T- and L-type voltage-gated calcium channels: their role in diabetic bladder dysfunction.

AIMS: We investigated the mechanisms of diabetic bladder dysfunction (BD) through analysis of the roles of L- and T-type voltage-gated calcium channels (VGCCs), with the ultimate goal of identifying potential drug targets for diabetic BD. METHODS: Bladder function of db/db (type 2 diabetes) and wild type (Wt) mice was evaluated by behavioral tests and in vivo cystometry. Contractile responses of bladder strips to carbachol were measured with or without pre-treatment with nifedipine (a L-type VGCC blocker) or mibefradil (a T-type VGCC blocker). Furthermore, the effects of mibefradil and nifedipine on the proliferation of human bladder smooth muscle cells (BSMCs) were studied. RESULTS: db/db mice had significantly increased voiding frequency, bladder weight, bladder compliance and capacity, and heightened contractile response to carbachol, compared to Wt mice. Nifedipine, but not mibefradil, dramatically suppressed bladder tissue contraction in Wt mice. Whereas nifedipine nearly completely inhibited bladder contraction in db/db mice, mibefradil "normalized" the heightened bladder contractility of db/db mice to the level of Wt mice. In culture, mibefradil, but not nifedipine, inhibited the proliferation of human BSMCs. CONCLUSION: Our results indicate that while L-type VGCCs play a major role in the contraction of both diabetic and non-diabetic bladders, T-type VGCCs are involved in the contraction of diabetic bladders and mediate BSMC proliferation. This study provides support for further investigations on the effect of blockade of T-type VGCC or combined blockade of both types of VGCCs in the treatment of diabetic BD.

Glutathione (GSH) and the GSH synthesis gene Gclm modulate plasma redox and vascular responses to acute diesel exhaust inhalation in mice.

CONTEXT: Inhalation of fine particulate matter (PM2.5) is associated with acute pulmonary inflammation and impairments in cardiovascular function. In many regions, PM2.5 is largely derived from diesel exhaust (DE), and these pathophysiological effects may be due in part to oxidative stress resulting from DE inhalation. The antioxidant glutathione (GSH) is important in limiting oxidative stress-induced vascular dysfunction. The rate-limiting enzyme in GSH synthesis is glutamate cysteine ligase and polymorphisms in its catalytic and modifier subunits (GCLC and GCLM) have been shown to influence vascular function and risk of myocardial infarction in humans. OBJECTIVE: We hypothesized that compromised de novo synthesis of GSH in Gclm⁻/⁺ mice would result in increased sensitivity to DE-induced lung inflammation and vascular effects. MATERIALS AND METHODS: WT and Gclm⁻/⁺ mice were exposed to DE via inhalation (300 µg/m³) for 6 h. Neutrophil influx into the lungs, plasma GSH redox potential, vascular reactivity of aortic rings and aortic nitric oxide (NO•) were measured. RESULTS: DE inhalation resulted in mild bronchoalveolar neutrophil influx in both genotypes. DE-induced effects on plasma GSH oxidation and acetylcholine (ACh)-relaxation of aortic rings were only observed in Gclm⁻/⁺ mice. Contrary to our hypothesis, DE exposure enhanced ACh-induced relaxation of aortic rings in Gclm⁻/⁺ mice. DISCUSSION AND CONCLUSION: THESE data support the hypothesis that genetic determinants of antioxidant capacity influence the biological effects of acute inhalation of DE. However, the acute effects of DE on the vasculature may be dependent on the location and types of vessels involved. Polymorphisms in GSH synthesis genes are common in humans and further investigations into these potential gene-environment interactions are warranted.

Genetics of erectile dysfunction.

PURPOSE: Erectile dysfunction affects 50% of men older than 40 years. Recently more attempts have been made to identify genetic predictors of this disease. We reviewed animal and human data on genes related to the development and increased risk of erectile dysfunction. MATERIALS AND METHODS: A literature search was performed using the PubMed® database. Articles addressing genes involved in erectile dysfunction were evaluated. RESULTS: The majority of studies used a candidate gene approach to investigate genetic polymorphisms of known pathways mediating erection/detumescence. Studies in human and animal models are available. Human studies often compared the frequency of a specifically predetermined genetic polymorphism in men with erectile dysfunction to that in matched controls in whom few genes were persistently replicated. Several gene expression profiling studies are available that targeted specific erectile dysfunction models. Currently, there are few human genome wide association studies of erectile dysfunction. CONCLUSIONS: Studies investigating the genetics of erectile dysfunction are mostly derived from animal models and candidate gene approaches. Candidate gene studies omit the greater portion of the genome, a problem that can be solved using a genome wide association study approach. The lack of persistently replicated results of candidate gene studies may be related to different patient ethnic backgrounds, variations in erectile dysfunction etiology and small sample sizes. Using strict inclusion/exclusion criteria for erectile dysfunction etiology and ethnicity in human studies may lead to improved understanding of the genetics of erectile dysfunction in specific populations.

