Rho-Mancing to Sensitize Calcium Signaling for Contraction in the Vasculature: Role of Rho Kinase.

Vascular smooth muscle contraction is an important physiological process contributing to cardiovascular homeostasis. The principal determinant of smooth muscle contraction is the intracellular free Ca2+ concentration, and phosphorylation of myosin light chain (MLC) by activated myosin light chain kinase (MLCK) in response to increased Ca2+ is the main pathway by which vasoconstrictor stimuli induce crossbridge cycling of myosin and actin filaments. A secondary pathway for vascular smooth muscle contraction that is not directly dependent on Ca2+ concentration, but rather mediating Ca2+ sensitization, is the RhoA/Rho kinase pathway. In response to contractile stimuli, the small GTPase RhoA activates its downstream effector Rho kinase which, in turn, promotes contraction via myosin light chain phosphatase (MLCP) inhibition. RhoA/Rho kinase-mediated MLCP inhibition occurs mainly by phosphorylation and inhibition of MYPT1, the regulatory subunit of MLCP, or by CPI-17-mediated inhibition of the catalytic subunit of MLCP. In this review, we describe the molecular mechanisms underlying the pivotal role exerted by Rho kinase on vascular smooth muscle contraction and discuss the main regulatory pathways for its activity.

Toll-like receptor 4 inhibition reduces vascular inflammation in spontaneously hypertensive rats.

AIMS: Hypertension is associated with increased levels of circulating cytokines and recent studies have shown that innate immunity contributes to hypertension. The mechanisms which hypertension stimulates immune response remain unclear, but may involve formation of neo-antigens that activate the immune system. Toll like receptor 4 (TLR4) is an innate immune receptor that binds a wide spectrum of exogenous (lipopolysaccharide) and endogenous ligands. TLR4 signaling leads to activation of nuclear factor kappa B (NFκB) and transcription of genes involved in inflammatory response. We previously demonstrated that TLR4 blockade reduces blood pressure and the augmented vascular contractility in spontaneously hypertensive rats (SHR). Here we hypothesized that inhibition of TLR4 ameliorates the vascular inflammatory process by a NFκB signaling pathway. MAIN METHODS: SHR and Wistar rats were treated with anti-TLR4 antibody (1µg/day) or unspecific IgG for 15days (i.p.). KEY FINDINGS: Anti-TLR4 treatment decreased production of reactive oxygen species and expression of IL-6 cytokine in mesenteric resistance arteries from SHR, when compared with IgG-treated SHR. Anti-TLR4 treatment also abolished the increased vascular reactivity to noradrenaline observed in IgG-treated SHR, as described before, and inhibition of NFκB decreased noradrenaline responses only in IgG-treated SHR. Mesenteric arteries from SHR treated with anti-TLR4 displayed decreased expression of MyD88, but not TRIF, key molecules in TLR4 signaling. Phosphorylation of p38 and NF-κB p65 were decreased in arteries from anti-TLR4-treated SHR versus IgG-treated SHR. SIGNIFICANCE: Together, these results suggest that TLR4 is a key player in hypertension and vascular inflammatory process by a NFκB signaling pathway.

Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain

O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2±2 vs 7.9±1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4±2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3±2 vs 7.5±2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1±2 vs 7.4±2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca2+/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.

Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain.

O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2 ± 2 vs 7.9 ± 1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4 ± 2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3 ± 2 vs 7.5 ± 2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1 ± 2 vs 7.4 ± 2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca(2+)/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.

Therapeutic implications of peptide interactions with G-protein-coupled receptors in diabetic vasculopathy.

The dramatic worldwide increase in the prevalence of diabetes has generated an attempt by the scientific community to identify strategies for its treatment and prevention. Vascular dysfunction is a hallmark of diabetes and frequently leads to the development of atherosclerosis, coronary disease-derived myocardial infarction, stroke, peripheral arterial disease and diabetic 'triopathy' (retinopathy, nephropathy and neuropathy). These vascular complications, developing in an increasingly younger cohort of patients with diabetes, contribute to morbidity and mortality. Despite the development of new anti-diabetic or anti-hyperglycaemic drugs, vascular complications remain to be a problem. This warrants a need for new therapeutic strategies to tackle diabetic vasculopathy. There is a growing body of evidence showing that peptide-binding G-protein-coupled receptors (peptide-binding GPCRs) play an important role in the pathophysiology of vascular dysfunction during diabetes. Thus, in this review, we discuss some of the peptide-binding GPCRs involved in the regulation of vascular function that have potential to be a therapeutic target in the treatment of diabetic vasculopathy.

