Norepinephrine-induced Contraction Research Articles

Circulating mitochondrial DNA and Toll-like receptor 9 are associated with vascular dysfunction in spontaneously hypertensive rats.

Immune system activation is a common feature of hypertension pathogenesis. However, the mechanisms that initiate this activation are not well understood. Innate immune system recognition and response to danger are becoming apparent in many cardiovascular diseases. Danger signals can arise from not only pathogens, but also damage-associated molecular patterns (DAMPs). Our first hypothesis was that the DAMP, mitochondrial DNA (mtDNA), which is recognized by Toll-like receptor 9 (TLR9), is elevated in the circulation of spontaneously hypertensive rats (SHR), and that the deoxyribonuclease enzymes responsible for its degradation have decreased activity in SHR. Based on these novel SHR phenotypes, we further hypothesized that (i) treatment of SHR with an inhibitory oligodinucleotide for TLR9 (ODN2088) would lower blood pressure and that (ii) treatment of normotensive rats with a TLR9-specific CpG oligonucleotide (ODN2395) would cause endothelial dysfunction and increase blood pressure. We observed that SHR have elevated circulating mtDNA and diminished deoxyribonuclease I and II activity. Additionally, treatment of SHR with ODN2088 lowered systolic blood pressure. On the other hand, treatment of normotensive rats with ODN2395 increased systolic blood pressure and rendered their arteries less sensitive to acetylcholine-induced relaxation and more sensitive to norepinephrine-induced contraction. This dysfunctional vasoreactivity was due to increased cyclooxygenase and p38 mitogen-activated protein kinase activation, increased reactive oxygen species generation, and reduced nitric oxide bioavailability. Circulating mtDNA and impaired deoxyribonuclease activity may lead to the activation of the innate immune system, via TLR9, and contribute to elevated arterial pressure and vascular dysfunction in SHR.

896 Additive contractions by thromboxane A2 and endothelin-1 are stronger than norepinephrineinduced contractions in human prostate smooth muscle

896 Additive contractions by thromboxane A2 and endothelin-1 are stronger than norepinephrineinduced contractions in human prostate smooth muscle

Intermedin inhibits norepinephrine-induced contraction of rat seminal vesicle

To study the effect of inter medin(IMD) on smooth muscle of rat seminal vesicles including the specific receptors and the signal pathways involved. The contraction of the seminal vesicle in response to norepinephrine (NE) and ADM2/IMD was studied by the organ bath method. The effects of antagonists for calcitonin gene related peptide (CGRP), adrenomedullin (ADM) and IMD receptors, and inhibitors of nitric oxide synthase, [L-NG-Nitroarginine Methyl Ester, L-NAME) and cAMP-dependent protein kinase (PKA), KT5720] were also investigated. The first overshoot, amplitude, frequency and basal tone were measured. There is no significant effect of IMD on the initial overshoot, frequency and the basal tone in the seminal vesicle contraction. Only the amplitude of the contraction induced by NE was inhibited by IMD. The IMD inhibitory actions on amplitude were completely blocked by hADM22-52 and L-NAME, but not by hCGRP8-37 or KT5720. Furthermore, the action was diminished by IMD17-47. The results demonstrated that the inhibitory action of IMD on NE-induced seminal vesicle contraction was mediated via the ADM receptor(s) and the nitric oxide production pathway, partially by the IMD receptor, but not by the CGRP receptor and the cAMP-PKA pathway.

MP43-14 EFFECT OF ANGIOTENSIN II TYPE 1 RECEPTOR BLOCKER, OLMESARTAN ON CORPUS CAVERNOSUM DAMAGES IN THE SPONTANEOUSLY HYPERTENSIVE RAT

INTRODUCTION AND OBJECTIVES: In this study, we focused on whether olmesartan, an angiotensin II receptor blocker, or nifedipine, an L-type calcium channel blocker, could prevent dysfunction of NO-induced relaxation in corpus cavernosum smooth muscle in the spontaneously hypertensive rat (SHR). METHODS: Twelve-week-old male SHRs, and Wistar rats as aged-matched normotensive controls were used in this study. The treatment groups were administered olmesartan (1 or 3mg/kg, per orally (p.o.); Olm1 or Olm3, respectively) or nifedipine (30mg/kg, p.o.; Nif) for six weeks daily. Penile function was evaluated by organ bath studies with norepinephrine-induced contractions and acetylcholineinduced relaxations. Penile cGMP concentrations and mRNA levels of eNOS and nNOS were investigated. RESULTS: The SHR showed significantly increased blood pressure (BP), norepinephrine-induced hyper-contractions, acetylcholine-induced hypo-relaxations, decreased cGMP concentrations, and decreased eNOS and nNOS mRNA levels in the penile tissue. Treatment with high dose of olmesartan significantly decreased BP, significantly increased the penile cGMP concentrations, partially normalized the eNOS and nNOS mRNA levels, significantly decreased the norepinephrine-induced contractions, and normalized the acetylcholineinduced relaxation compared to the SHR. Nifedipine significantly decreased BP, increased cGMP, and normalized the hyper-contractions and hypo-relaxations observed in the SHR group, while the mRNA levels of eNOS and nNOS were significantly lower compared to the Wistar. CONCLUSIONS: Our results indicate that the dysfunction in the SHR penile tissue could be better reversed by olmesartan treatment and to a slightly lesser extent by nifedipine treatment.

