Biomedical subjects

R C Webb

Publications and source records attributed to R C Webb.

At least 19 recordsLinked to original sources

PubMed2006

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.

Acetophenones↗

PubMed2005

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.

Animals↗

PubMed2004

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.

Administration, Topical↗

PubMed2003

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.

Animals↗

PubMed2003

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.

Humans↗

PubMed2001

RhoA/Rho-kinase, vascular changes, and hypertension.

Hypertension, the result of a sustained increase in vascular peripheral resistance, is partly due to vascular remodeling and increased vasoconstrictor sensitivity. Stimulation of heterotrimeric G-protein-coupled receptors by various contractile agonists activates intracellular signaling molecules to result in an increase in cytosolic Ca++ and the subsequent phosphorylation of myosin light chain by Ca++/calmodulin-dependent myosin light chain kinase. Additionally, a portion of alpha-adrenergic, serotonergic, and endothelin-1-induced contraction is partially mediated by the calcium-independent activation of the small G-protein RhoA and of a downstream target, Rho-kinase. Isolated arteries from hypertensive animals have been shown to have an increased contractile sensitivity to various agonists and to exhibit evidence of remodeling. Recent data suggest that some of these vascular changes may be mediated by increased activity of RhoA/Rho-kinase, potentially introducing a novel therapeutic approach for the treatment of hypertension.

Animals↗

PubMed2001

Differential salt-sensitivity in the pathogenesis of renal damage in SHR and stroke prone SHR.

The spontaneously hypertensive rat (SHR) and the stroke prone SHR (SHRsp) display contrasting susceptibilities to the development of the severe hypertensive lesions of malignant nephrosclerosis, both with aging and after the provision of a high salt intake on the background of a Japanese style "stroke prone" rodent diet. The SHR is relatively resistant, whereas the SHRsp is markedly susceptible. The responsible mechanisms remain controversial. Blood pressure (BP) radiotelemetry was used to investigate the interrelationship between salt intake, systolic BP, and renal damage in 8- to 12-week-old male SHR and SHRsp given a standard North American style diet for 6 weeks, a standard diet plus 1% NaCl as drinking water for 6 weeks, or an 8% NaCl diet plus tap water for 4 weeks. After 4 weeks, BP was significantly greater in the SHRsp compared to the SHR and was significantly more sensitive to supplemental salt in the SHRsp than in SHR. Average systolic pressures during week 5 (after 4 weeks on standard diet plus tap water, standard diet plus 1% NaCl, and 8% NaCl diet plus tap water) were 188.0 +/- 3.0 mm Hg, 207.3 +/- 5.6 mm Hg, and 226 +/- 9.4 mm Hg in SHRsp compared with 171.4 +/- 3.8 mm Hg, 180.6 +/- 3.8 mm Hg, and 190.3 +/- 5.0 mm Hg in SHR. In the absence of supplemental NaCl, both strains exhibited minimal evidence of hypertensive renal damage until about 16 weeks of age. A high salt intake resulted in the development of lesions of malignant nephrosclerosis (fibrinoid necrosis and thrombosis of small vessels and glomeruli) in the SHRsp but not in the SHR; semiquantitative histologic renal damage scores in SHRsp versus SHR being 10.4 +/- 2.0 versus 0.7 +/- 0.2 after 6 weeks of standard diet plus 1% NaCl, and 32.1 +/- 2.5 versus 0.7 +/- 0.4 after 4 weeks of 8% NaCl diet plus tap water; P < .001 for both comparisons. The development of more severe hypertension in salt-supplemented SHRsp could only partly account for the severity of renal damage in SHRsp, the increase in which was disproportionate to the increase in absolute BP. However, the rate of increase of BP was greater in the SHRsp and this might have contributed to the greater renal damage observed in the SHRsp. These data indicate that the contrasting genetic susceptibility to renal damage between SHR and SHRsp is mediated, at least in part, by a differential BP salt sensitivity.

Animals↗

PubMed2001

Increased membrane sphingomyelin and arachidonic acid in stroke-prone spontaneously hypertensive rats.

