Human platelets are constantly regulated by activating and inhibitory effectors. Thrombin,the most potent platelet agonist, induces signaling through the protease-activated receptors(PARs) 1 and 4 which in turn convey their signal by coupling to G-proteins. Nitric oxide (NO)is a potent platelet inhibitor continuously formed by the endothelium exerting its effect byincreasing cGMP through activation of soluble guanylyl cyclase (sGC). The purpose of thiswork has been to investigate how NO would affect platelets already activated by PARagonists.To examine the different contributions of the PAR1- and PAR4-signals, the selectiveagonist peptides SFLLRN and AYPGKF-NH2 were utilized. Aggregation, Ca2+-mobilization andphosphorylation of threonine 696 in myosin phosphatase target subunit 1 (MYPT1) wereanalyzed. Intriguingly PAR1-, but not PAR4-, agonist provoked aggregation was rapidlyreversed upon NO exposure. PAR-agonist induced Ca2+-mobilization was markedly reducedafter exposure to NO, however this Ca2+-suppression did not cause the disaggregation ofPAR1-agonist evoked platelet aggregation. The reversal of aggregation was suspected to becaused by a cGMP-mediated inhibition of the Rho-kinase/ROCK-signaling pathway. This wassupported by Westen blot analysis where a marked decrease of MYPT1 phosphorylationcompared to basal levels could be observed. In conclusion, NO was found to reverse humanplatelet aggregation evoked by PAR1-activation by inhibition of a Rho-kinase/ROCK-signalingpathway.

Rationale: Vascular remodeling and right-ventricular (RV) dysfunction are features of refractory pulmonary hypertension (PHT) in human neonates. These features are replicated in rats chronically exposed to hypoxia (13% O2), in which increased pulmonary vascular resistance (PVR) was acutely normalized by Y-27632, a Rho-kinase (ROCK) inhibitor, but not by inhaled nitric oxide. Objective: To examine the reversing effects of sustained ROCK inhibition on haemodynamic (RV dysfunction and increased PVR) and structural (RV hypertrophy and arterial wall remodeling) changes of chronic hypoxic PHT. Methods: Rat pups were exposed to air or hypoxia from birth for up to 21 days and received Y-27632 (15 mg/kg/b.i.d.) or vehicle from day 14. Results: Y-27632 normalised RV dysfunction and reversed remodeling secondary to chronic hypoxia. These changes were accompanied by increased apoptosis of smooth muscle and attenuated endothelin-1 expression in pulmonary arteries. Conclusion: ROCK inhibitors hold promise as a rescue therapy for refractory PHT in neonates.

Rationale: Vascular remodeling and right-ventricular (RV) dysfunction are features of refractory pulmonary hypertension (PHT) in human neonates. These features are replicated in rats chronically exposed to hypoxia (13% O2), in which increased pulmonary vascular resistance (PVR) was acutely normalized by Y-27632, a Rho-kinase (ROCK) inhibitor, but not by inhaled nitric oxide. Objective: To examine the reversing effects of sustained ROCK inhibition on haemodynamic (RV dysfunction and increased PVR) and structural (RV hypertrophy and arterial wall remodeling) changes of chronic hypoxic PHT. Methods: Rat pups were exposed to air or hypoxia from birth for up to 21 days and received Y-27632 (15 mg/kg/b.i.d.) or vehicle from day 14. Results: Y-27632 normalised RV dysfunction and reversed remodeling secondary to chronic hypoxia. These changes were accompanied by increased apoptosis of smooth muscle and attenuated endothelin-1 expression in pulmonary arteries. Conclusion: ROCK inhibitors hold promise as a rescue therapy for refractory PHT in neonates.

