Thomas M. Mills

Antagonism of Rho-kinase stimulates rat penile erection via a nitric oxide-independent pathway.

Relaxation of the smooth muscle cells in the cavernosal arterioles and sinuses results in increased blood flow into the penis, raising corpus cavernosum pressure to culminate in penile erection. Nitric oxide, released from non-adrenergic/non-cholinergic nerves, is considered the principle stimulator of cavernosal smooth muscle relaxation, however, the inhibition of vasoconstrictors (that is, norepinephrine and endothelin-1, refs. 5–9) cannot be ignored as a potential regulator of penile erection. The calcium-sensitizing ρ-A/Rho-kinase pathway may play a synergistic role in cavernosal vasoconstriction to maintain penile flaccidity. Rho-kinase is known to inhibit myosin light chain phosphatase, and to directly phosphorylate myosin light-chain (in solution), altogether resulting in a net increase in activated myosin and the promotion of cellular contraction. Although Rho-kinase protein and mRNA have been detected in cavernosal tissue, the role of Rho-kinase in the regulation of cavernosal tone is unknown. Using pharmacologic antagonism (Y-27632, ref. 13, 18), we examined the role of Rho-kinase in cavernosal tone, based on the hypothesis that antagonism of Rho-kinase results in increased corpus cavernosum pressure, initiating the erectile response independently of nitric oxide. Our finding, that Rho-kinase antagonism stimulates rat penile erection independently of nitric oxide, introduces a potential alternate avenue for the treatment of erectile dysfunction.

Isoelectric focusing technology quantifies protein signaling in 25 cells

A previously undescribed isoelectric focusing technology allows cell signaling to be quantitatively assessed in <25 cells. High-resolution capillary isoelectric focusing allows isoforms and individual phosphorylation forms to be resolved, often to baseline, in a 400-nl capillary. Key to the method is photochemical capture of the resolved protein forms. Once immobilized, the proteins can be probed with specific antibodies flowed through the capillary. Antibodies bound to their targets are detected by chemiluminescence. Because chemiluminescent substrates are flowed through the capillary during detection, localized substrate depletion is overcome, giving excellent linearity of response across several orders of magnitude. By analyzing pan-specific antibody signals from individual resolved forms of a protein, each of these can be quantified, without the problems associated with using multiple antibodies with different binding avidities to detect individual protein forms.

The human sexuality education of physicians in North American medical schools

Individuals seeking treatment for sexual problems frequently would like to turn to a source they consider knowledgeable and worthy of respect, their doctor. The objective was to assess how well the 125 schools of medicine in the United States and the 16 in Canada prepare physicians to diagnose and treat sexual problems. A prospective cohort study was carried out. The main outcome results were description of the medical educational experiences, teaching time, specific subject areas, clinical programs, clerkships, continuing education programs in the domain of human sexuality in North American medical schools. The results were as follows. There were 101 survey responses (71.6%) of a potential of 141 medical schools (74% of United States and 50% of Canadian medical schools). A total of 84 respondents (83.2%) for sexuality education used a lecture format. A single discipline was responsible for this teaching in 32 (31.7%) schools, but a multidisciplinary team was responsible in 64 (63.4%) schools (five schools failed to respond to the question). The majority (54.1%) of the schools provided 3–10 h of education. Causes of sexual dysfunction (94.1%), its treatment (85.2%) altered sexual identification (79.2%) and issues of sexuality in illness or disability (69.3%) were included in the curriculum of 96 respondents. Only 43 (42.6%) schools offered clinical programs, which included a focus on treating patients with sexual problems and dysfunctions, and 56 (55.5%) provided the students in their clerkships with supervision in dealing with sexual issues. In conclusion, expansion of human sexuality education in medical schools may be necessary to meet the public demand of an informed health provider.

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.

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.

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.

Adeno-associated viral gene transfer of dominant negative RhoA enhances erectile function in rats.

We previously reported the inhibition of Rho-kinase to result in increased intracavernosal pressure (ICP) in an in vivo rat model of erection. Expression of an upstream activator of Rho-kinase, RhoA, has been demonstrated in the penile vasculature; however, the functional role of RhoA in the regulation of erection remains unknown. We used adeno-associated viral gene transfer of a dominant negative RhoA mutant (T19NRhoA) into rat cavernosum to test the hypothesis that RhoA activation is physiologically important for maintenance of the non-erect state and inhibition of this pathway leads to erection. Anesthetized, male, Sprague-Dawley rats transfected with the T19NRhoA mutant exhibited an elevated baseline ICP/mean arterial pressure (MAP) and nerve stimulation-induced ICP/MAP as compared with beta-galactosidase-transfected controls. The novel findings of this study demonstrate a functional role of RhoA in maintaining the flaccid penis and provide support for the inhibition of RhoA as a potential therapy for the enhancement of erectile function.