Altered bladder function in elastin-deficient mice at baseline and in response to partial bladder outlet obstruction.

OBJECTIVE: • To examine functional and molecular changes of the bladders from elastin-haploinsufficient mice (Eln(+/-) ) at baseline as well as in response to partial bladder outlet obstruction (pBOO). MATERIALS AND METHODS: • Female Eln(+/-) and wild type (Wt) mice (3-4 months old) were studied. • The bladder elastin content was quantified by measuring desmosine. • Mice were divided into two groups to undergo surgery to create pBOO or to undergo sham surgery. Three days after surgery, bladder function was evaluated by in vivo cystometry, and the contractile response of bladder strips exposed to electrical field stimulation (EFS) and carbachol was examined by ex vivo myography. RESULTS: • The Eln(+/-) -sham mice had a 33.6% decrease in bladder elastin compared with Wt-sham mice. • Cystometry showed significantly decreased bladder compliance and capacity in Eln(+/-) -sham vs Wt-sham mice; pBOO increased bladder compliance and capacity to a greater extent in Eln(+/-) mice compared with Wt mice. • Bladder strips from Eln(+/-) -sham mice showed a significantly heightened contractile response to both EFS and carbachol compared with Wt-sham mice. • A significantly increased contractile response to carbachol was detected in Wt-pBOO vs Wt-sham but not between Eln(+/-) -pBOO and Eln(+/-) -sham mice. CONCLUSION: • The results that elastin-deficient mice had decreased bladder compliance and capacity and increased bladder contractility; and that Wt-pBOO mice showed an enhanced contractile response to carbachol, but Eln(+/-) -pBOO mice did not, suggest that elastin is critical for normal bladder function and is involved in bladder response to pBOO.

Common pitfalls in some of the experimental studies in erectile function and dysfunction: a consensus article.

INTRODUCTION: Experimental studies investigating physiology of erectile function and pathophysiology erectile dysfunction employ several in vitro and in vivo techniques. As the field of sexual medicine expanding, the proper conduct of such techniques is becoming an even more important necessity than before. AIM: This review article aims to guide scientists, particularly young researchers and new comers in the field, toward employment of these techniques in an appropriate, timely, and competent fashion. METHODS: The authors reviewed the existing available published articles on the following topics: intracavernosal pressure measurements, cavernous nerve injury models, nitric oxide-cyclic guanosine monophosphate pathway, hypertension- and smoking-induced erectile dysfunction models, and stem cells. RESULTS: The authors present a consensus on how to best perform these models and techniques and also highlight the pitfalls. CONCLUSIONS: The authors hope that this article will assist and encourage young scientists in the field and that similar articles covering other important models will be also available to them soon.

Helper-dependent adenovirus is superior to first-generation adenovirus for expressing transgenes in atherosclerosis-prone arteries.

OBJECTIVE: Vascular gene transfer is a powerful tool for investigating and treating vascular diseases; however, its utility is limited by brevity of transgene expression and vector-associated inflammation. Helper-dependent adenovirus (HDAd), an advanced-generation adenovirus that lacks all viral genes, is superior to first-generation adenovirus (FGAd) in normal rabbit arteries. We compared HDAd to FGAd in arteries of cholesterol-fed rabbits, a model of early atherogenesis in which transgene expression might be decreased, and inflammation increased. METHODS AND RESULTS: Carotid arteries of chow- and cholesterol-fed rabbits were infused with FGAd, HDAd, or medium. HDAd expressed a transgene at least as well in arteries of cholesterol-fed rabbits as in arteries of chow-fed rabbits and expressed more durably than FGAd. In arteries of cholesterol-fed rabbits, HDAd stimulated less intimal growth, lipid deposition, and inflammation than FGAd. Neither vector affected phenylephrine-induced contraction or nitroprusside-mediated relaxation; however, both vectors decreased maximal acetylcholine-stimulated vasorelaxation. The relative absence of intimal growth in HDAd arteries could interfere with the utility of this model for testing atheroprotective genes; however, both coinfusion of FGAd and extension of cholesterol feeding yielded larger intimal lesions, on which atheroprotective genes could be tested. CONCLUSION: HDAd is superior to FGAd for expression of transgenes in atherosclerosis-prone arteries.