MP47-14 PIOGLITAZONE IS PROTECTIVE AGAINST POST-PROSTATECTOMY ERECTILE DYSFUNCTION

INTRODUCTION AND OBJECTIVES: Penile smooth muscle tone is finely tuned by neurogenic stimuli. Alteration of neurogenic regulation could cause an imbalance in favour of contractile stimuli and compromise erection. We have previously observed that calcium-activated potassium channel (KCa) stimulation enhances vasodilatory capacity of PDE5 inhibitors in human penile arteries. The aim of this work was to evaluate the impact of KCa modulation on the neurogenic regulation of cavernosal smooth muscle tone in rats and humans. METHODS: Rat corpus cavernosum (RCC) was obtained from thirty 12-16 weeks old male Sprague-Dawley rats. Human corpus cavernosum (HCC) strips were obtained from 20 patients with erectile dysfunction (ED) who gave informed consent at the time of penile prosthesis implantation. RCC and HCC were mounted in organ chambers and responses to electrical field stimulation (EFS) were evaluated. RESULTS: EFS caused reproducible, frequency-dependent contractions in RCC. Blockade of KCa with tertaethylammonium (TEA) markedly potentiated neurogenic contractions (16Hz, 67.2 3.2% vs. 105.6 4.6% at 5mM, p<0.001) but did not modify norepinephrineinduced contractions. By using specific KCa subtype blockers, largeconductance KCa (BK) appeared as responsible for modulation of neurogenic contractions. Stimulation of BK with NS1619 significantly inhibited EFS-induced contractions in RCC (67.5 5.4% vs. 39.3 5.8%, p<0.01 at 30mM). BK stimulation also caused a significant potentiation of EFS-induced relaxations in phenylephrine-contracted RCC treated with guanethidine and atropine (2 Hz, 31.8 6.9% vs. 64.9 6.7%, p<0.01). Similar results were obtained in HCC where TEA caused concentration-dependent enhancement of EFS-induced contractions (35.3 3.4% vs. 118.5 32.0% at 5mM, p<0.01) while BK activation with NS1619 (30mM) inhibited neurogenic contractions and potentiated neurogenic relaxations. CONCLUSIONS: BK participates in regulation of neurogenic control of cavernosal smooth muscle tone. Pharmacological stimulation of these channel would favor relaxant responses over contractile input in rat and human cavernosal tissue. Thus, BK stimulation could be a reasonable strategy for treating ED mainly in situations involving imbalanced regulation of neurogenic control.

MP47-13 CALCIUM-ACTIVATED POTASSIUM CHANNEL (KCA) STIMULATION MODULATES NEUROGENIC CONTRACTION AND FAVOURS NEUROGENIC RELAXATION IN RAT AND HUMAN CORPUS CAVERNOSUM