Yes / Abstract Recent evidence suggests that the predominant astrocyte glutamate transporter, GLT-1/ Excitatory Amino Acid Transporter 2 (EAAT2) is associated with mitochondria. We used primary cultures of mouse astrocytes to assess co-localization of GLT-1 with mitochondria, and tested whether the interaction was dependent on neurons, actin polymerization or the kinesin adaptor, TRAK2. Mouse primary astrocytes were transfected with constructs expressing V5-tagged GLT-1, pDsRed1-Mito with and without dominant negative TRAK2. Astrocytes were visualized using confocal microscopy and co-localization was quantified using Volocity software. Image analysis of confocal z-stacks revealed no co-localization between mitochondria and GLT-1 in pure astrocyte cultures. Co-culture of astrocytes with primary mouse cortical neurons revealed more mitochondria in processes and a positive correlation between mitochondria and GLT-1. This co-localization was not further enhanced after neuronal depolarization induced by 1 h treatment with 15 mM K+. In pure astrocytes, a rho kinase inhibitor, Y27632 caused the distribution of mitochondria to astrocyte processes without enhancing GLT-1/mitochondrial co-localization, however, in co-cultures, Y27632 abolished mitochondrial: GLT-1 co-localization. Disrupting potential mitochondrial: kinesin interactions using dominant negative TRAK2 did not alter GLT-1 distribution or GLT-1: mitochondrial co-localization. We conclude that the association between GLT-1 and mitochondria is modest, is driven by synaptic activity and dependent on polymerized actin filaments. Mitochondria have limited co-localization with the glutamate transporter GLT-1 in primary astrocytes in culture. Few mitochondria are in the fine processes where GLT-1 is abundant. It is necessary to culture astrocytes with neurones to drive a significant level of co-localization, but co-localization is not further altered by depolarization, manipulating sodium ion gradients or Na/K ATPase activity.

Infecções por Plasmodium sp. podem levar a um quadro respiratório grave, com complicações pulmonares denominadas lesão pulmonar aguda e síndrome do desconforto respiratório agudo (LPA/SDRA). Inflamação aguda, lesão do endotélio alveolar e do parênquima pulmonar, disfunção e aumento da permeabilidade da barreira alvéolo-capilar e, consequente, formação de edema, caracterizam esta síndrome. O modelo experimental, que utiliza o parasita murino Plasmodium berghei ANKA e camundongos da linhagem DBA/2, é empregado no estudo de mediadores imunológicos e fatores que propiciam o estabelecimento das lesões pulmonares associados à LPA/SDRA. Diversos estímulos podem atuar diretamente no aumento da permeabilidade endotelial por meio da desestabilização dos microtúbulos, rearranjo dos microfilamentos de actina e contração das células endoteliais, via sinalização de Rho-GTPases, causando disfunção da barreira endotelial. Desta forma, este trabalho tem como objetivo avaliar as alterações do citoesqueleto em células endoteliais primárias pulmonares de camundongos DBA/2 (CEPP-DBA/2), as vias de sinalização das principais Rho-GTPases e o estresse oxidativo, causados pela presença de eritrócitos parasitados com esquizontes de P. berghei ANKA (EP-PbA). As CEPP-DBA/2 foram estimuladas com TNF, VEGF ou IFNγ, em diferentes tempos de exposição, seguido da incubação com EP-PbA. Assim, foram realizados ensaios de imunofluorescência para análise do rearranjo de microfilamentos de actina e da desestabilização de microtúbulos. As vias de sinalização das Rho-GTPases foram avaliadas por Western blot, para as expressões proteicas de RhoA, Cdc42 e MLC. Além disso, ensaio fluorométrico foi realizado para detectar a produção de espécies reativas de oxigênio, resultantes do estímulo com