findings suggest that NO can relax cerebral artery myocytes by blocking IP3R‐mediated activation of TRPM4. RO1HL091905; F31HL094145–01 (AG)

Linaclotide: A new drug for the treatment of chronic constipation and irritable bowel syndrome with constipation.

Linaclotide is the first member of a novel class of drugs to be extensively evaluated for the treatment of chronic constipation (CC) and irritable bowel syndrome with constipation (IBS-C). To provide a comprehensive overview of the current state of knowledge on linaclotide, its pharmacological properties, mode of action and efficacy in clinical trials to date. We conducted a systematic review of the literature. The survey revealed that linaclotide is a minimally absorbed, 14-amino acid peptide which acts in the intestinal lumen on guanylate cyclase-C (GC-C). This results in generation of cyclic guanosine monophosphate (cGMP), which stimulates chloride secretion, resulting in increased luminal fluid secretion and an acceleration of intestinal transit. In animal models, linaclotide also decreased visceral hypersensitivity. Linaclotide softened stool and increased transit in CC and in IBS-C. Phase II and phase III clinical studies established efficacy of linaclotide in CC (linaclotide 145 µg daily approved in the United States for CC) and in IBS-C (linaclotide 290 µg daily US Food and Drug Administration-approved for IBS-C, with favourable recommendation for the European Medicines Agency Committee for Medicinal Products for Human Use (CHMP). Linaclotide showed a favourable safety profile, and the main treatment-emerging adverse event was diarrhea, leading to discontinuation rates of up to 5%. Linaclotide is an important addition to the therapeutic possibilities for treating IBS-C and CC.

Transcriptional and post-transcriptional regulation of cGMP-dependent protein kinase (PKG-I): pathophysiological significance.

The ability of the endothelium to produce nitric oxide, which induces generation of cyclic guanosine monophosphate (cGMP) that activates cGMP-dependent protein kinase (PKG-I), in vascular smooth muscle cells (VSMCs), is essential for the maintenance of vascular homeostasis. Yet, disturbance of this nitric oxide/cGMP/PKG-I pathway has been shown to play an important role in many cardiovascular diseases. In the last two decades, in vitro and in vivo models of vascular injury have shown that PKG-I is suppressed following nitric oxide, cGMP, cytokine, and growth factor stimulation. The molecular basis for these changes in PKG-I expression is still poorly understood, and they are likely to be mediated by a number of processes, including changes in gene transcription, mRNA stability, protein synthesis, or protein degradation. Emerging studies have begun to define mechanisms responsible for changes in PKG-I expression and have identified cis- and trans-acting regulatory elements, with a plausible role being attributed to post-translational control of PKG-I protein levels. This review will focus mainly on recent advances in understanding of the regulation of PKG-I expression in VSMCs, with an emphasis on the physiological and pathological significance of PKG-I down-regulation in VSMCs in certain circumstances.

C-Type Natriuretic Peptide Protects the Retinal Pigment Epithelium against Advanced Glycation End Product–Induced Barrier Dysfunction

In diabetic retinopathy, vision loss is usually secondary to macular edema, which is thought to depend on the functional integrity of the blood-retina barrier. The levels of advanced glycation end products in the vitreous correlate with the progression of diabetic retinopathy. Natriuretic peptides (NP) are expressed in the eye and their receptors are present in the retinal pigment epithelium (RPE). Here, we investigated the effect of glycated-albumin (Glyc-alb), an advanced glycation end product model, on RPE-barrier function and the ability of NP to suppress this response. Transepithelial electrical resistance (TEER) measurements were used to assess the barrier function of ARPE-19 and human fetal RPE (hfRPE) monolayers. The monolayers were treated with 0.1-100 μg/ml Glyc-alb in the absence or presence of 1 pM to 100 nM apical atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), or C-type natriuretic peptide (CNP). Glyc-alb induced a significant reduction in TEER within 2 hours. This response was concentration-dependent (EC(50)= 2.3 μg/ml) with a maximal reduction of 40 ± 2% for ARPE-19 and 27 ± 7% for hfRPE at 100 μg/ml 6 hours post-treatment. One hour pretreatment with ANP, BNP, or CNP blocked the reduction in TEER induced by Glyc-alb (100 μg/ml). The suppression of the Glyc-alb response by NP was dependent on the generation of cyclic guanosine monophosphate and exhibited a rank order of agonist potency consistent with the activation of natriuretic-peptide-receptor-2 (NPR2) subtype (CNP >> BNP ≥ ANP). Our data demonstrate that Glyc-alb is effective in reducing RPE-barrier function, and this response is suppressed by NP. Moreover, these studies support the idea that NPR2 agonists can be potential candidates for treating retinal edema in diabetic patients.