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research II1 Apr 2014MP47-13 CALCIUM-ACTIVATED POTASSIUM CHANNEL (KCA) STIMULATION MODULATES NEUROGENIC CONTRACTION AND FAVOURS NEUROGENIC RELAXATION IN RAT AND HUMAN CORPUS CAVERNOSUM Juan I. Martínez-Salamanca, José M. La Fuente, Argentina Fernández, Eduardo Martínez-Salamanca, Joaquín Carballido, and Javier Angulo Juan I. Martínez-SalamancaJuan I. Martínez-Salamanca More articles by this author , José M. La FuenteJosé M. La Fuente More articles by this author , Argentina FernándezArgentina Fernández More articles by this author , Eduardo Martínez-SalamancaEduardo Martínez-Salamanca More articles by this author , Joaquín CarballidoJoaquín Carballido More articles by this author , and Javier AnguloJavier Angulo More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1466AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail Introduction and Objectives Penile smooth muscle tone is finely tuned by neurogenic stimuli. Alteration of neurogenic regulation could cause an imbalance in favour of contractile stimuli and compromise erection. We have previously observed that calcium-activated potassium channel (KCa) stimulation enhances vasodilatory capacity of PDE5 inhibitors in human penile arteries. The aim of this work was to evaluate the impact of KCa modulation on the neurogenic regulation of cavernosal smooth muscle tone in rats and humans. Methods Rat corpus cavernosum (RCC) was obtained from thirty 12-16 weeks old male Sprague-Dawley rats. Human corpus cavernosum (HCC) strips were obtained from 20 patients with erectile dysfunction (ED) who gave informed consent at the time of penile prosthesis implantation. RCC and HCC were mounted in organ chambers and responses to electrical field stimulation (EFS) were evaluated. Results EFS caused reproducible, frequency-dependent contractions in RCC. Blockade of KCa with tertaethylammonium (TEA) markedly potentiated neurogenic contractions (16Hz, 67.2±3.2% vs. 105.6±4.6% at 5mM, p<0.001) but did not modify norepinephrine-induced contractions. By using specific KCa subtype blockers, large-conductance KCa (BK) appeared as responsible for modulation of neurogenic contractions. Stimulation of BK with NS1619 significantly inhibited EFS-induced contractions in RCC (67.5±5.4% vs. 39.3±5.8%, p<0.01 at 30μM). BK stimulation also caused a significant potentiation of EFS-induced relaxations in phenylephrine-contracted RCC treated with guanethidine and atropine (2 Hz, 31.8±6.9% vs. 64.9±6.7%, p<0.01). Similar results were obtained in HCC where TEA caused concentration-dependent enhancement of EFS-induced contractions (35.3±3.4% vs. 118.5±32.0% at 5mM, p<0.01) while BK activation with NS1619 (30μM) inhibited neurogenic contractions and potentiated neurogenic relaxations. Conclusions BK participates in regulation of neurogenic control of cavernosal smooth muscle tone. Pharmacological stimulation of these channel would favor relaxant responses over contractile input in rat and human cavernosal tissue. Thus, BK stimulation could be a reasonable strategy for treating ED mainly in situations involving imbalanced regulation of neurogenic control. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e523 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Juan I. Martínez-Salamanca More articles by this author José M. La Fuente More articles by this author Argentina Fernández More articles by this author Eduardo Martínez-Salamanca More articles by this author Joaquín Carballido More articles by this author Javier Angulo More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Contribution of Ca2+-Dependent Cl−Channels to Norepinephrine-Induced Contraction of Femoral Artery Is Replaced by Increasing EDCF Contribution during Ageing

The activation of Ca2+-dependent Cl− channels during norepinephrine-induced contraction of vascular smooth muscle was suggested to depolarize cell membrane and to increase Ca2+ entry. Hypertension and ageing are associated with altered Ca2+ handling including possible activation of Ca2+-dependent Cl− channels. Our study was aimed to determine Ca2+-dependent Cl− channels contribution to norepinephrine-induced contraction during hypertension and ageing. Norepinephrine-induced concentration-response curves of femoral arteries from 6- and 12-month-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were recorded using wire myograph. Pretreatment with Ca2+-dependent Cl- channel inhibitor indanyloxyacetic acid 94 [R(+)-IAA-94](IAA) attenuated norepinephrine-induced contraction in all groups, but relatively more in WKY than SHR arteries. The attenuation of norepinephrine-induced contraction after Ca2+-dependent Cl− channels blockade was partially reduced in 12-month-old WKY rats, but substantially diminished in 12-month-old SHR. IAA effect was enhanced after NO synthase inhibition but decreased by ageing. In 20-month-old WKY rats norepinephrine-induced contraction was not affected by IAA but was almost abolished after cyclooxygenase inhibition by indomethacin or niflumic acid. In conclusion, contribution of Ca2+-dependent Cl− channels to norepinephrine-induced contraction diminished with age, hypertension development, and/or NO synthesis inhibition. Ca2+-dependent Cl− channels are important for maintenance of normal vascular tone while their inactivation/closing might be a pathological mechanism.

Effect of an angiotensin II receptor blocker and a calcium channel blocker on hypertension associated penile dysfunction in a rat model.