eritrócitos parasitados. CEPP-DBA/2 estimuladas por EP-PbA, VEGF, TNF ou IFNγ, em associação ou não, apresentaram alterações morfológicas nos microfilamentos de actina e aumento dos espaços interendoteliais. Imagens de imunofluorescência também mostram desestabilização de microtúbulos e desfosforilação de FAK, causadas por EP-PbA. Os ensaios de permeabilidade validam que os eritrócitos parasitados com formas maduras de P. berghei induziram aumento da permeabilidade microvascular nas CEPP-DBA/2. Além disso, estas células, estimuladas com EP-PbA, demonstraram elevada produção de espécies reativas de oxigênio (EROs), o que pode estar contribuindo com o desenvolvimento de estresse oxidativo e com a injúria endotelial, assim como, com o aumento da permeabilidade vascular. O mais interessante é que estas alterações endoteliais podem estar relacionadas ao aumento da razão RhoA/Cdc42, da expressão proteica de MLC fosforilada e do sinal de ativação de RhoA. Em conjunto, estes resultados mostram envolvimento dos eritrócitos parasitados com esquizontes de Plasmodium berghei ANKA na desorganização do citoesqueleto e na disfunção da barreira alvéolo-capilar, via RhoA/Rho-kinase, o que pode estar contribuindo com a patogênese da LPA/SDRA associada à malária. / Infections by Plasmodium sp. can lead to a serious respiratory condition with pulmonary complications, named acute lung injury and acute respiratory distress syndrome (ALI/ARDS). Acute inflammation, alveolar endothelium and lung parenchyma injuries, dysfunction and increased permeability of the pulmonary alveolar-capillary barrier and consequent formation of edema characterize this syndrome. Several stimuli can directly increase endothelial permeability through actin microfilaments rearrangement, via Rho- GTPases signaling, leading to endothelial barrier dysfunction. DBA/2 mice infected with Plasmodium berghei ANKA develop ALI/ARDS similar to that observed in humans. The purpose of this research was to assess cytoskeletal changes in DBA/2 mice primary microvascular lung endothelial cells (PMLEC), verify the signaling pathways of the Rho- GTPases and analyze the oxidative stress on these cells in the presence of P. berghei ANKA-infected red blood cells (PbA-iRBC). PMLEC were stimulated by TNF, VEGF or IFNγ followed by incubation with PbA-iRBC. Immunofluorescence assays were performed to analyze actin microfilaments rearrangement and microtubules destabilization. Western blot for RhoA, Cdc42 and MLC proteins were conducted to assess alterations in signaling pathways of Rho-GTPases. In addition, a fluorimetric assay was performed to detect the production of reactive oxygen species resulting from PbA-iRBC stimulus. P. berghei ANKA, VEGF, TNF and IFNγ stimuli, in association or not, caused morphological disturbances in actin microfilaments of PMLEC and an increase of intercellular spaces. Moreover, immunofluorescence images showed microtubules destabilization and FAK dephosphorylation in these cells, caused by PbA-iRBC. The permeability assay showed that PbA-iRBC induced an increase of microvascular permeability in PMLEC. In addition, PMLEC stimulated by PbA-iRBC, showed elevated production of ROS, which may be contributing to oxidative stress and increasing the damage of endothelial cells, as well as an increase of vascular permeability. Interestingly, these endothelial changes may be related to the increased RhoA/Cdc42 protein expressions ratio, augmented protein expression of phosphorylated MLC and RhoA activation signal. Taken together, these data demonstrate the involvement of P. berghei ANKA-infected red blood cells in cytoskeleton disorganization and alveolar-capillary barrier dysfunction, through of RhoA / Rho-kinase signaling pathway, which may contribute to ALI/ARDS pathogenesis.