Characterization of erectile function in elastin haploinsufficicent mice.

INTRODUCTION: Elastin fibers confer passive recoil to many tissues including the lung, skin, and arteries. In the penis, elastin is present in sinusoids, arterioles, and in the tunica albuginea. Although decreased penile elastin has been reported in men with erectile dysfunction, the exact role of elastin in physiologic processes integral to erection remains speculative. AIM: The aim of this study was to characterize erectile function in elastin-deficient mice. METHODS: Elastin haploinsufficient mice (Eln(+/-) ) and aged match Eln(+/+) (Wt) mice were used. Cavernosum was removed from some mice for quantification of elastin, collagen, and smooth muscle actin. Ex vivo assessment of contractile force generation was performed by myography. In vivo assessment of intracorporal pressure normalized to mean arterial pressure in response to electrical stimulation of the cavernosal nerve was measured. Veno-occlusive function was determined by cavernosography. MAIN OUTCOME MEASURES: The main outcome measures of this study were the in vitro and in vivo assessment of cavernosal vasoreactivity, veno-occlusive function and erection in mice deficient in elastin. RESULTS: Eln (+/-) mice exhibited ∼33% less penile elastin than Wt mice, with no change in collagen. Cavernosal tissue from Eln(+/-) mice has a significantly heightened contractile response, explained in part by increased smooth muscle cell content. Veno-occlusive function was significantly altered in Eln(+/-) mice. Interestingly, erectile function was impaired only at submaximal voltage (1 V) stimulation (there was no impairment during the higher 2-V stimulus). CONCLUSIONS: Eln (+/-) mice display a cavernosal phenotype consistent with developmental changes attributable to the loss of elastin. These alterations confer a degree of altered erectile function that is able to be overridden by maximal stimulatory input. Altogether, these data suggest that elastin is important for erectile function.

Anatomy, physiology, and pathophysiology of erectile dysfunction.

INTRODUCTION: Significant scientific advances during the past 3 decades have deepened our understanding of the physiology and pathophysiology of penile erection. A critical evaluation of the current state of knowledge is essential to provide perspective for future research and development of new therapies. AIM: To develop an evidence-based, state-of-the-art consensus report on the anatomy, physiology, and pathophysiology of erectile dysfunction (ED). METHODS: Consensus process over a period of 16 months, representing the opinions of 12 experts from seven countries. MAIN OUTCOME MEASURE: Expert opinion was based on the grading of scientific and evidence-based medical literature, internal committee discussion, public presentation, and debate. RESULTS: ED occurs from multifaceted, complex mechanisms that can involve disruptions in neural, vascular, and hormonal signaling. Research on central neural regulation of penile erection is progressing rapidly with the identification of key neurotransmitters and the association of neural structures with both spinal and supraspinal pathways that regulate sexual function. In parallel to advances in cardiovascular physiology, the most extensive efforts in the physiology of penile erection have focused on elucidating mechanisms that regulate the functions of the endothelium and vascular smooth muscle of the corpus cavernosum. Major health concerns such as atherosclerosis, hyperlipidemia, hypertension, diabetes, and metabolic syndrome (MetS) have become well integrated into the investigation of ED. CONCLUSIONS: Despite the efficacy of current therapies, they remain insufficient to address growing patient populations, such as those with diabetes and MetS. In addition, increasing awareness of the adverse side effects of commonly prescribed medications on sexual function provides a rationale for developing new treatment strategies that minimize the likelihood of causing sexual dysfunction. Many basic questions with regard to erectile function remain unanswered and further laboratory and clinical studies are necessary.

Transcriptional profiling of human cavernosal endothelial cells reveals distinctive cell adhesion phenotype and role for claudin 11 in vascular barrier function.