Possible effect of olmesartan, an angiotensin II receptor blocker (ARB), or nifedipine, an L-type calcium channel blocker, on penile dysfunction in the spontaneously hypertensive rat (SHR) was investigated in this study. Twelve-week-old male SHRs were treated with olmesartan (1 or 3 mg/kg, per orally (p.o.)) or nifedipine (30 mg/kg, p.o.) once a day for 6 weeks. Wistar rats and SHRs with vehicle treatment were used as controls. Penile cGMP and malondialdehyde concentrations, and mRNA levels of endothelial and neuronal NO synthase (eNOS and nNOS) were measured. Penile function was evaluated by organ bath studies with norepinephrine-induced contractions and acetylcholine-induced relaxations. The SHR showed significantly increased blood pressure, decreased cGMP concentrations, increased malondialdehyde concentrations, decreased eNOS and nNOS mRNA levels, norepinephrine-induced hyper-contractions, and acetylcholine-induced hypo-relaxations in the penile tissue compared to the Wistar rat. Both nifedipine and olmesartan significantly decreased blood pressure, increased cGMP and normalized the hyper-contractions and hypo-relaxations observed in the SHR group. However, not nifedipine but olmesartan improved the malondialdehyde concentrations and increased mRNA levels of eNOS and nNOS in the penis. Our results indicate that the hypertension-associated penile dysfunction might be treated with ARBs such as olmesartan better than calcium channel blockers, such as nifedipine.

The receptor antagonist picotamide inhibits adrenergic and thromboxane-induced contraction of hyperplastic human prostate smooth muscle

Inhibition of prostate smooth muscle contraction is an important strategy for medical treatment of lower urinary tract symptoms (LUTS). Besides α1-adrenoceptors, prostate smooth muscle contraction is induced by activation of thromboxane (TXA2) receptors (TXA2-R). Here, we examined the effects of the TXA2-R antagonist picotamide on contraction of human prostate tissue. Prostate tissues were obtained from radical prostatectomy. The effects of picotamide (300 μM), L-665,240 (3 μM), and seratrodast (3 μM) on U46619-, electric field stimulation- (EFS-), phenylephrine-, and norepinephrine-induced contractions were studied in organ baths. Expression of TXA2-R and TXA2 synthase (TXS) was examined by fluorescence stainings. Picotamide, seratrodast, and L-655,240 inhibited concentration-dependent contractions induced by the TXA2 analog U46619. Picotamide, but not seratrodast or L-655,240, inhibited frequency-dependent contractions induced by EFS. Picotamide inhibited concentration-dependent contractions induced by norepinephrine or by the selective α1-adrenoceptor agonist phenylephrine. In prostate strips, where only submaximal contraction by a low dose of phenylephrine was induced, application of U46619 raised tone to maximum phenylephrine-induced tension. Immunoreactivity for TXA2-R and TXS was observed in the stroma and in epithelial cells of glands. Colocalization of both immunoreactivites was observed with the smooth muscle markers calponin and α-smooth muscle actin, with the epithelial marker pan-cytokeratin, and with prostate-specific antigen in the stroma and glands. The receptor antagonist picotamide inhibits α1-adrenergic, TXA2-mediated, and EFS-induced contractions in the human prostate. To the best of our knowledge, this is the first antagonist able to inhibit two different contraction systems in the prostate.

Monoclonal Antibody against α-Actinin 4 from Human Umbilical Vein Endothelial Cells Inhibits Endothelium-Dependent Vasorelaxation

Background: This study was attempted to identify new molecules expressed on the plasma membrane of human umbilical vein endothelial cells (HUVECs) using monoclonal antibody-based proteomics technology and to determine the effect of the identified antibody on vascular reactivity. Methods: Twenty-two antibodies were developed from rats inoculated with HUVECs, and their effects were determined by observing vascular reactivity. Results: Among the 22 antibodies, the C-7 antibody significantly inhibited endothelium-dependent vasorelaxation in response to acetylcholine (ACh) but not to histamine. Moreover, the C-7 antibody did not affect norepinephrine-induced contraction in either the endothelium-intact or -denuded aorta. A proteomics study involving immunoprecipitation of the C-7 antibody with biotinylated HUVECs showed that this antibody binds to plasma membrane proteins corresponding to immunoglobulin heavy chain (VHDJ region), chaperonin-containing T-complex polypeptide 1 and α-actinin 4. The muscarinic M3 ACh receptor and α-actinin 4 were colocalized on the plasma membrane of HUVECs, and the colocalization was found to increase in response to ACh and was inhibited by pretreatment with the C-7 antibody. Conclusions: These results demonstrate that monoclonal C-7 antibody exerts an inhibitory effect on endothelium-dependent vasorelaxation induced by ACh and that this response may at least partially result from the inhibition of α-actinin 4.