Introduction : Chez les malades porteurs d’une broncho-pneumopathie chronique obstructive (BPCO) modérée à sévère, l’hypoxie induit à la fois une augmentation du tonus vasculaire pulmonaire et la survenue d’un remodelage tissulaire touchant les trois tuniques des vaisseaux pulmonaires. Des données récentes montraient que la voie RhoA/Rho-kinase était impliquée dans le dysfonctionnement endothélial et dans la vasoconstriction induite par l’endothéline-1 (ET-1) chez les sujets tabagiques et les patients porteurs d’une hyperension artérielle pulmonaire (HTAP). Objectifs : Le but de ce travail était de clarifier le rôle de la voie RhoA/Rho-kinase dans la vasodilatation pulmonaire dépendante de l’endothélium et la vasoconstriction pulmonaire induite par l’ET-1, ainsi que les différents niveaux d’interaction entre la voie RhoA/Rho-kinase et celle de la NO synthase endothéliale (NOS-3) chez les patients BPCO post-tabagiques. Matériels et Méthodes : Les prélèvements d’artères pulmonaires et de tissus pulmonaire provenaient des patients porteurs d’un cancer broncho-pulmonaire, opérés pour une lobectomie ou pneumonectomie. L’expression protéique de la NOS-3, des récepteurs de l’ET-1 (ET-A et ET-B), des Rho-kinases (ROCK- 1 et ROCK-2), de la petite protéine G RhoA (sous sa forme activée, liée au GTP, ou sous sa forme inactivée), et de la forme phosphorylée de la sous-unité pMYPT-1 ont été évaluées par immunohistochimie, western blot, et immunoprécipitation. Les activités enzymatiques de la NO synthase et de la Rho-kinase, ainsi que la concentration du GMPc ont été mesurées par la méthode Elisa. L’expression des ARNm de l’ET-1, ET-A, et ET-B a été quantifiée par RT-PCR. Résultats : La relaxation dépendante de l’endothélium des vaisseaux pulmonaires était nettement réduite chez les BPCO par rapport aux témoins (P < 0,05). De façon plus surprenante, nous avions retrouvé la même différence significative entre le groupe témoin et le groupe des patients tabagiques sans TVO. La vasoconstriction pulmonaire en réponse à l’ET-1 était augmentée chez les patients BPCO par rapport aux deux autres groupes (P < 0,05). L’expression protéique de la NOS-3 ainsi que les expressions en ARNm de l’ET-1 et des récepteurs ET-A et ET-B n’étaient pas significativement différentes entre les trois groupes (P > 0,05). L’expression de la RhoA-GTP, ROCK-1, ROCK-2, et pMYPT-1 était augmentée chez les patients BPCO mais également chez les fumeurs sans TVO (P < 0,01 et P < 0,05). L’activité de la NOS-3 était diminuée chez les BPCO avec et sans hypoxémie (P < 0,01, P < 0,05). La concentration du GMPc dans les tissus pulmonaires et dans les artères pulmonaires était diminuée de façon parallèle chez les patients BPCO hypoxémiques par rapport aux BPCO non-hypoxémiques. En revanche, l’activité des Rho-kinases n’était augmentée que chez les patients BPCO (P < 0,001) et ne semblait pas être augmentée chez les fumeurs sans TVO par rapport aux témoins. Conclusion : Notre étude a montré que la voie de signalisation RhoA/Rho-kinase module la fonction endothéliale en diminuant l’expression et l’activité de la NO synthase et en favorisant l’effet de l’ET-1, ce qui aboutit à une augmentation du tonus vasculaire pulmonaire des patients porteurs d’une BPCO post-tabagique mais également chez des fumeurs sans TVO / Background : In patients with moderate to severe chronic obstructive pulmonary disease (COPD), hypoxia induces an increase of pulmonary vascular tonus and vascular remodeling. Recent data showed that RhoA/Rho-kinase pathway had been involved in endothelial dysfunction and vasoconstriction induced by endothelin-1 (ET-1) in smokers and in patients with pulmonary hypertension (PA). Objective : The aims of study were to clarify the role of RhoA/Rho-kinase pathway in pulmonary vasodilatation endothelium dependent and the interaction between Rho-A/Rho-kinase and endothelial NOS (NOS-3) signalization in cigarette smoke-induced COPD patients. Material and Methods : Lung tissues and pulmonary arteries from patients with broncho-pulmonary cancer, operated for lobectomy or pneumonectomy. Protein expression of NOS-3, ET-1 receptors (ET-A and ET-B), Rho-kinase (ROCK-1 and ROCK-2), a small protein G RhoA (active form combined to GTP or inactive forme), and phosphorylated form of pMYPT-1 subunit had been evaluated by immunohistochemistry, western blot, and pull-down assay. Enzyme activity of NO synthase, Rho-kinase, and the concentration of cGMP had been measured by Elisa. mRNA expression had been quantified by RT-PCR. Results : Endothelium-dependent relaxation of pulmonary vessels was clearly reduced in COPD patients in comparison with control subjects (P < 0.05). Surprisingly, we founded the significant difference of relaxation between smokers without airflow obstruction and control subjects. Pulmonary vasoconstriction in response to ET-1 was increased in COPD patients as compared to two other groups (P < 0.05). Protein