To determine specific molecular features of endothelial cells (ECs) relevant to the physiological process of penile erection we compared gene expression of human EC derived from corpus cavernosum of men with and without erectile dysfunction (HCCEC) to coronary artery (HCAEC) and umbilical vein (HUVEC) using Affymetrix GeneChip microarrays and GeneSifter software. Genes differentially expressed across samples were partitioned around medoids to identify genes with highest expression in HCCEC. A total of 190 genes/transcripts were highly expressed only in HCCEC. Gene Ontology classification indicated cavernosal enrichment in genes related to cell adhesion, extracellular matrix, pattern specification and organogenesis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed high expression of genes relating to ECM-receptor interaction, focal adhesions, and cytokine-cytokine receptor interaction. Real-time PCR confirmed expression differences in cadherins 2 and 11, claudin 11 (CLDN11), desmoplakin, and versican. CLDN11, a component of tight junctions not previously described in ECs, was highly expressed only in HCCEC and its knockdown by siRNA significantly reduced transendothelial electrical resistance in HCCEC. Overall, cavernosal ECs exhibited a transcriptional profile encoding matrix and adhesion proteins that regulate structural and functional characteristics of blood vessels. Contribution of the tight junction protein CLDN11 to barrier function in endothelial cells is novel and may reflect hemodynamic requirements of the corpus cavernosum.

Diabetes, obesity and erectile dysfunction: field overview and research priorities.

PURPOSE: We provide an overview of basic, clinical and epidemiological research in the field of erectile dysfunction and important research priorities presented at the 2009 National Institute of Diabetes and Digestive and Kidney Diseases symposium on Urological Complications of Diabetes and Obesity. MATERIALS AND METHODS: Experts in molecular biology, physiology, pharmacology, clinical trials, epidemiology and urological surgery highlighted current knowledge on erectile dysfunction associated with diabetes mellitus and obesity. RESULTS: Predictable associations between erectile dysfunction, and poor diabetic control and modifiable risk factors, including body mass index, have not yet been translated into randomized trials in the United States. The relationship between erectile dysfunction and metabolic syndrome, and surrogate markers for erectile dysfunction requires further investigation. Basic research aimed at discovering disease mechanisms and therapeutic targets has focused on autonomic neuropathy, vascular dysfunction, smooth muscle contractile function and matrix. However, significant gaps exist in regard to the integration of molecular, cellular and functional data. Animal models of type 2 diabetes and obesity associated erectile dysfunction require investigation because most basic science studies have used rodent models of type 1 diabetes. CONCLUSIONS: Studies are needed to synthesize a systems biology understanding of erectile function/dysfunction, and characterize and disseminate rodent models of erectile dysfunction associated with type 2 diabetes and obesity. Clinical studies are needed of promising intervention and prevention strategies. Leveraging existing and future cohort phenotypes, and biological samples is needed for risk factor analysis, biomarker discovery and genome wide association studies.

Type 1 and Type 2 diabetic-erectile dysfunction: same diagnosis (ICD-9), different disease?

INTRODUCTION: Although hyperglycemia is a common defining feature of both type 1 and type 2 diabetes, many unique characteristics distinguish these diseases, including insulin and lipid levels, obesity status, and inflammatory agent profiles. In the laboratory, the presence of erectile dysfunction (ED) has been established in animal models of both type 1 and type 2 diabetes. AIM: The purpose of this study was to determine whether unique mechanisms underlie ED in type 1 vs. type 2 diabetic animal models. MAIN OUTCOME MEASURES: Many mechanisms can underlie ED, including impaired dilatory signaling, heightened contractile sensitivity, and veno-occlusive disorder. METHODS: Using PubMed, the literature was mined to evaluate what is known about which mechanism underlie ED in type 1 vs. type 2 diabetic animal models. RESULTS: Impaired cavernosal vasodilation has been established in type 1 diabetic rodents. This dysfunction appears to be mediated by a severe defect in non-adrenergic-non-cholinergic nerve signaling, as well as impairment in penile endothelial function. In contrast, type 2 diabetic animals appear to have minimal impairment in parasympathetic-mediated dilatory function, but do have evidence of endothelial dysfunction. Type 2 diabetic models also exhibit a significant and striking increase in cavernosal contractile sensitivity, and a significant veno-occlusive disorder, neither of which is consistently reported in type 1 diabetic animals. CONCLUSIONS: With the distinct mechanisms underlying the ED phenotype in animal models of type 1 and type 2 diabetes, tailoring therapeutic treatments for diabetic-ED to the specific mechanisms underlying this disease complication may be warranted. Further examination of mechanisms underlying ED in diabetic human patients may thus lead to significant changes in the way urologists diagnose, code, and treat diabetic-ED.