Streptozotocin-induced vascular and biochemical changes in rats: Effects of rosiglitazone vs. metformin

The aim was to investigate rosiglitazone and metformin effects on some vascular and biochemical changes associated with streptozotocin (55mg/kg; i.p.)-induced hyperglycaemia in rats. Isolated aortas were used to evaluate their reactivity towards norepinephrine, acetylcholine, and sodium nitroprusside. Blood samples were used to assess the biochemical changes of some parameters viz., plasma lipid peroxides and nitric oxide levels and erythrocytic glutathione peroxidase activity. Hyperglycaemic animals orally received rosiglitazone (0.5mg/kg) or metformin (150mg/kg) daily for 2weeks and their effects were determined 24h after the last dose. Our results revealed that streptozotocin-induced hyperglycaemia is associated with impaired vascular reactivity towards various agents, increased lipid peroxides level, decreased glutathione peroxidise activity, and decreased nitric oxide level. Both drugs further decreased norepinephrine-induced contraction and improved acetylcholine- and sodium nitroprusside-induced relaxations. Rosiglitazone restored the alterations in all tested biochemical parameters while metformin restored only glutathione peroxidise activity. In conclusion both drugs show beneficial effects against the vascular dysfunction associated with hyperglycaemia which might be related to their euglycaemic activity in addition to anti-oxidant property of rosiglitazone and a direct effect of metformin on vascular smooth muscle.

Artemisia copa aqueous extract as vasorelaxant and hypotensive agent

Ethnopharmacological relevanceArtemisia copa Phil. (Asteraceae) is a medicinal plant commonly used in traditional medicine in Argentina. Aim of the studyThe vasorelaxant and hypotensive activities of the aqueous extract of Artemisia copa have been investigated. Materials and methodsThe in vitro effect of the extract and isolated compounds from Artemisia copa was investigated using isolated rat aortic rings. The acute effect caused by the intravenous (i.v.) infusion (0.1–300mg/kg) on blood pressure and heart rate was evaluated in spontaneous hypertensive rats. In addition, a phytochemical analysis of the extract was performed by HPLC. ResultsArtemisia copa had a relaxant effect in endothelium-intact aortic rings that had been pre-contracted with 10−7M phenylephrine (Emax=96.7±1.3%, EC50=1.1mg/ml), 10−5M 5-hydroxytriptamine (Emax=96.7±3.5%, EC50=1.5mg/ml) and 80mM KCl (Emax=97.9± 4.4%, EC50=1.6mg/ml). In denuded aortic rings contracted by phenylephrine, a similar pattern was observed (Emax=92.7±6.5%, EC50=1.8mg/ml). l-NAME, indomethacin, tetraethylammonium and glibenclamide were not able to block the relaxation induced by the extract. Nevertheless, the pre-treatment with Artemisia copa attenuated the CaCl2-induced contraction in a concentration-dependent manner (Emax: 86% of inhibition for 3mg/ml and 52% de-inhibition for 1mg/ml). This pre-treatment also induced a significant attenuation of the norepinephrine-induced contraction in a concentration-dependent manner (Emax: 72.7% of inhibition for 3mg/ml and 27% de inhibition for 1mg/ml) in a Ca2+ free medium. Upon analyzing the composition of the extract, the presence of p-coumaric acid, isovitexin, luteolin and chrysoeriol were found. Luteolin (CE50: 1.5μg/ml), chrysoeriol (CE50: 13.2μg/ml) and p-coumaric acid (CE50: 95.2μg/ml), isolated from the aqueous extract, caused dilatation of thoracic aortic rings pre-contracted with phenylephrine. Artemisia copa administered i.v. also induced a decrease in the mean arterial pressure but did not affect the heart rate in hypertensive rats. ConclusionsThe aqueous extract of Artemisia copa proved to have vasorelaxing and hypotensive effects through the inhibition of Ca2+ influx via membranous calcium channels and intracellular stores. The presence of luteolin, chrysoeriol and p-coumaric acid found in this plant could be involved in this effect.

Effects of dietary flaxseed on atherosclerotic plaque regression

Dietary flaxseed can retard the progression of atherosclerotic plaques. However, it remains unclear whether these antiatherogenic effects extend to plaque regression. In the present study, the therapeutic potential of dietary flaxseed on atherosclerotic plaque regression and vascular contractile function was evaluated using a novel rabbit model. Rabbits were randomly assigned to receive either a regular diet for 12 wk (group I) or a 1% cholesterol-supplemented diet for 4 wk followed by a regular diet for 8 wk (group II). The remaining experimental animals were treated as in group II but were fed for an additional 14 wk with either a regular diet (group III) or a 10% flaxseed-supplemented diet (group IV). Animals in group II showed clear evidence of plaque growth stabilization. Their vessels also exhibited significantly lower norepinephrine-induced contraction and an impaired relaxation response to acetylcholine compared with animals in group I. Dietary flaxseed supplementation resulted in a significant ≈40% reduction in plaque formation (P = 0.033). Animals in both groups II and III displayed improved contraction and endothelium-dependent vessel relaxation. Dietary flaxseed is a valuable strategy to accelerate the regression of atherosclerotic plaques; however, flaxseed intervention did not demonstrate a clear beneficial effect on the vessel contractile response and endothelium-dependent vasorelaxation.