expression of NOS-3 and mRNA expression of ET-1 and ET-A/ET-B receptors were not significant different between three groups (P > 0.05). Expression of GTP-Rho-A, ROCK-1, ROCK-2, and pMYPT-1 were increased in COPD and in smokers without airway obstruction (P < 0.01 and P < 0.05). NOS-3 activity was reduced in COPD patients with and without hypoxemia (P < 0.01 and P < 0.05). The concentration of cGMP in lung tissues and pulmonary arteries were reduced in hypoxemic COPD patients in comparison with non-hypoxemic COPD patients. Whereas, Rho-kinase activity had been increased predominantly in COPD patients (P < 0.001) than in smokers without COPD as compared to control subjects. Conclusion : The present study showed that RhoA/Rho-kinase pathway modulated the endothelial function by decreasing the expression and activity of NO synthase. It facilitated the effect of ET-1 in pulmonary vasoconstriction. These phenomena produced an increase of pulmonary vascular tonus in cigarette smoke-induced COPD and in smokers without airway obstruction

La cholestase est l'une des manifestations les plus courantes des lésions induitespar des médicaments. Dans 40% des cas elle n’est pas prévisible; une meilleure prédictibilité représente donc un défi majeur. Tout d'abord, nous avons démontré que les cellules hépatiques humaines HepaRG différenciées sont un modèle approprié pour étudier la cholestase induite par les médicaments en comparant la localisation et l’activité des transporteurs d'influx et d'efflux avec les hépatocytes humains primaires. Tous les transporteurs d'efflux et d’influx testés ont été correctement localisés au niveau des membranes canaliculaire (BSEP, MRP2, MDR1 et MDR3) et basolatéral (NTCP, MRP3) et sont fonctionnels. En outre, ces cellules expriment également les enzymes qui métabolisent les acides biliaires (ABs) et ont la capacité de les synthétiser et de les conjuguer avec la taurine, la glycine et le sulfate, à un taux similaire à celui des hépatocytes primaires. Des changements ont été observés sur la répartition des ABs totaux après traitements de cellules HepaRG par un médicament cholestatique, la cyclosporine A (CsA), de manière concentration- dépendante. L'inhibition de l'efflux et de l'influx de taurocholate a été observée après 15 min et 1 h respectivement. Ces premiers effets ont été associés à la dérégulation de la voie des cPKC et l'induction d’un stress du réticulum endoplasmique puis d’un stress oxydant. Nous avons également montré pour la première fois une accumulation intracellulaire d’ABs endogènes avec un médicament cholestatique in vitro. En outre, notre travail apporte des preuves que la motilité des canalicules biliaires (BC) est indispensable à la clairance des ABs. La voie ROCK et le complexe actomyosine sont fortement impliqués. Nous avons fourni la première démonstration que la voie ROCK et les dynamiques des BC sont des cibles majeures des composés cholestatiques. Nos données devraient contribuer à l'élaboration de méthodes de screening pour la prédiction précoce des effets secondaires induits par les médicaments cholestatiques. / Cholestasis is one of the most common manifestations of drug-induced liver injury (DILI). Since up to now it is unpredictable in 40% of all cases its accurate prediction represents a major challenge. First, we validated that differentiated HepaRG human liver cells are a suitable in vitro model to study drug-induced cholestasis, by comparing localization of influx and efflux transporters and their functional activity in these cells and primary human hepatocytes. All tested influx and efflux transporters were correctly localized to canalicular (BSEP, MRP2, MDR1, and MDR3) or basolateral (NTCP, MRP3) membrane domains and were functional. In addition, the HepaRG cell line also exhibits bile acids (BAs) metabolizing enzymes and has the capacity to synthesize BAs and to further amidate these BAs with taurine and glycine as well as sulfate, at a rate similar to that of primary hepatocytes. Concentration- dependent changes were observed in total BAs disposition after treatment of HepaRG cells by the cholestatic drug cyclosporine A (CsA). Inhibition of efflux and uptake of taurocholate was evidenced as early as 15 min and 1 h respectively. These early effects were associated with deregulation of cPKC pathway and induction of endoplasmic reticulum stress that preceded generation of oxidative stress. We also showed for the first time intracellular accumulation of endogenous BAs by a cholestatic drug in vitro. In addition, our work brings evidences that motility of bile canaliculi (BC) is essential for BAs clearance where ROCK pathway and actomyosin complex are highly implicated. We provided the first demonstration that ROCK pathway and BC dynamics are major targets of cholestatic compounds. Our data should help in the development of screening methods for early prediction of drug-induced cholestatic side effects.