Mitochondrial-derived N-formyl peptides: novel links between trauma, vascular collapse and sepsis.

Sepsis is a major cause of mortality and morbidity in trauma patients despite aggressive treatment. Traumatic injury may trigger infective or non-infective systemic inflammatory response syndrome (SIRS) and sepsis. Sepsis and SIRS are accompanied by an inability to regulate the inflammatory response but the cause of this perturbation is still unknown. The major pathophysiological characteristic of sepsis is the vascular collapse (i.e., loss of control of vascular tone); however, at the cellular level the final mediator of extreme vasodilatation has yet to be identified. After trauma, cellular injury releases endogenous damage-associated molecular patterns (DAMPs) that activate the innate immune system. Mitochondrial DAMPs express at least two molecular signatures, N-formyl peptides and mitochondrial DNA that act on formyl peptide receptors (FPRs) and Toll-like receptor 9, respectively. N-Formyl peptides are potent immunocyte activators and, once released in the circulation, they induce modulation of vascular tone by cellular mechanisms that are not completely understood. We have observed that N-formyl peptides from bacterial (FMLP) and mitochondrial (FMIT) sources induce FPR-mediated vasodilatation in resistance arteries. Accordingly, we propose that tissue and cellular trauma induces the release of N-formyl peptides from mitochondria triggering inflammation and vascular collapse via activation of FPR and contributing to the development of sepsis. The proposed hypothesis provides clinically significant information linking trauma, mitochondrial N-formyl peptides and inflammation to vascular collapse and sepsis. If our hypothesis is true, it may lead to new strategies in the management of sepsis that can help clinicians effectively manage non-infectious and infectious inflammatory responses.

The adipokine chemerin augments vascular reactivity to contractile stimuli via activation of the MEK-ERK1/2 pathway.

AIMS: Cytokines interfere with signaling pathways and mediators of vascular contraction. Endothelin-1 (ET-1) plays a major role on vascular dysfunction in conditions characterized by increased circulating levels of adipokines. In the present study we tested the hypothesis that the adipokine chemerin increases vascular contractile responses via activation of ET-1/ET-1 receptors-mediated pathways. MAIN METHODS: Male, 10-12 week-old Wistar rats were used. Endothelium-intact and endothelium-denuded aortic rings were incubated with chemerin (0.5 ng/mL or 5 ng/mL, for 1 or 24h), and isometric contraction was recorded. Protein expression was determined by Western blotting. KEY FINDINGS: Constrictor responses to phenylephrine (PE) and ET-1 were increased in vessels treated for 1h with chemerin. Chemerin incubation for 24h decreased PE contractile response whereas it increased the sensitivity to ET-1. Endothelium removal significantly potentiated chemerin effects on vascular contractile responses to PE and ET-1. Incubation with either an ERK1/2 inhibitor (PD98059) or ETA antagonist (BQ123) abolished chemerin effects on PE- and ET-1-induced vasoconstriction. Phosphorylation of MEK1/2 and ERK1/2 was significantly increased in vessels treated with chemerin for 1 and 24h. Phosphorylation of these proteins was further increased in vessels incubated with ET-1 plus chemerin. ET-1 increased MEK1/2, ERK1/2 and MKP1 protein expression to values observed in vessels treated with chemerin. SIGNIFICANCE: Chemerin increases contractile responses to PE and ET-1 via ERK1/2 activation. Our study contributes to a better understanding of the mechanisms by which the adipose tissue affects vascular function and, consequently, the vascular alterations present in obesity and related diseases.

Increased cavernosal relaxation by Phoneutria nigriventer toxin, PnTx2-6, via activation at NO/cGMP signaling.