BACKGROUND: Cell membrane composition and fluidity are altered in hypertension. Previous reports suggest arachidonic acid, a metabolically active fatty acid, is increased in the membranes of hypertensive animals compared to control. This increase in unsaturated fatty acids does not explain the observed reduction in fluidity in hypertensive rats, suggesting some other factors affecting fluidity may be present. It has been suggested that the metabolism of sphingomyelin is altered in genetic hypertension. We hypothesized that membrane sphingomyelin content is increased in hypertensive animals. PROCEDURES: Stroke-prone spontaneously hypertensive rats (n = 8) were compared with Wistar-Kyoto rats (n = 8). Erythrocyte membranes were prepared and the lipids extracted and separated. Fatty acid methyl esters were produced, identified, and quantified by gas chromatography-mass spectrometry; membrane lipid content was also assessed. RESULTS: The concentration of sphingomyelin was higher in the membrane of the hypertensive rats (45.7+/-6 v 22.4+/-2 microg/mg of protein) compared to control. The previously observed increase in membrane arachidonic acid content was observed in hypertensive animals when compared to control (130+/-32 v 40+/-3 microg/mg of protein). However, this difference was confined to the phosphatidylinositol (18+/-4 v 6.5+/-1.5 microg/mg of protein) and free fatty acid (2.1+/-0.4 v 0.6+/-0.1 microg/mg of protein) fractions. CONCLUSION: We hypothesize that reports of reduced membrane fluidity observed in hypertension may be due to an increase in the proportion of sphingomyelin in the cell membrane.

Animals↗

PubMed2001

RhoA/Rho-kinase: a novel player in the regulation of penile erection.

Current research has centered around the role of nitric oxide in the stimulation of cavernosal vasodilation and erection. However, recent evidence from our lab details the importance of endogenous vasoconstrictor mechanisms in maintaining a flaccid penile state, and further demonstrates that the inhibition of endogenous vasoconstriction is sufficient to stimulate erection in a rat model. In this article, we suggest inhibition of endogenous vasoconstriction as a potential therapeutic avenue in the treatment of erectile dysfunction. We also speculate on potential physiologic mechanisms by which endogenous vasoconstriction is inhibited in order for arousal-initiated vasorelaxation, and erection, to occur.

Enzyme Inhibitors↗

PubMed2001

Decreased penile erection in DOCA-salt and stroke prone-spontaneously hypertensive rats.

Numerous etiological studies have established a positive clinical association between hypertension and erectile dysfunction. However, to date, the mechanism underlying this dysfunction remains to be established. In this study, we demonstrate the presence of erectile dysfunction in two rat models of hypertension, and hypothesize that increased vasoconstrictor signaling via Rho-kinase contributes to the decreased erectile response. We found deoxycorticosterone-salt and stroke prone-spontaneously hypertensive rats to exhibit a decreased erectile response, recorded as intracavernosal pressure/mean arterial pressure (ICP/MAP) upon electrical stimulation of the major pelvic ganglion. As previously shown, inhibition of Rho-kinase activity by intracavernosal injection of the selective inhibitor, Y-27632, resulted in an increase in ICP/MAP. However, Y-27632 was significantly less effective at increasing ICP/MAP in the hypertensive as compared to normotensive rats. Additionally, intracavernosal injection of Y-27632 potentiated the voltage-stimulated increase in ICP/MAP in both hypertensive and normotensive rats, but was less effective at potentiating the voltage-mediated erectile response in the hypertensive rats. Altogether, our data demonstrate a decreased erectile response in a mineralocorticoid and genetic model of hypertension, and suggest the role of increased cell signaling by Rho-kinase in the vasoconstrictor activity of erectile dysfunction associated with hypertension.

Amides↗

PubMed2001

Microtubule depolymerization facilitates contraction of vascular smooth muscle via increased activation of RhoA/Rho-kinase.

The microtubule network is in a dynamic equilibrium between free and polymerized tubulin, with depolymerization resulting in increased cellular contractility (1-4). Originally, microtubule depolymerization was thought to facilitate contractile responses via the release of an internal, mechanical opposition to contraction. However, recent evidence suggests that depolymerization may also lead to the enhanced activity of various intracellular signaling proteins. The precise signaling pathway by which microtubule depolymerization facilitates vascular smooth muscle contraction is unknown. In non-vascular cells, depolymerization initiates stress fiber formation via increased activity of the small G-protein, RhoA (5-7). The role of this signaling candidate in a calcium-sensitizing contractile pathway is well established. We and others have found it tempting to speculate that RhoA mediates a contractile pathway enhanced by microtubule depolymerization. We further hypothesize the involvement of microtubule depolymerization (via RhoA and Rho-kinase) in the regulation of vascular smooth muscle contraction, with evidence of potential augmentations of this pathway contributing to the increased vasoconstrictor sensitivity seen in various hypertensive animal models.