Review type 2 diabetes mellitus and erectile dysfunction.

INTRODUCTION: Diabetes mellitus (DM) is a major risk factor for the development of erectile dysfunction (ED). Although most diabetic ED cases are in patients with type 2 diabetes (T2DM), the majority of basic science studies examining mechanisms of diabetic ED have been conducted in animal models of type 1 diabetes. AIM: Recently, however, clinical and laboratory-based studies have uncovered some key underlying factors of T2DM-associated ED, which we have compiled in this review of T2DM ED. MAIN OUTCOME MEASURES: The outcomes discussed in this review include major mechanisms underlying T2DM, discussing both clinical and basic science studies. METHODS: We conducted an extensive search of pertinent clinical and basic science literature using PUBMED. RESULTS: Mechanisms causing ED in T2DM are multifactorial and often lead to resistance to current therapy. Systemic effects of hyperglycemia and hypogonadism contribute to the development of impaired vasodilatory signaling, smooth muscle cell hypercontractility, and veno-occlusive disorder in T2DM ED. CONCLUSIONS: Understanding the different causes for ED in T2DM patients may allow targeted therapy for improved erectile function.

Strain differences in susceptibility to in vivo erectile dysfunction following 6 weeks of induced hyperglycemia in the mouse.

INTRODUCTION: With the large-scale availability of transgenic and knockout mouse models, the use of mice may greatly facilitate the examination of the mechanisms underlying diabetic erectile dysfunction (ED). Although in vitro studies of the mouse cavernosum show impairment of vasoreactivity, to date, no studies have demonstrated the in vivo impairment of erectile function in diabetic mice. AIM: To establish whether mouse models of type I diabetes exhibit in vivo ED. METHODS: Hyperglycemia was induced by injection with streptozotocin (STZ, 125 mg/kg x 2 days) in two mouse strains, C57BLKS (BKS) and BALB/c. Six weeks after injection, the cavernosum was removed from some mice for the in vitro assessment of the endothelium and nerve-mediated dilatory responses of the cavernosal strips. The in vivo assessment of intracorporal pressure normalized to mean arterial pressure, in response to the electrical stimulation of the cavernosal nerve, was performed in the remaining mice. MAIN OUTCOME MEASURES: The main outcome measure of this study was the in vivo assessment of erectile function following diabetic induction in mice. RESULTS: Despite similar levels of sustained hyperglycemia following STZ injection, the phenotype of diabetic ED was observed only in BKS and not BALB/c mice. The cavernosum from diabetic BKS mice showed decreased endothelium-dependent dilation in response to acetylcholine (ACh), as well as impaired parasympathetic nerve-mediated relaxation. There was no change in ACh or nerve-mediated relaxation in the cavernousum from diabetic vs. control BALB/c mice. Further, in vivo physiologic assessment of erectile activity revealed a significant decrease in erectile function in diabetic BKS but not in BALB/c mice. CONCLUSION: Together these data first established in vivo ED in a mouse model of type I diabetes (BKS mouse) and importantly demonstrated that certain inbred strains may be protected from hyperglycemia-induced erectile impairment. Further study of the strain-dependent effects may offer important clues into the mechanisms of ED as it relates to type I diabetes.

Subclinical Vascular Disease and Subsequent Erectile Dysfunction: The Multiethnic Study of Atherosclerosis (MESA).