Erectile dysfunction (ED) mechanisms in diabetic patients are multifactorial and often lead to resistance to current therapy. Animal toxins have been used as pharmacological tools to study penile erection. Human accidents involving the venom of Phoneutria nigriventer spider are characterized by priapism. We hypothesize that PnTx2-6 potentiates cavernosal relaxation in diabetic mice by increasing cyclic guanosine monophosphate (cGMP). This effect is neuronal nitric oxide synthase (nNOS) dependent. Cavernosal strips were contracted with phenylephrine (10(-5) M) and relaxed by electrical field stimulation (20 V, 1-32 Hz) in the presence or absence of PnTx2-6 (10(-8) M). Cavernosal strips from nNOS- and endothelial nitric oxide synthase (eNOS)-knockout (KO) mice, besides nNOS inhibitor (10(-5) M), were used to evaluate the role of this enzyme in the potentiation effect evoked by PnTx2-6. Tissue cGMP levels were determined after stimulation with PnTx2-6 in presence or absence of N-nitro-L-arginine methyl ester (L-NAME) (10(-4) M) and ω-conotoxin GVIA (10(-6) M), an N-type calcium channel inhibitor. Results showed that PnTx2-6 enhanced cavernosal relaxation in diabetic mice (65%) and eNOS KO mice, but not in nNOS KO mice. The toxin effect in the cavernosal relaxation was abolished by nNOS inhibitor. cGMP levels are increased by PnTx2-6, however, L-NAME abolished this enhancement as well as ω-conotoxin GVIA. We conclude that PnTx2-6 facilitates penile relaxation in diabetic mice through a mechanism dependent on nNOS, probably via increasing nitric oxide/cGMP production.

STIM1/Orai1-mediated store-operated Ca2+ entry: the tip of the iceberg

Highly efficient mechanisms regulate intracellular calcium (Ca2+) levels. The recent discovery of new components linking intracellular Ca2+ stores to plasma membrane Ca2+ entry channels has brought new insight into the understanding of Ca2+ homeostasis. Stromal interaction molecule 1 (STIM1) was identified as a Ca2+ sensor essential for Ca2+ store depletion-triggered Ca2+ influx. Orai1 was recognized as being an essential component for the Ca2+ release-activated Ca2+ (CRAC) channel. Together, these proteins participate in store-operated Ca2+ channel function. Defective regulation of intracellular Ca2+ is a hallmark of several diseases. In this review, we focus on Ca2+ regulation by the STIM1/Orai1 pathway and review evidence that implicates STIM1/Orai1 in several pathological conditions including cardiovascular and pulmonary diseases, among others.

STIM1/Orai1-mediated store-operated Ca2+ entry: the tip of the iceberg.

Highly efficient mechanisms regulate intracellular calcium (Ca2+) levels. The recent discovery of new components linking intracellular Ca2+ stores to plasma membrane Ca2+ entry channels has brought new insight into the understanding of Ca2+ homeostasis. Stromal interaction molecule 1 (STIM1) was identified as a Ca2+ sensor essential for Ca2+ store depletion-triggered Ca2+ influx. Orai1 was recognized as being an essential component for the Ca2+ release-activated Ca2+ (CRAC) channel. Together, these proteins participate in store-operated Ca2+ channel function. Defective regulation of intracellular Ca2+ is a hallmark of several diseases. In this review, we focus on Ca2+ regulation by the STIM1/Orai1 pathway and review evidence that implicates STIM1/Orai1 in several pathological conditions including cardiovascular and pulmonary diseases, among others.

A key role for Na+/K+-ATPase in the endothelium-dependent oscillatory activity of mouse small mesenteric arteries

Oscillatory contractile activity is an inherent property of blood vessels. Various cellular mechanisms have been proposed to contribute to oscillatory activity. Mouse small mesenteric arteries display a unique low frequency contractile oscillatory activity (1 cycle every 10-12 min) upon phenylephrine stimulation. Our objective was to identify mechanisms involved in this peculiar oscillatory activity. First-order mesenteric arteries were mounted in tissue baths for isometric force measurement. The oscillatory activity was observed only in vessels with endothelium, but it was not blocked by L-NAME (100 µM) or indomethacin (10 µM), ruling out the participation of nitric oxide and prostacyclin, respectively, in this phenomenon. Oscillatory activity was not observed in vessels contracted with K+ (90 mM) or after stimulation with phenylephrine plus 10 mM K+. Ouabain (1 to 10 µM, an Na+/K+-ATPase inhibitor), but not K+ channel antagonists [tetraethylammonium (100 µM, a nonselective K+ channel blocker), Tram-34 (10 µM, blocker of intermediate conductance K+ channels) or UCL-1684 (0.1 µM, a small conductance K+ channel blocker)], inhibited the oscillatory activity. The contractile activity was also abolished when experiments were performed at 20°C or in K+-free medium. Taken together, these results demonstrate that Na+/K+-ATPase is a potential source of these oscillations. The presence of α-1 and α-2 Na+/K+-ATPase isoforms was confirmed in murine mesenteric arteries by Western blot. Chronic infusion of mice with ouabain did not abolish oscillatory contraction, but up-regulated vascular Na+/K+-ATPase expression and increased blood pressure. Together, these observations suggest that the Na+/K+ pump plays a major role in the oscillatory activity of murine small mesenteric arteries.