The metanephric kidney consists of two types of epithelia; the Wolffian duct-derived ureteric bud and the nephrogenic components that originate from mesenchymal-toepithelial transitions in the metanephric mesenchyme. The ureteric bud forms when inductive signals from the metanephric mesenchyme stimulates the evagination of an epithelial tube from the Wolffian duct into the mesenchyme. Reciprocal signalling between the ureteric bud and the metanephric mesenchyme regulates the branching of the ureteric bud and the induction of nephron formation. Inductive and inhibitory signalling of ureteric bud growth and branching has been shown by several protein families, however, the mechanical aspects of ureteric bud branching and nephrogenesis are largely unknown. I investigated the roles of Rac1-GTPase and Rho-kinase during kidney development. These proteins are important regulators of the cytoskeleton where Rac1 is a promoter of actin filament polymerisation and Rho-kinase directly stimulates the formation and contraction of actin-myosin stress fibres. Using a cell-permeable inhibitor, Rac1 was inhibited with no effects on nephron formation or subsequent segmentation and patterning. Inhibition of active Rac1 significantly reduced the level of ureteric bud branching and also resulted in lower proliferation rates. Rho-kinase was similarly targeted using two inhibitors. Rho-kinase inhibition had important effects on nephron formation and nephron maturation. Inhibition of Rhokinase resulted in decreased levels of nephron formation and severely morphologically abnormal nephrons. The formation of apical-basal polarity was disturbed as was the development of the visceral and parietal epithelia; precursors of the renal corpuscle. Inhibition of Rho-kinase led to abnormal formation of the proximal-distal axis and abnormal segmentation of the nephron. The effects of Rho-kinase inhibition were partially mimicked by direct targeting of actin-myosin contractions using a myosin-ATPase inhibitor. This demonstrated that Rho-kinase is necessary during multiple stages of nephrogenesis and maturation, at least in part, as a result of its ability to regulate actin-myosin contraction. These results show that Rac1 and Rho-kinase play important roles during several aspects of kidney development and highlights the significance of further investigating the mechanisms involved during kidney organogenesis.