BACKGROUND: The association between subclinical cardiovascular disease and subsequent development of erectile dysfunction (ED) remains poorly described. HYPOTHESIS: Among multiple subclinical atherosclerosis and vascular dysfunction measurements, coronary artery calcium (CAC) score best predicts ED. METHODS: After excluding participants taking ED medications at baseline, we studied 1862 men age 45 to 84 years free of known cardiovascular disease from the Multi-Ethnic Study of Atherosclerosis (MESA) with comprehensive baseline subclinical vascular disease phenotyping and ED status assessed at MESA visit 5 (9.4 ± 0.5 years after baseline) using a standardized question on ED symptoms. Multivariable logistic regression was used to assess the associations between baseline measures of vascular disease (atherosclerosis domain: CAC, carotid intima-media thickness, carotid plaque, ankle-brachial index; vascular stiffness/function domain: aortic stiffness, carotid stiffness, brachial flow-mediated dilation) and ED symptoms at follow-up. RESULTS: Mean baseline age was 59.5 ± 9 years, and 839 participants (45%) reported ED symptoms at follow-up. Compared with symptom-free individuals, participants with ED had higher baseline prevalence of CAC score >100 (36.4% vs 17.2%), carotid intima-media thickness Z score >75th percentile (35.3% vs 16.6%), carotid plaque score ≥2 (39% vs 21.1%), carotid distensibility <25th percentile (34.6% vs 17.1%), aortic distensibility <25th percentile (34.2% vs 18.7%), and brachial flow-mediated dilation <25th percentile (28.4% vs 21.3%); all P < 0.01. Only CAC >100 (odds ratio: 1.43, 95% confidence interval: 1.09-1.88) and carotid plaque score ≥2 (odds ratio: 1.33, 95% confidence interval: 1.02-1.73) were significantly associated with ED. CONCLUSIONS: Subclinical vascular disease is common in men who later self-report ED. Early detection of subclinical atherosclerosis, particularly advanced CAC and carotid plaque, may provide opportunities for predicting the onset of subsequent vascular ED.

Vasodilator-stimulated phosphoprotein regulates proliferation and growth inhibition by nitric oxide in vascular smooth muscle cells.

OBJECTIVE: Vasodilator-stimulated phosphoprotein (VASP) was identified as a substrate for cGMP-dependent protein kinase (PKG) and cAMP-dependent protein kinase (PKA). It is preferentially phosphorylated at serine239 by PKG, whereas serine157 is a preferred phosphorylation site for PKA. In addition, serine157 is phosphorylated by PKC in response to serum. We have investigated the effects of VASP and VASP phosphorylation at serine157 and serine239 on smooth muscle cell (SMC) proliferation and nitric oxide (NO)-mediated growth inhibition. METHODS AND RESULTS: Aortic SMCs derived from VASP-deficient mice were transduced with retroviral vectors encoding either wild-type VASP or VASP mutants (S157A-VASP and S239A-VASP), in which serine157 and serine239, respectively, were replaced by a nonphosphorylatable amino acid, alanine. Expression of wt-VASP and S239A-VASP significantly increased proliferation, whereas expression of S157A-VASP was inhibitory. Expression of S239A-VASP rendered SMCs less sensitive to growth inhibition by the NO donor, S-nitroso-n-acetylpenicillamine, when compared with cells expressing wt-VASP. Similar effects were observed in cultured rat SMCs in which wt-VASP, S157A-VASP, and S239A-VASP were expressed. CONCLUSIONS: Our data suggest that VASP phosphorylation at serine157 is required for the growth-stimulatory effect of VASP in SMCs, whereas VASP phosphorylation at serine239 is involved in the growth inhibitory effects of NO on SMCs.

Nitric oxide inhibits RhoA/Rho-kinase signaling to cause penile erection.

The RhoA/Rho-kinase pathway mediates vasoconstriction in the cavernosal circulation. Inhibition of this pathway leads to penile erection in the in vivo rat model. These studies examined the hypothesis that nitric oxide (NO) inhibits RhoA/Rho-kinase signaling as part of normal erection. The results show that NO causes increased intracavernosal pressure and that this response is potentiated by prior treatment with a threshold dose of the Rho-kinase inhibitor, (+)-(R)-trans-4-(1-Aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride, monohydrate (Y-27632). These results support the hypothesis that NO inhibits Rho-kinase-induced cavernosal vasoconstriction during erection.

Microtubule depolymerization facilitates contraction of rat aorta via activation of Rho-kinase.