Ramipril Sensitizes Platelets to Nitric Oxide: Implications for Therapy in High-Risk Patients

Using 2 sequential studies in HOPE (Heart Outcomes Prevention Evaluation) study-type patients, the aims of this study were: 1) to test the hypothesis that ramipril improves platelet nitric oxide (NO) responsiveness: and 2) to explore biochemical and physiological effects of ramipril in a cohort selected on the basis of platelet NO resistance. Ramipril prevents cardiovascular events, but the bases for these effects remain uncertain. NO resistance at both the platelet and vascular levels is present in a substantial proportion of patients with diabetes or ischemic heart disease and is an independent risk factor for cardiovascular events. Study 1 was a double-blind, randomized comparison of ramipril (10 mg) with placebo in a cohort of patients (n = 119) with ischemic heart disease or diabetes plus additional coronary risk factor(s), in which effects on platelet responsiveness to NO were compared. Study 2 was a subsequent short-term evaluation of the effects of ramipril in a cohort of subjects (n = 19) with impaired platelet NO responsiveness in whom additional mechanistic data were sought. In study 1, ramipril therapy increased platelet responsiveness to NO relative to the extent of aggregation (p < 0.001), but this effect occurred primarily in patients with severely impaired baseline NO responsiveness (n = 41). In study 2, ramipril also improved platelet NO responsiveness (p < 0.01), and this improvement was correlated directly with increased NO-stimulated platelet generation of cyclic guanosine monophosphate (p < 0.02) but not with changes in plasma thrombospondin-1 levels. Ramipril ameliorates platelet NO resistance in HOPE study-type patients, with associated increases in soluble guanylate cyclase responsiveness to NO. This effect is likely to contribute to treatment benefit and define patients in whom ramipril therapy is particularly effective.

Emerging role of PKA/eNOS pathway in therapeutic angiogenesis for ischaemic tissue diseases

Although an abundant amount of research has been devoted to the study of angiogenesis, its precise mechanisms are incompletely understood. Numerous clinical trials focused on therapeutic angiogenesis for the treatment of tissue ischaemia have not been as successful as those of preclinical studies. Thus, additional studies are needed to better understand critical molecular mechanisms regulating ischaemic neovascularization to identify novel therapeutic agents. Nitric oxide (NO) plays a central role in ischaemic neovascularization through the generation of cyclic guanosine monophosphate (cGMP) and the activation of several other signalling responses. Accumulated evidence suggests that endothelial protein kinase A/endothelial NO synthase (PKA/eNOS) signalling may play an important role in ischaemic disorders by promoting neovascularization. This review highlights recent advances in the role of the PKA/eNOS and NO-cGMP-kinase cascade pathway in ischaemic neovascularization. We also discuss molecular relationships of PKA/eNOS with other angiogenic pathways and explore the possibility of activation of the NO/nitrite endocrine system as potential therapeutic targets for ischaemic angiogenesis.