807 THE EFFECT OF HYDROXYFASUDIL ON PENILE DYSFUNCTION IN THE MALE SPONTANEOUSLY HYPERTENSIVE RAT

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research (I)1 Apr 2013807 THE EFFECT OF HYDROXYFASUDIL ON PENILE DYSFUNCTION IN THE MALE SPONTANEOUSLY HYPERTENSIVE RAT Motoaki Saito, Shogo Shimizu, and Shuhei Tomita Motoaki SaitoMotoaki Saito Yonago, Japan More articles by this author , Shogo ShimizuShogo Shimizu Yonago, Japan More articles by this author , and Shuhei TomitaShuhei Tomita Yonago, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.373AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Hypertension represents a major risk factor for erectile dysfunction. Although the etiology of hypertension-induced erectile dysfunction is multifactorial and still unknown, Rho-Rho kinase pathway is one of the key factors. We investigated whether administration of hydroxyfasudil, a Rho kinase inhibitor could alternate dysfunction of NO-induced relaxation in corpus cavernosum smooth muscle in the SHR (spontaneously hypertensive rat). METHODS Twelve-week-old male SHRs were treated with hydroxyfasudil (3 or 10 mg/kg, i.p.) once a day for 6 weeks. Wistar rats and SHRs treated with vehicle were used as age-matched controls. Penile cGMP concentrations and Rho kinase activities were determined, and penile function was estimated by organ bath studies with norepinephrine-induced contractions and acetylcholine-induced relaxations. The participation mRNA levels of eNOS and participation protein levels of eNOS and phosphorylated eNOS were investigated by quantitative real-time PCR methods and immunoblot analysis, respectively. RESULTS The SHR showed significantly decreased cGMP concentrations, increased Rho kinase activities, norepinephrine-induced hyper-contractions, and acetylcholine-induced hypo-relaxations in the penile tissue. Treatment with hydroxyfasudil significantly improved the decreased penile cGMP concentrations, the increased Rho kinase activities, the increased norepinephrine-induced contractions, and the decreased acetylcholine-induced relaxation in a dose-dependent manner. Although there were no significant differences in expression protein levels of eNOS among any of the groups, down-regulation of eNOS mRNAs as well as phosphorylated eNOS were significantly ameliorated after treatment with hydroxyfasudil. CONCLUSIONS Our data suggest that hydroxyfasudil ameliorates hypertension-associated dysfunction of NO-induced relaxation in corpus cavernosum smooth muscle possibly via inhibition of the Rho-Rho kinase pathway and activation of NO-eNOS pathway in the SHR. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e332 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Motoaki Saito Yonago, Japan More articles by this author Shogo Shimizu Yonago, Japan More articles by this author Shuhei Tomita Yonago, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Lipid Emulsions Enhance the Norepinephrine-Mediated Reversal of Local Anesthetic-Induced Vasodilation at Toxic Doses

PurposeIntravenous lipid emulsions have been used to treat the systemic toxicity of local anesthetics. The goal of this in vitro study was to examine the effects of lipid emulsions on the norepinephrine-mediated reversal of vasodilation induced by high doses of levobupivacaine, ropivacaine, and mepivacaine in isolated endothelium-denuded rat aorta, and to determine whether such effects are associated with the lipid solubility of local anesthetics.Materials and MethodsThe effects of lipid emulsions (0.30, 0.49, 1.40, and 2.61%) on norepinephrine concentration-responses in high-dose local anesthetic (6×10-4 M levobupivacaine, 2×10-3 M ropivacaine, and 7×10-3 M mepivacaine)-induced vasodilation of isolated aorta precontracted with 60 mM KCl were assessed. The effects of lipid emulsions on local anesthetic- and diltiazem-induced vasodilation in isolated aorta precontracted with phenylephrine were also assessed.ResultsLipid emulsions (0.30%) enhanced norepinephrine-induced contraction in levobupivacaine-induced vasodilation, whereas 1.40 and 2.61% lipid emulsions enhanced norepinephrine-induced contraction in both ropivacaine- and mepivacaine-induced vasodilation, respectively. Lipid emulsions (0.20, 0.49 and 1.40%) inhibited vasodilation induced by levobupivacaine and ropivacaine, whereas 1.40 and 2.61% lipid emulsions slightly attenuated mepivacaine (3×10-3 M)-induced vasodilation. In addition, lipid emulsions attenuated diltiazem-induced vasodilation. Lipid emulsions enhanced norepinephrine-induced contraction in endothelium-denuded aorta without pretreatment with local anesthetics.ConclusionTaken together, these results suggest that lipid emulsions enhance the norepinephrine-mediated reversal of local anesthetic-induced vasodilation at toxic anesthetic doses and inhibit local anesthetic-induced vasodilation in a manner correlated with the lipid solubility of a particular local anesthetic.