Animals↗

PubMed2001

Impaired ceramide signalling in spontaneously hypertensive rat vascular smooth muscle: a possible mechanism for augmented cell proliferation.

OBJECTIVES: In hypertension, the vascular wall undergoes morphological changes that alter mechanical responses to vasoactive substances. Ceramide is a recently identified second messenger synthesized in response to cytokines such as tumour necrosis factor alpha (TNF-alpha). It has been previously demonstrated that vascular smooth muscle cells (VSMC) from genetically hypertensive rats proliferate at a higher rate than those of normotensive origin. We tested the hypothesis that the ceramide pathway is impaired in VSMC from spontaneously hypertensive rats (SHR). DESIGN: VSMC were isolated from aortae of SHR and from Wistar-Kyoto (WKY) rats. Ceramide levels were measured under baseline and agonist-stimulated conditions and cell proliferation was monitored. METHODS: Cell proliferation was determined by cell counting. Ceramide levels were determined via radioactive labelling, high-performance thin-layer chromatography and phosphorimaging. Relative mRNA levels of neutral sphingomyelinase were determined using semi-quantitative polymerase chain reaction (PCR). RESULTS: Basal ceramide levels in untreated cells were lower in cells from SHR compared to WKY rats. During chronic treatment with TNF-alpha, ceramide levels increased in WKY rat cells but remained unchanged in cells from SHR. TNF-alpha treatment had an inhibitory effect on WKY rat VSMC proliferation, but stimulated proliferation in cells from SHR. Short-term incubation with TNF-alpha resulted in a greater increase in ceramide in cells from WKY rats than those from SHR. Semiquantitative PCR analysis indicated that neutral sphingomyelinase mRNA may be reduced in SHR VSMC. CONCLUSIONS: We conclude that ceramide synthesis is impaired in vascular smooth muscle from SHR and may contribute to increased VSMC proliferation in hypertension.

Animals↗

PubMed2001

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.

Animals↗

PubMed2001

Mineralocorticoids upregulate arterial contraction to epidermal growth factor.

The present studies test the hypothesis that contraction to EGF is dependent on mineralocorticoids and/or an elevation in systolic blood pressure (SBP). Endothelium-denuded thoracic aortas from sham normotensive, N(omega)-nitro-L-arginine (L-NNA) hypertensive, Wistar-Kyoto (WKY), and spontaneously hypertensive rats (SHR) were used in isolated tissue-bath experiments. Maximal contraction to epidermal growth factor [EGF; percentage of phenylephrine (PE; 10 umol/l)-induced contraction] was greater in strips from L-NNA (32 +/- 5%) and SHR (53 +/- 8%) rats compared with sham and WKY rats (17 +/- 1 and 12 +/- 4%, respectively). Wistar-Furth rats became only mildly hypertensive when given DOCA salt (134 +/- 6 mmHg) compared with Wistar rats (176 +/- 9 mmHg), but aortas from both strains had a similarly enhanced contraction to EGF (approximately 9 times the maximal contraction of sham aorta). Furthermore, in vitro incubation of aortas from Wistar and Wistar-Furth rats with aldosterone (10 nmol/l) increased EGF-receptor mRNA expression by >50%. These data indicate that arterial contraction to EGF may occur independent of hypertension and be stimulated by mineralocorticoids.

Aldosterone↗

PubMed2001

Spironolactone reduces cerebral infarct size and EGF-receptor mRNA in stroke-prone rats.

Remodeling of the cerebral vasculature contributes to the pathogenesis of cerebral ischemia. Remodeling is caused by increased smooth muscle proliferation and may be due to an increase in the responsiveness of vascular cells to epidermal growth factor (EGF). Aldosterone is a risk factor for stroke, and the literature suggests it may play a role in increasing the expression of the receptor for EGF (EGFR). We hypothesized that mRNA for the EGF-stimulated pathway would be elevated in the vasculature of stroke-prone spontaneously hypertensive rats (SHRSP) and that this and experimental ischemic cerebral infract size would be reduced by aldosterone inhibition with spironolactone. We found that spironolactone treatment reduced the size of cerebral infarcts after middle cerebral artery occlusion in SHRSP (51.69 +/- 3.60 vs. 22.00 +/- 6.69% of hemisphere-infarcted SHRSP vs. SHRSP + spironolactone P < 0.05). Expression of EGF and EGFR mRNA was higher in cerebral vessels and aorta from adult SHRSP compared with Wistar-Kyoto rats. Only the expression of EGFR mRNA was elevated in the young SHRSP. Spironolactone reduced the EGFR mRNA expression in the aorta (1.09 +/- 0.25 vs. 0.56 +/- 0.11 phosphorimage units SHRSP vs. SHRSP + spironolactone P < 0.05) but had no effect on EGF mRNA. In vitro incubation of aorta with aldosterone +/- spironolactone produced similar results, suggesting a direct effect of aldosterone. Thus spironolactone may reduce the size of cerebral infarcts via a reduction in the expression of the EGFR mRNA, leading to reduced remodeling.