Urodilatin increases renal dopamine uptake: intracellular network involved

Dopamine and urodilatin promote natriuresis and diuresis through a common pathway that involves reversible deactivation of renal Na+, K+-ATPase. We have reported that urodilatin enhances dopamine uptake in outer renal cortex through the natriuretic peptide type A receptor. Moreover, urodilatin enhances dopamine-induced inhibition of Na+, K+-ATPase activity. The objective of the present work was to investigate the intracellular signals involved in urodilatin effects on dopamine uptake in renal cortex of kidney rats. We show that urodilatin-elicited increase in ³H-dopamine was blunted by methylene blue (10 μM), a non-specific guanylate cyclase inhibitor, and by phorbol-12-myristate-13-acetate (1 μM), a particulate guanylate cyclase inhibitor, but not by 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one (10 μM), a specific soluble guanylate cyclase inhibitor; therefore the involvement of particulate guanylate cyclase on urodilatin mediated dopamine uptake was confirmed. Cyclic guanosine monophosphate and proteinkinase G were also implicated in the signaling pathway, since urodilatin effects were mimicked by the analog 125 μM 8-Br-cGMP and blocked by the proteinkinase G-specific inhibitor, KT-5823 (1 μM). In conclusion, urodilatin increases dopamine uptake in renal cortex stimulating natriuretic peptide type A receptor, which signals through particulate guanylate cyclase activation, cyclic guanosine monophosphate generation, and proteinkinase G activation. Dopamine and urodilatin may achieve their effects through a common pathway that involves deactivation of renal Na+, K+-ATPase, reinforcing their natriuretic and diuretic properties.

Modulating cGMP in heart failure by novel natriuretic peptides

An emerging concept in cardiorenal therapeutics is the design and synthesis of chimeric drugs that are single-chemical entities that combine structural and biological components of different molecules resulting in novel agents possessing highly desirable properties. Such a strategy optimizes favorable actions of a least two different molecules and often reduces unwanted adverse effects. We recently reported the design, synthesis and biological actions of a novel chimeric natriuretic peptide (NP) fusing the 22-amino acid (AA) sequence of mature C-type natriuretic peptide (CNP), a member of the NP family that is highly conserved among species, with the 15-AA C-terminus of Dendroaspis natriuretic peptide (DNP) that was isolated from the venom of the eastern green mamba snake (Dendroaspis angusticeps). The rationale of the design of this novel chimeric NP was based upon our knowledge that CNP, principally an endothelial cell derived peptide, has favorable venorelaxing properties that would unload the heart in states of volume overload but when compared to atrial NP (ANP), B-type NP (BNP) or urodilatin (URO), which are marked arterial vasodilators, would be less hypotensive thus better preserving renal perfusion pressure. Such hemodynamic properties are a consequence of CNP activation of the NP receptor-B (NPR-B) that is highly expressed in veins as compared to arteries. In contrast, ANP and BNP which function via the NP receptor-A (NPR-A) principally vasorelax arteries where NPR-A is highly expressed. A therapeutic limitation of the use of CNP in volume overload states is that in contrast to ANP and BNP, CNP lacks renal actions limiting its utility in states of sodium and water retention or altered glomerular function. The second principle in the design of CD-NP was based upon our understanding of DNP. Indeed, previous studies from our laboratory have demonstrated that DNP is potently natriuretic and diuretic but also markedly hypotensive. More recently, studies have established that DNP like ANP and BNP is a ligand for NPR-A. Further, Lisy et al have reported that the 15-AA C-terminus of DNP possessed mild natriuretic and diuretic actions, which provided the rationale for the hypothesis that the C-terminus of DNP in a chimeric design would provide CNP with the ability to activate NPR-A and transform CNP into a natriuretic and diuretic peptide lacking significant hypotensive actions. Indeed, in recent studies in cells overexpressing either NPR-A or NPR-B, CD-NP activated both NPR-B and NPR-A with predominate stimulation being NPR-B. Specifically, CD-NP was able to activate in cells expressing either NPR-A or NPR-B the generation of cyclic guanosine monophosphate (cGMP) that is the second messenger for both NP receptors. Here we will review both preclinical development and the first clinical studies including first in humans and studies in human heart failure.

Saphenous vein harvest with the Mayo extraluminal dissector: Is endothelial function preserved?