Vascular reactivity of arteria femoralis in adult and aged spontaneously hypertensive and Wistar-Kyoto rats

The relationship of age and hypertension on endothelial dysfunction and increased responses to vasoconstrictor stimuli. Hypertension is a disease accompanied by endothelial dysfunction and is characterized by an impaired vascular reactivity and enhanced activity of sympathetic nervous system. In our experiment, we used spontaneously hypertensive rats representing model of essential hypertension and the Wistar-Kyoto rats as normotensive strain. Femoral arteries of adult and aged rats were put into the chamber of Mulvany-Halpern isometric myograph. As the nutrient solution, the modified Krebs-Henseleit solution having temperature 37 °C and bubbled with O2 was used. After 30 minutes stabilization of blood vessels, a dose-dependent curve of norepinephrine response was recorded (concentrations 3x10-8 M, 10-7 M, 3x10-7 M, 10-6 M, 3x10-6 M, 10-5 M, 3x10-5 M, 10-4 M), followed by a dose-dependent curve of acetylcholine response (concentrations 3x10-8 M, 10-7 M, 3x10-7 M, 10-6 M, 3x10-6 M). Our experiments recorded an increased reactivity to contraction stimuli in spontaneously hypertensive animals. Vascular reactivity to norepinephrine at 5 month and 12 month old rats from the same group was not significantly affected. Our experiments on the other hand, did not record a reduced endothelium-dependent relaxation in hypertensive compared to normotensive animals, neither in different age groups. Increased norepinephrine-induced contraction occurs even before development of reduced acetylcholine-induced relaxation in SHR rats. We predict that in our experiment hypertension plays a bigger role in the development of endothelial dysfunction than aging (Fig. 2, Ref. 22).

Hydroxyfasudil ameliorates penile dysfunction in the male spontaneously hypertensive rat

Hypertension represents a major risk factor for erectile dysfunction. Although the etiology of hypertension-induced erectile dysfunction is multifactorial and still unknown, Rho–Rho kinase pathway is one of the key factors. To investigate whether administration of hydroxyfasudil, a Rho kinase inhibitor could prevent dysfunction of NO-induced relaxation in corpus cavernosum smooth muscle in the SHR (spontaneously hypertensive rat), twelve-week-old male SHRs were treated with hydroxyfasudil (3 or 10mg/kg, i.p.) once a day for 6weeks. Wistar rats and SHRs treatment with vehicle were used as age-matched controls. Penile cGMP concentrations and Rho kinase activities were determined, and penile function was estimated by organ bath studies with norepinephrine-induced contractions and acetylcholine-induced relaxations. The participation mRNA levels of eNOS and participation protein levels of eNOS and phosphorylated eNOS were investigated by quantitative real-time PCR methods and immunoblot analysis, respectively. The SHR showed significantly decreased cGMP concentrations, increased Rho kinase activities, norepinephrine-induced hyper-contractions, and acetylcholine-induced hypo-relaxations in the penile tissue. Treatment with hydroxyfasudil significantly improved the decreased penile cGMP concentrations, the increased Rho kinase activities, the increased norepinephrine-induced contractions, and the decreased acetylcholine-induced relaxation in a dose-dependent manner. Although there were no significant differences in expression protein levels of eNOS among any of the groups, down-regulation of eNOS mRNAs as well as phosphorylated eNOS were significantly ameliorated after treatment with hydroxyfasudil. Our data suggest that hydroxyfasudil ameliorates hypertension-associated dysfunction of NO-induced relaxation in corpus cavernosum smooth muscle possibly via inhibition of the Rho–Rho kinase pathway and activation of NO–eNOS pathway in the SHR.