A key role for Na+/K+-ATPase in the endothelium-dependent oscillatory activity of mouse small mesenteric arteries.

Oscillatory contractile activity is an inherent property of blood vessels. Various cellular mechanisms have been proposed to contribute to oscillatory activity. Mouse small mesenteric arteries display a unique low frequency contractile oscillatory activity (1 cycle every 10-12 min) upon phenylephrine stimulation. Our objective was to identify mechanisms involved in this peculiar oscillatory activity. First-order mesenteric arteries were mounted in tissue baths for isometric force measurement. The oscillatory activity was observed only in vessels with endothelium, but it was not blocked by L-NAME (100 microM) or indomethacin (10 microM), ruling out the participation of nitric oxide and prostacyclin, respectively, in this phenomenon. Oscillatory activity was not observed in vessels contracted with K+ (90 mM) or after stimulation with phenylephrine plus 10 mM K+. Ouabain (1 to 10 microM, an Na+/K+-ATPase inhibitor), but not K+ channel antagonists [tetraethylammonium (100 microM, a nonselective K+ channel blocker), Tram-34 (10 microM, blocker of intermediate conductance K+ channels) or UCL-1684 (0.1 microM, a small conductance K+ channel blocker)], inhibited the oscillatory activity. The contractile activity was also abolished when experiments were performed at 20 degrees C or in K+-free medium. Taken together, these results demonstrate that Na+/K+-ATPase is a potential source of these oscillations. The presence of alpha-1 and alpha-2 Na+/K+-ATPase isoforms was confirmed in murine mesenteric arteries by Western blot. Chronic infusion of mice with ouabain did not abolish oscillatory contraction, but up-regulated vascular Na+/K+-ATPase expression and increased blood pressure. Together, these observations suggest that the Na+/K+ pump plays a major role in the oscillatory activity of murine small mesenteric arteries.

Tx2-6 toxin of the Phoneutria nigriventer spider potentiates rat erectile function.

The venom of the spider Phoneutria nigriventer contains several toxins that have bioactivity in mammals and insects. Accidents involving humans are characterized by various symptoms including penile erection. Here we investigated the action of Tx2-6, a toxin purified from the P. nigriventer spider venom that causes priapism in rats and mice. Erectile function was evaluated through changes in intracavernosal pressure/mean arterial pressure ratio (ICP/MAP) during electrical stimulation of the major pelvic ganglion (MPG) of normotensive and deoxycorticosterone-acetate (DOCA)-salt hypertensive rats. Nitric oxide (NO) release was detected in cavernosum slices with fluorescent dye (DAF-FM) and confocal microscopy. The effect of Tx2-6 was also characterized after intracavernosal injection of a non-selective nitric oxide synthase (NOS) inhibitor, L-NAME. Subcutaneous or intravenous injection of Tx2-6 potentiated the elevation of ICP/MAP induced by ganglionic stimulation. L-NAME inhibited penile erection and treatment with Tx2-6 was unable to reverse this inhibition. Tx2-6 treatment induced a significant increase of NO release in cavernosum tissue. Attenuated erectile function of DOCA-salt hypertensive rats was fully restored after toxin injection. Tx2-6 enhanced erectile function in normotensive and DOCA-salt hypertensive rats, via the NO pathway. Our studies suggest that Tx2-6 could be important for development of new pharmacological agents for treatment of erectile dysfunction.

Increased reactive oxygen species contributes to kidney injury in mineralocorticoid hypertensive rats.