Despite a significantly lower incidence of wound infection rates and compelling evidence that there is no difference in the degree of preservation of histologic architecture, vascular tone, or vasomotor reactivity of veins harvested with the Mayo dissector (Johnson and Johnson, Langhorne, PA) and a similar graft patency compared with the open method, use of the Mayo dissector still remains limited, probably because of a lack of evidence regarding the functional quality of the vein. In modern practice, although endoscopic vein harvest is gaining popularity, the Mayo dissector (Figure 1) still remains a satisfactory and inexpensive, albeit underused, alternative for vein harvesting, and hence we sought to examine the functional quality of the veins harvested with the Mayo extraluminal dissector. Nitric oxide (NO), a vasoactive molecule produced from the endothelium, is a marker of endothelial function. NO is associated with a diminished incidence of neointimal proliferation, producing these effects through stimulation of soluble guanylate cyclase and generation of cyclic guanosine monophosphate (cGMP). To compare the functional ability of harvested saphenous veins with the Mayo stripper and conventional open dissection, we measured cGMP production ex vivo after stimulation with specific activators of the NO–cGMP axis.

NO Signaling Through cGMP in Renal Tissue Fibrosis and Beyond: Key Pathway and Novel Therapeutic Target

Nitric oxide (NO) produced by endothelial NO synthase (NOS) in low concentrations is a unique messenger molecule with key homeostatic functions concerning the prevention of pathological vascular and tissue changes such as increases in blood pressure, platelet degranulation, mononuclear cell infiltration, cell proliferation and extracellular matrix protein accumulation. This is in contrast to high levels of NO derived from inducible NOS which act as detrimental effector molecules and free radicals in immune response. Deficiency in NO's protective signaling actions is a major characteristic in numerous experimental and human disease situations. The main function of the NO signaling pathway is activation of the soluble guanylate cyclase (sGC) enzyme with subsequent generation of cyclic guanosine monophosphate (cGMP) as a second messenger and downstream mediator. In the past, attempts to overcome deficiency in endothelial NO effects were focused primarily on increasing the supply with the NO precursor L-arginine or on the use of directly NO-releasing compounds. The clinical impact of these strategies, however, was rather limited. Recent state-of-the-art studies have revealed that NO signaling is highly regulated at the transcriptional level and that deficiency in NO signaling correlates closely with pathological changes. In parallel efforts, novel pharmacological compounds which specifically enhance NO/cGMP signaling have been developed and have demonstrated remarkable efficacy in experimental disease settings. In this review, we summarize the current state of knowledge on the impairment of NO/cGMP signaling and about its pharmacological stimulation. In the first part, experimental renal fibrosis, i.e. the tandem rat model of acute anti-thy1 glomerulonephritis and progressive anti-thy1 renal fibrosis will serve as a paradigm for introducing this new and exciting field. In the second part, we will address the most recent findings on NO signaling in non-renal diseases. Together, these results point out that deficiency in NO/cGMP is a common key pathway as well as a novel therapeutic target in a number of diseases.

Follistatin induction by nitric oxide through cyclic GMP: a tightly regulated signaling pathway that controls myoblast fusion

The mechanism of skeletal myoblast fusion is not well understood. We show that endogenous nitric oxide (NO) generation is required for myoblast fusion both in embryonic myoblasts and in satellite cells. The effect of NO is concentration and time dependent, being evident only at the onset of differentiation, and direct on the fusion process itself. The action of NO is mediated through a tightly regulated activation of guanylate cyclase and generation of cyclic guanosine monophosphate (cGMP), so much so that deregulation of cGMP signaling leads to a fusion-induced hypertrophy of satellite-derived myotubes and embryonic muscles, and to the acquisition of fusion competence by myogenic precursors in the presomitic mesoderm. NO and cGMP induce expression of follistatin, and this secreted protein mediates their action in myogenesis. These results establish a hitherto unappreciated role of NO and cGMP in regulating myoblast fusion and elucidate their mechanism of action, providing a direct link with follistatin, which is a key player in myogenesis.