4-Substitutedphthalazines and phthalazinones: synthesis, characterization and β-adrenergic blocking activity

Novel 4-(4-bromophenyl)phthalazine and phthalazinone derivatives connected through 2-propanol spacer to N-substituted piperazine residue were synthesized. All the new compounds were screened for their effect on β-adrenergic blocking activity on the norepinephrine-induced precontracted aortic ring module. Most test compounds displayed appreciable β-adrenolytic activity compared to propranolol as a reference standard. The results have shown that compounds 3a, 3d, 3e and 7c displayed appreciable inhibition of norepinephrine-induced aortic ring contraction. A novel 4-(4-bromophenyl)phthalazine and phthalazinone derivatives connected through 2-propanol spacer to N-substituted piperazine residue were synthesized. All new compounds were screened for their effect on β-adrenergic blocking activity on the norepinephrine-induced precontracted aortic ring module.

Arteriolar insulin resistance in a rat model of polycystic ovary syndrome

To investigate the vascular dysfunction caused by insulin resistance in polycystic ovary syndrome (PCOS) and the effectiveness of vitamin D in an animal model. Controlled experimental animal study. Animal laboratory at a university research institute. Thirty female Wistar rats. Rats were divided into groups at age 21-28 weeks. Twenty of them were subjected to dihydrotestosterone (DHT) treatment (83 μg/d); ten of them also received parallel vitamin D treatment (120 ng/100 g/wk). Oral glucose tolerance tests with insulin level measurements were performed. Gracilis arterioles were tested for their contractility as well as their nitric oxide (NO)-dependent and insulin-induced dilation using pressure arteriography. Several physiologic parameters, glucose metabolism, and pressure arteriography. DHT treatment increased the passive diameter of resistance arterioles, lowered norepinephrine-induced contraction (30.1 ± 4.7% vs. 8.7 ± 3.6%) and reduced acetylcholine-induced (122.0 ± 2.9% vs. 48.0 ± 1.4%) and insulin-induced (at 30 mU/mL: 21.7 ± 5.3 vs. 9.8 ± 5.6%) dilation. Vitamin D treatment restored insulin relaxation and norepinephrine-induced contractility; in contrast, it failed to alter NO-dependent relaxation. In DHT-treated rats, in addition to metabolically proven insulin resistance, decreased insulin-induced vasorelaxation was observed and was improved by vitamin D treatment without affecting NO-dependent relaxation. The reduction in insulin-induced dilation of arterioles is an important as yet undescribed pathway of vascular damage in PCOS and might explain the clinical effectiveness of vitamin D treatment.

Vascular Dysfunction in Sepsis

The mechanisms contributing to sepsis vascular dysfunction are not well known. We tested the hypothesis that peroxynitrite scavenging ameliorates sepsis-induced macrovascular and microvascular dysfunction. Male Sprague-Dawley rats were killed 48 h after cecal ligation (n = 15) and puncture or sham procedure (n = 15). Their aortas and mesenteric vessels were mounted in organ baths for isometric tension recording. We studied contraction in resting vessels (norepinephrine 1 nM-10 μM and 10 nM-10 μM) and endothelium-dependent relaxation (acetylcholine, 10 nM-10 μM and 1 nM-10 μM) for aortas and microvessels, respectively. Vascular rings were preincubated for 30 min with the superoxide scavenger Cu-Zn-superoxide dismutase (SOD) (100 U/mL), the SOD mimetic and peroxynitrite scavenger tempol (10 M), the NO synthase inhibitor N-nitro-l-arginine methyl ester (10 M), or the peroxynitrite decomposition catalyst manganese tetrakis(4-N-methylpyridyl)porphyrin (MnTMPyP) (10 M). Fluorescence to 3-nitrotyrosine, oxidized dihydroethidium, and NOS2 was assessed in vascular tissue. Vascular NOS2, endothelial nitric oxide synthase (NOS1), NADPH-oxidase-1 (NOX-1), and SOD expression was analyzed by reverse transcription-polymerase chain reaction. Sepsis induced (i) in macrovessels, impairment of norepinephrine-induced contractions; (ii) in microvessels, impairment in norepinephrine-induced contractions and acetylcholine-induced relaxations; (iii) aortic and microvascular tissue increased reactivity to 3-nitrotyrosine, oxidized dihydroethidium, NOS2, and increased expression of NOS2, as well as increased expression of NOX-1 in microvascular tissue. Contractile responses in aortic and microvascular rings improved by ex vivo treatment with MnTMPyP and tempol, whereas vascular relaxation in microvessels improved only with MnTMPyP. Peroxynitrite scavenging protects from vascular dysfunction in sepsis.