Hypertension is associated with increased reactive oxygen species (ROS). Renal ROS production and their effects on renal function have never been investigated in mineralocorticoid hypertensive rats. In this study we hypothesized that increased ROS production in kidneys from deoxycorticosterone (DOCA)-salt rats contributes to adverse renal morphological changes and impaired renal function in DOCA-salt hypertensive rats. We also determined whether ROS-induced renal injury was dependent on blood pressure. DOCA-salt hypertensive rats exhibited a marked increase in blood pressure, renal ROS production, glomerular and tubular lesions, and microalbuminuria compared to sham rats. Treatment of DOCA-salt hypertensive rats with apocynin for 28 days resulted in attenuation of systolic blood pressure and improvement of renal morphology. Renal superoxide level in DOCA-salt rats was 215% of sham-operated rats and it was significantly decreased to 140% with apocynin treatment. Urinary protein level was decreased from 27 +/- 3 mg/day in DOCA-salt hypertensive rats to 9 +/- 2 mg/day. 28 days of Vitamin E treatment also reduced renal injury in regard to urinary protein level and renal morphology but had no effect on blood pressure in DOCA-salt rats. Increased urinary 8-isoprostane, a marker for oxidative stress, in DOCA-salt hypertensive rats (55 +/- 8 ng/day) was diminished by vitamin E treatment (24 +/- 6 ng/day). These data suggest that renal injury characteristic of mineralocorticoid hypertension is associated with oxidative stress and is partly independent of blood pressure.

Rho-kinase and RGS-containing RhoGEFs as molecular targets for the treatment of erectile dysfunction.

Erectile dysfunction (ED) is a highly prevalent and often under-treated condition. Erection is basically a spinal reflex that can be initiated by recruitment of penile afferents but also by visual, olfactory and imaginary stimuli. The generated nervous signals will influence the balance between contractile and relaxant factors, which control the degree of contraction of penile corporal cavernosal smooth muscles and, thus, determine the erectile state of the penis. The different steps involved in neurotransmission, impulse propagation and intracellular transduction of neural signals may be changed in different types of ED. Recent studies have revealed important roles for the small GTPase RhoA and its effector, Rho-kinase in regulating cavernosal smooth muscle tone. The RhoA/Rho-kinase pathway modulates the level of phosphorylation of the myosin light chain, mainly through inhibition of myosin phosphatase, and contributes to agonist-induced Ca(2+)-sensitization in smooth muscle contraction. Changes in this pathway may contribute to ED in various patient subgroups (e.g. hypertension, diabetes, hypogonadism). This review summarizes the importance of Rho-kinase signaling in the erectile response and introduces the evidence pointing to RGS-containing Rho-guanine nucleotide exchange factors (GEFs) as critical mediators of RhoA-GTPase activation in cavernosal smooth muscle and its possible compartmentalization in the caveolae. In addition, we suggest that the design of selective inhibitors of these GEFs might represent a novel class of pharmacological agents to treat ED.

Topical application of a Rho-kinase inhibitor in rats causes penile erection.

Studies from this laboratory have demonstrated that RhoA/Rho-kinase signaling mediates vasoconstriction in the penile circulation of the rat and that erection results from inhibition of this activity with Y-27632. In prior animal studies, Y-27632 was administered to the rats by intracavernous injection. To determine if topical application of the Rho-kinase inhibitor is an effective mode of delivery, Y-27632 was applied to the surface of the tunica albuginea or to the glans penis and surrounding skin in intact or castrated rats. Both sites of drug administration resulted in a marked increase in the erectile response both with and without stimulation of the autonomic innervation of the penile vasculature. Although high doses of the drug were found to reduce systemic blood pressure, topical administration of the Rho-kinase inhibitor, in appropriate doses, may have clinical value for the treatment erectile dysfunction.

Vasoconstriction, RhoA/Rho-kinase and the erectile response.

Recent studies have suggested that contraction of the smooth muscle in the cavernosal arterioles and in the walls of the cavernosal sinuses is maintained by the RhoA/Rho-kinase signaling pathway. However, this contraction activity must be overcome to permit the vasorelaxation essential for erection. We postulate that nitric oxide (NO) causes erection primarily by inhibiting the RhoA/Rho-kinase pathway. The following will discuss evidence in support of the important role of Rho-kinase-mediated vasoconstriction in the nonerect penis and how NO overrides this Rho-kinase-mediated vasoconstriction to permit vasodilation and erection.

The ups and downs of Rho-kinase and penile erection: upstream regulators and downstream substrates of rho-kinase and their potential role in the erectile response.