Circulating Endothelial Microparticles Are Associated with Vascular Dysfunction in Patients with End-Stage Renal Failure

Endothelial dysfunction and arterial stiffness are major determinants of cardiovascular risk in patients with end-stage renal failure (ESRF). Microparticles are membrane fragments shed from damaged or activated cells. Because microparticles can affect endothelial cells, this study investigated the relationship between circulating microparticles and arterial dysfunction in patients with ESRF and identified the cellular origin of microparticles associated with these alterations. Flow cytometry analysis of platelet-free plasma from 44 patients with ESRF indicated that circulating levels of Annexin V+ microparticles were increased compared with 32 healthy subjects, as were levels of microparticles derived from endothelial cells (three-fold), platelets (16.5-fold), and erythrocytes (1.6-fold). However, when arterial function was evaluated noninvasively in patients with ESRF, only endothelial microparticle levels correlated highly with loss of flow-mediated dilation (r = -0.543; P = 0.004), increased aortic pulse wave velocity (r = 0.642, P < 0.0001), and increased common carotid artery augmentation index (r = 0.463, P = 0.0017), whereas platelet-derived, erythrocyte-derived, and Annexin V+ microparticle levels did not. In vitro, microparticles from patients with ESRF impaired endothelium-dependent relaxations and cyclic guanosine monophosphate generation, whereas microparticles from healthy subjects did not. Moreover, in vitro endothelial dysfunction correlated with endothelial-derived (r = 0.891; P = 0.003) but not platelet-derived microparticle concentrations. In fact, endothelial microparticles alone decreased endothelial nitric oxide release by 59 +/- 7% (P = 0.025). This study suggests that circulating microparticles of endothelial origin are tightly associated with endothelial dysfunction and arterial dysfunction in ESRF.

Novel therapeutic option in hypertensive crisis: sildenafil augments nitroprusside-induced hypotension

Sodium nitroprusside (SNP) produces hypotension by increasing nitric oxide-mediated generation of cyclic guanosine monophosphate (cGMP), which causes vasodilatation. On the other hand, sildenafil, a phosphodiesterase type 5 inhibitor (PDE5), increases cGMP by inhibiting its degradation by PDE5. Hence, theoretically, the two should be complementary. We present a case where the two agents were used together to manage a hypertensive crisis. This, to the best of our knowledge, is the first such case report worldwide.

The role of nitric oxide (NO) in control of LHRH release that mediates gonadotropin release and sexual behavior.

Nitric oxide (NO) plays a crucial role in reproduction at every level in the organism. In the brain, it activates the release of luteinizing hormone-releasing hormone (LHRH). The axons of the LHRH neurons project to the mating centers in the brain stem and by efferent pathways, evoke the lordosis reflex in female rats. In males, there is activation of NOergic terminals that release NO in the corpora cavernosa penis to induce erection by generation of cyclic guanosine monophosphate (cGMP). NO also activates the release of LHRH which reaches the pituitary and activates the release of gonadotropins by activating neural NO synthase (NOS) in the pituitary gland. Follicle stimulating hormone (FSH)RH selectively releases FSH also by activating NOS. Leptin releases LHRH by activating NOS to release FSH and LH with the same potency as LHRH. These actions are mediated by specific receptors on the gonadotropes for LHRH, FSHRH and leptin. The responsiveness of the pituitary is controlled by gonadal steroids. In the gonad, NO plays an important role inducing ovulation and in causing luteolysis; whereas in the reproductive tract, it relaxes uterine muscle via cGMP and constricts it by prostaglandins.

Serum levels of human growth hormone during different penile conditions in the cavernous and systemic blood of healthy men and patients with erectile dysfunction

Objectives. To detect changes in growth hormone (GH) serum levels during different penile conditions in the cavernous and systemic blood of patients with erectile dysfunction and compare them with the course of GH registered in healthy men. It has been suggested that human GH is involved in sexual maturation and plays a regulatory role in male reproductive function. Deficiency may result in fatigability, loss of sexual desire and erection, or oligospermia or azoospermia. It is assumed that the biologic effects of GH include insulin-like growth factor 1-mediated stimulation of endothelial nitric oxide formation. It has recently been demonstrated that GH serum levels in the systemic and cavernous blood of healthy men increases during developing penile erection. Methods. Thirty-five healthy adult men and 45 patients with erectile dysfunction of either organogenic or psychogenic etiology were exposed to visual and tactile erotic stimuli to elicit penile tumescence and, in the group of healthy subjects, rigidity. Whole blood was simultaneously aspirated from the corpus cavernosum and the cubital vein during the different functional conditions of the penis. Serum levels of GH were determined by means of an immunoradiometric assay. Results. In the healthy subjects, systemic GH serum levels significantly increased during penile tumescence, followed by a transient decline from tumescence to rigidity and detumescence. In the unselected patients, the mean GH levels during penile flaccidity were determined to be about sevenfold lower than those registered in the blood of the healthy men. During penile tumescence, the mean increase in the GH levels in the systemic and cavernous blood of psychogenic patients was comparable to that seen in healthy men, but, in the group of organogenic patients, this increase was found to be negligible. Conclusions. We believe our data provide strong evidence that GH may be of major importance in the maintenance of male erectile capability—probably through a stimulating effect on cyclic guanosine monophosphate generation in human cavernous smooth muscle—and that a decline in GH release may contribute to the manifestation of erectile dysfunction.