Aldosterone↗

PubMed2001

Effect of Rho-kinase inhibition on vasoconstriction in the penile circulation.

A recent report from this laboratory (Chitaley K, Wingard C, Webb R, Branam H, Stopper V, Lewis R, and Mills T. Nature Medicine 7: 119-122, 2001) showed that inhibition of Rho-kinase increased the erectile response (intracavernosal pressure and mean arterial pressure) by a process that does not require nitric oxide or cGMP. The present study investigated whether vasoconstrictor agents, which are active in the penis, act via the Rho-kinase pathway. Western analysis revealed RhoA and Rho-kinase protein in the penis. Treatment with the selective Rho-kinase inhibitor Y-27632 significantly increased the magnitude of the erectile response. Intracavernous administration of endothelin-1 (ET-1; 50 pmol) or methoxamine (10 microg/kg) reduced the erectile response to autonomic stimulation. If Y-27632 was given before ET-1 or methoxamine, the vasoconstrictor effect was reduced, and intracavernosal pressure and mean arterial pressure remained elevated. However, when given after methoxamine, Y-27632 had a reduced vasodilatory effect, and Y-27632 had no vasodilatory effect when given after ET-1. These findings suggest that ET-1 and methoxamine increase Rho-kinase activity in the cavernous circulation and support the hypothesis that the vasoconstriction that maintains the penis in the nonerect state is mediated, in part, by the Rho-kinase pathway.

Amides↗

PubMed2001

Increased dilator response to heptanol and octanol in aorta from DOCA-salt-hypertensive rats.

This study tests the hypothesis that contractile responses in aortae of hypertensive rats are more dependent on gap junctional communication compared to those from normotensive rats. The experimental approach was pharmacological, using inhibitors of gap junctional activity (heptanol and octanol). Two models of experimental hypertension were characterized: (1) mineralocorticoid (DOCA)-hypertensive rats and (2) stroke-prone spontaneous hypertensive rats (SHRSP). Vessels from DOCA-hypertensive rats showed a greater relaxation to heptanol and octanol, particularly when precontracted with phenylephrine, compared to sham-operated animals. Octanol-induced relaxation in aortic segments from SHRSP did not differ from normotensive values regardless of the agonist used to cause contraction. These results suggest that in DOCA hypertension, gap junctional communication and voltage-operated calcium channels are differentially regulated, which could explain in part the changes in vascular reactivity observed in mineralocorticoid hypertension.

Animals↗

PubMed2001

Enhanced relaxation to the rho-kinase inhibitor Y-27632 in mesenteric arteries from mineralocorticoid hypertensive rats.

Increased vasoconstriction is characteristic of hypertension. In this study, we tested the hypothesis that changes in vascular responses during mineralocorticoid hypertension may be due to increased activation of the Rho/Rho-kinase pathway. To test this, relaxation responses to the Rho-kinase inhibitor Y-27632 were determined by measuring isometric force in deendothelialized mesenteric arteries from mineralocorticoid-hypertensive rats and sham-operated controls. Following agonist-induced contraction by serotonin (5-HT, 5-hydroxytryptamine), arteries from hypertensive rats demonstrated a greater relaxation to the Rho-kinase inhibitor Y-27632 (65 +/- 5% vs. 28 +/- 10%). Treatment with an EC50 concentration of Y-27632 following a KCl-induced contraction caused minimal relaxation of arteries in both groups of animals. These findings suggest that augmented Rho-kinase activity in the vasculature of mineralocorticoid hypertensive rats may contribute to the enhanced vascular reactivity of agonist-mediated stimuli characteristic of this model of hypertension.

Amides↗