In the absence of arousal stimuli, the activity of the Rho-kinase-mediated signaling pathway promotes vasoconstriction of the cavernosal arterioles and sinuses, keeping the penis in the nonerect state. Upon sexual arousal or during nocturnal tumescence, nitric oxide (NO), released from nonadrenergic/noncholinergic nerves or from local endothelial cells, induces cavernosal vasodilation, resulting in an elevation in blood flow and intracavernosal pressure to initiate the erectile response. Although NO is thought to be the principal stimulator of penile erection, the signaling mechanism(s) of NO-mediated cavernosal vasodilation is unknown. In this article, we will consider the novel hypothesis that NO induces penile erection through the inhibition of endogenous Rho-kinase-mediated vasoconstriction. Additionally, we will look downstream of Rho-kinase, introducing a potential role for various substrates in the mechanism of Rho-kinase-mediated constriction in the cavernosal vasculature.

NADH/NADPH oxidase and enhanced superoxide production in the mineralocorticoid hypertensive rat.

We previously reported increased aortic reactive oxygen species (ROS) production in mineralocorticoid (deoxycorticosterone acetate [DOCA]-salt) hypertensive rats. In the present study, we tested the hypothesis that NADH/NADPH oxidase is responsible for increased ROS production, namely superoxide (O(2-)), in aorta from the DOCA-salt rat. Treatment of aortic rings from DOCA-salt rats with the NO synthase inhibitor N-nitro-L-arginine and the xanthine oxidase inhibitor allopurinol did not significantly change O(2-) production. Furthermore, de-endothelialization of aorta from DOCA-salt rats did not affect O(2-) production compared with that of sham-operated rats. Thus, xanthine oxidase and uncoupled endothelial NO synthase were not responsible for increased O(2-) production in the DOCA-salt rats. In contrast, treatment with the NADPH oxidase inhibitor apocynin significantly decreased O(2-) production in aortic rings from DOCA-salt rats compared with sham-operated rats. Moreover, long-term administration of apocynin (in drinking water, 1.5 mmol/L, 28 days) to DOCA-salt rats significantly decreased systolic blood pressure compared with that of rats treated with DOCA-salt alone. Furthermore, O(2-) production in aortic rings from DOCA-salt rats treated with apocynin for 28 days was reduced compared with that of untreated DOCA-salt rats. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated that DOCA-salt rats have significantly greater mRNA levels of the NADPH oxidase subunit p22phox than do sham-operated rats. These findings suggest that NADPH oxidase is increased and is responsible for increased O(2-) production and possibly contributes to increased blood pressure in the DOCA-salt hypertensive rat.

Angiotensin receptors: signaling, vascular pathophysiology, and interactions with ceramide.

Angiotensin II (ANG II) is a pleiotropic vasoactive peptide that binds to two distinct receptors: the ANG II type 1 (AT(1)) and type 2 (AT(2)) receptors. Activation of the renin-angiotensin system (RAS) results in vascular hypertrophy, vasoconstriction, salt and water retention, and hypertension. These effects are mediated predominantly by AT(1) receptors. Paradoxically, other ANG II-mediated effects, including cell death, vasodilation, and natriuresis, are mediated by AT(2) receptor activation. Our understanding of ANG II signaling mechanisms remains incomplete. AT(1) receptor activation triggers a variety of intracellular systems, including tyrosine kinase-induced protein phosphorylation, production of arachidonic acid metabolites, alteration of reactive oxidant species activities, and fluxes in intracellular Ca(2+) concentrations. AT(2) receptor activation leads to stimulation of bradykinin, nitric oxide production, and prostaglandin metabolism, which are, in large part, opposite to the effects of the AT(1) receptor. The signaling pathways of ANG II receptor activation are a focus of intense investigative effort. We critically appraise the literature on the signaling mechanisms whereby AT(1) and AT(2) receptors elicit their respective actions. We also consider the recently reported interaction between ANG II and ceramide, a lipid second messenger that mediates cytokine receptor activation. Finally, we discuss the potential physiological cross talk that may be operative between the angiotensin receptor subtypes in relation to health and cardiovascular disease. This may be clinically relevant, inasmuch as inhibitors of the RAS are increasingly used in treatment of hypertension and coronary heart disease, where activation of the RAS is recognized.