NO Inhibits Stretch-induced MAPK Activity by Cytoskeletal Disruption

Mesangial cells (MC) grown on extracellular matrix protein-coated plates and exposed to cyclic strain/relaxation proliferate and produce extracellular matrix protein, providing an in vitro model of signaling in stretched MC. Intracellular transduction of mechanical strain involves mitogen-activated protein kinases, and we have shown that p42/44 mitogen-activated protein kinase (extracellular signal-regulated kinase (ERK)) is activated by cyclic strain in MC. In vivo studies show that increased production of nitric oxide (NO) in the remnant kidney limits glomerular injury without reducing glomerular capillary pressure, and we have observed that NO attenuates stretch-induced ERK activity in MC via generation of cyclic guanosine monophosphate (cGMP). Accordingly, we sought to determine whether NO affects strain-induced ERK activity after strain and how this is mediated. Strain-induced ERK activity was dependent on time and magnitude of stretch and was maximal after 10 min at -27 kilopascals. Actin cytoskeleton disruption with cytochalasin D abrogated this. The non-metabolizable cGMP analogue 8-bromo cyclic GMP (8-Br-cGMP) dose-dependently attenuated strain-induced ERK activity. Cytoskeletal stabilization with jasplakinolide prevented this inhibitory effect of 8-Br-cGMP. Cyclic strain increased nuclear translocation of phospho-ERK by immunofluorescent microscopy, again attenuated by 8-Br-cGMP. Jasplakinolide prevented the inhibitory effect of 8-Br-cGMP on activated ERK nuclear translocation after strain. Strain increased ERK-dependent AP-1 nuclear protein binding, which was attenuated by cytochalasin D and 8-Br-cGMP. These data indicate that cGMP can inhibit cyclic strain-induced ERK activity, nuclear translocation, and AP-1 nuclear protein binding. Cytoskeletal disruption leads to the same effect, whereas cytoskeleton stabilization reverses the effect of 8-Br-cGMP. Thus, NO inhibits strain-induced ERK activity by cytoskeletal destabilization.

Generation of cyclic guanosine monophosphate by heme oxygenases in the human testis--a regulatory role for carbon monoxide in Sertoli cells?

Previous studies have demonstrated that cGMP is produced by nitric oxide-mediated activation of soluble guanylyl cyclase (sGC) in seminiferous tubules of the human testis. It is not known, however, whether carbon monoxide (CO), another activator of sGC, is also involved in testicular function. To address this issue, testicular probes from 65- to 75-yr-old men have been examined. The CO-generating enzyme, heme oxygenase-1 (HO-1), could be localized by immunohistochemical and immunoblot analyses to Sertoli cells. In these cells, HO-1 is detectable in adluminal cell compartments, whereas sGC immunoreactivity is distributed exclusively in basal compartments. Treatments of isolated tubules with either sodium arsenite, known to induce HO-1, or hematin, an HO substrate, resulted in 4.4- and 1.8-fold, respectively, increases in cGMP levels. ODQ, a specific sGC inhibitor, inhibited completely the sodium arsenite-stimulated cGMP production. Moreover, the HO inhibitor zinc protoporphyrin-IX and the CO scavenger hemoglobin both significantly reduced (77% or 46% of control, respectively) tubular cGMP generation. These findings, demonstrating for the first time a link between HO-1 activity in Sertoli cells and sGC-dependent cGMP production in seminiferous tubules, suggest a functional role of CO in the human testis.