Cerebral parenchymal arterioles (PAs) play a critical role in assuring appropriate blood flow and perfusion pressure within the brain. PAs are unique in contrast to upstream pial arteries, as defined by their critical roles in neurovascular coupling, distinct sensitivities to vasoconstrictors, and enhanced myogenic responsiveness. Dysfunction of these blood vessels is implicated in numerous cardiovascular diseases. However, treatments are limited due to incomplete understanding of the fundamental control mechanisms at this level of the circulation. One of the key elements within most vascular networks, including the cerebral circulation, is the presence of myogenic tone, an intrinsic process whereby resistance arteries constrict and reduce their diameter in response to elevated arterial pressure. This process is centrally involved in the ability of the brain to maintain nearly constant blood flow over a broad range of systemic blood pressures. The overall goal of this dissertation was to investigate the unique mechanisms of myogenic tone regulation in the cerebral microcirculation. To reveal the contributions of various signaling factors in this process, measurements of diameter, intracellular Ca2+ concentration ([Ca2+]i), membrane potential and ion channel activity were performed. Initial work determined that two purinergic G protein-coupled receptors, P2Y4 and P2Y6 receptors, play a unique role in mediating pressure-induced vasoconstriction of PAs in a ligand-independent manner. Moreover, a particular transient receptor potential (TRP) channel in the melastatin subfamily, i.e. TRPM4, was also identified as a mediator of PA myogenic responses. Notably, the observations that inhibiting TRPM4 channels substantially reduces P2Y receptor-mediated depolarization and vasoconstriction, and that P2Y receptor ligands markedly activate TRPM4 currents provide definitive evidence that this ion channel functions as an important link between mechano-sensitive P2Y receptor activation and the myogenic response in PAs. Next, the signaling cascades that mediate stretch-induced TRPM4 activation in PA myocytes were explored. Interestingly, these experiments determined that the RhoA/Rho kinase signaling pathway is involved in this mechanism by facilitating pressure-induced, P2Y receptor-mediated stimulation of TRPM4 channels, leading to subsequent smooth muscle depolarization, [Ca2+]i increase and contraction. Since Rho kinase is generally accepted as a 'Ca2+-sensitization' mediator, the present, contrasting observations point to an underappreciated role of RhoA/Rho kinase signaling in the excitation-contraction mechanisms within the cerebral microcirculation. Overall, this dissertation provides evidence that myogenic regulation of cerebral PAs is mediated by mechano-sensitive P2Y receptors, which initiate the RhoA/Rho kinase signaling pathway, subsequent TRPM4 channel opening, and concomitant depolarization and contraction of arteriolar smooth muscle cells. Revealing the unique mechanochemical coupling mechanisms in the cerebral microcirculation may lead to development of innovative therapeutic strategies for prevention and treatment of microvascular pathologies in the brain.

Nitric oxide (NO) is the principal mediator of erectile function. NO is released from the nerves and endothelium of small arteries in the penis and diffuses into surrounding smooth muscle to vasodilate through activation of soluble guanylate cyclase (sGC). Erectile dysfunction (ED) occurs in 50% of men between the ages of 40 and 70. It is likely that the pathology of ED results from impairment of NO formation or bioavailability in penile tissue. Iatrogenic nerve damage occurring during prostatectomy can attenuate neurotransmission and release of vasodilators from cavernosal nerves. Oxidative stress from chronic conditions such as diabetes and cardiovascular disease generates reactive oxygen species that can oxidize NO and decrease the molecule's bioavailability. The "gold standard" treatment for ED involves use of oral PDE-5 inhibitors that rely on an intact NO-signaling mechanism for efficacy. Although these therapies are easy to use, they are not effective in many patients suffering from ED associated with pathological conditions of decreased NO bioavailability. Rho-kinase inhibitors, sGC stimulators and sGC activators offer three new interventions that may demonstrate efficacy in treating ED associated with low NO bioavailability. Our results suggest that erectile responses to Rho-kinase inhibitors are not modulated by muscarinic receptor blockade, soluble guanylate cyclase inhibition or cavernosal nerve injury in the rat and that Rho-kinase inhibitors are additive and do not potentiate the endogenous NO-mediated erectile response. Our results with BAY 41-8543 show that this sGC stimulator has significant erectile activity and can potentiate erectile responses to low levels of exogenous and endogenously released NO. These results suggest that BAY 41-8543 would be useful in the treatment of ED occurring following nerve damage from prostatectomy. The sGC activator BAY 60-2770 has very potent erectile activity that is enhanced significantly in conditions of oxidative stress when erectile responses to endogenous NO or sGC stimulators are severely diminished. In oxidizing conditions erectile activity of sGC activators may be enhanced further with concomitant PDE-5 inhibitor therapy, providing evidence that sGC activators may be used alone and in combination with existing treatments to improve erectile function in patients who are non-responsive to standard therapeutic options for ED. / acase@tulane.edu