This study tests the hypothesis that microtubule (MT) depolymerization facilitates contraction of rat aorta via activation of Rho-kinase. Aortic rings from Sprague-Dawley rats were placed in a muscle bath for the measurement of isometric force generation. Bath temperature was decreased from 37 to 10-20 degrees C (30 min), inducing MT depolymerization. Some vessels were treated with nocodazole (10(-5) M) or colchicine (10(-8)-10(-5) M) to stabilize the MTs in the depolymerized state, and the remaining vessels were treated with dimethyl sulfoxide (DMSO: vehicle). Warming of vessels to 37 degrees C induced a significantly greater contraction in nocodazole- and colchicine-treated vessels as compared with controls, and this increase was blocked by pretreatment with taxol (10(-5) M; a MT stabilizing agent) [force (mg): NOC 1159 +/- 93; COL 1138 +/- 69; DMSO 578 +/- 14; TAX + NOC 526 +/- 43; TAX + COL 538 +/- 90]. Following the sustained contraction in response to rewarming, Rho-kinase inhibition with Y-27632 (10(-5) M) relaxed nocodazole- and colchicine-treated rings to a significantly greater extent as compared to DMSO-treated vessels (percent relaxation: NOC 64 +/- 2; COL 65 +/- 5; DMSO 33 +/- 5). These results support the hypothesis that MT depolymerization facilitates contraction of rat aorta via activation of Rho-kinase.

[Effect of topical application of a Rho-kinase inhibitor on the erectile response in rats].

OBJECTIVE: To investigate the effect of Rho-kinase inhibitor applied topically on the penile erection and systemic circulation of rats. METHODS: Y-27632 was applied to the surface of the tunica albuginea or to the penile skin of rats, and the changes of CCP/MAP were observed continuously. RESULTS: Both methods of drug administration resulted in a marked increase in the erectile response both with and without stimulation of the innervation of the penile vasculature. Some doses of the drug were also found to reduce systemic blood pressure. CONCLUSION: Inhibitors of Rho-kinase may represent a new and promising method of treatment for erectile dysfunction.

Glutathione (GSH) and the GSH synthesis gene Gclm modulate vascular reactivity in mice.

Oxidative stress has been implicated in the development of vascular disease and in the promotion of endothelial dysfunction via the reduction in bioavailable nitric oxide (NO()). Glutathione (GSH) is a tripeptide thiol antioxidant that is utilized by glutathione peroxidase (GPx) to scavenge reactive oxygen species such as hydrogen peroxide and phospholipid hydroperoxides. Relatively frequent single-nucleotide polymorphisms (SNPs) within the 5' promoters of the GSH synthesis genes GCLC and GCLM are associated with impaired vasomotor function, as measured by decreased acetylcholine-stimulated coronary artery dilation, and with increased risk of myocardial infarction. Although the influence of genetic knockdown of GPx on vascular function has been investigated in mice, no work to date has been published on the role of genetic knockdown of GSH synthesis genes on vascular reactivity. We therefore investigated the effects of targeted disruption of Gclm in mice and the subsequent depletion of GSH on vascular reactivity, NO() production, aortic nitrotyrosine protein modification, and whole-genome transcriptional responses as measured by DNA microarray. Gclm(-/+) and Gclm(-/-) mice had 72 and 12%, respectively, of wild-type (WT) aortic GSH content. Gclm(-/+) mice had a significant impairment in acetylcholine (ACh)-induced relaxation in aortic rings as well as increased aortic nitrotyrosine protein modification. Surprisingly, Gclm(-/-) aortas showed enhanced relaxation compared to Gclm(-/+) aortas, as well as increased NO() production. Although aortic rings from Gclm(-/-) mice had enhanced ACh relaxation, they had a significantly increased sensitivity to phenylephrine (PE)-induced contraction. Alternatively, the PE response of Gclm(-/+) aortas was nearly identical to that of their WT littermates. To examine the role of NO() or other potential endothelium-derived factors in differentially regulating vasomotor activity, we incubated aortic rings with the NO() synthase inhibitor L-NAME or physically removed the endothelium before PE treatment. L-NAME treatment and endothelium removal enhanced PE-induced contraction in WT and Gclm(-/+) mice, but this effect was severely diminished in Gclm(-/-) mice, indicating a potentially unique role for GSH in mediating vessel contraction. Whole-genome assessment of aortic mRNA in Gclm(-/-) and WT mice revealed altered expression of genes within the canonical Ca(2+) signaling pathway, which may have a role in mediating these observed functional effects. These findings provide additional evidence that the de novo synthesis of GSH can influence vascular reactivity and provide insights regarding possible mechanisms by which SNPs within GCLM and GCLC influence the risk of developing vascular diseases in humans.