Identification and measurement by gas chromatography-mass spectrometry of norethindrone and metabolites in human urine and blood

Abstract Urinary metabolites of norethindrone (17β-hydroxy-17α-ethynyl-4-estren-3-one) in the free, sulfate, and glueuronide fractions were identified by gas chromatography-mass spectrometry following treatment of a volunteer with a 25 mg dose of norethindrone. Compounds identified in the three fractions were norethindrone, the four ring A reduced isomers, a reduced and hydroxylated metabolite, and ethynyl estradiol (17α-ethynylestra-l,3,5(10)-triene-3, 17β-diol). Plasma free, sulfate and glucuronide conjugated metabolites of norethindrone were identified and quantified by mass fragmentography. Blood metabolites identified in the three fractions 3 h following oral administration of 25 mg of norethindrone were unchanged drug, and ring A reduced metabolites 17α-ethynyl-5α-estrane-3α,17β-diol, 17α-ethynyl-5β-estrane-3α,17β-diol, 17α-ethynyl-5β-estrane-3β, 17β-diol, and 17β-hydroxy-17α-ethynyl-5β-estrane-3-one. These plasma metabolites were also quantified using mass fragmentography in blood obtained from a volunteer undergoing daily treatment with 2 mg norethindrone, 0.1 mg mestranol (17α-ethynylestra-1,3,5(10)-triene-3,17β-diol-3-methyl ether) (Orth-o-Novum, 2 mg).

Captopril treatment reverses erectile dysfunction in male stroke prone spontaneously hypertensive rats.

The involvement of antihypertensive therapy in the pathology of hypertension associated male erectile dysfunction is unclear. Stroke prone spontaneously hypertensive rats (SHRSP) were treated chronically with the angiotensin converting enzyme (ACE) inhibitor captopril or placebo, normotensive rats served as controls. Mean arterial and intracavernosal pressure were measured during the induction of erection by autonomic ganglion stimulation. SHRSP-placebo treated rats were hypertensive and had a blunted erectile response. Captopril treatment returned both the blood pressure and erectile response to control levels. Therefore, ACE inhibitor therapy may not be responsible for the erectile dysfunction observed in treated hypertensive subjects.

Animal Models for Study of Polycystic Ovaries and Ovarian Atresia

In the human, polycystic ovaries are generally accompanied by normal or elevated levels of serum LH, normal or slightly depressed FSH and by high levels of circulating estrogens and androgens. If the excess androgen secretion is reduced by one of several methods, ovulatory cycles are usually restored. Several animal model systems have been proposed for the study of the pathophysiology of the polycystic ovarian syndrome. These include neonatal androgenization, hCG administration to hypothyroid rats, injection of estradiol valerate and maintaining animals in constant light. In a model developed in this laboratory, pubertal or adult rats were treated with the weak androgen, dehydroepiandrosterone (DHA), to induce polycystic ovaries. This treatment also altered the blood levels of LH and FSH but the effect on gonadotropins and on the formation of the degenerative follicles was fully reversed following discontinuation of the androgen injections. The polycystic ovaries of the DHA-treated animals were steroidogenically more active than controls raising the possibility that the DHA was acting directly on the ovary in addition to an action on the pituitary-hypothalamus axis. In order to study the direct effect of androgens on the ovary, another animal model was developed in which immature, hypophysectomized rats were injected with pregnant mare serum gonadotropin (PMSG) to initiate follicular growth followed by a single injection of dihydrotestosterone (DHT). The androgen caused follicular atresia and decreased the number of ova shed in response to ovulation induction with hCG. The suppressive effects of DHT were entirely prevented by concomitant treatment with estradiol. The studies with DHT were continued using another batch of PMSG, but the DHT-induced increase in the rat of atresia and suppression of induced ovulation were no longer seen. However, when this same batch of PMSG was given with estrogen or with the antiandrogen flutamide, there was less atresia and the growth of follicles was actually enhanced. Based on these studies, it was postulated that the second batch of PMSG had greater LH activity than the first preparation and that the LH has stimulated endogenous androgen production. The ovarian follicles which appeared to be most susceptible to this DHT effect were small to medium in size and had a low capacity to synthesize estrogen. This possibility was confirmed in another animal model system in which immature rats were injected with PMSG and 4 separate injections of DHT and then sacrificed at several time points over the next 8 days.(ABSTRACT TRUNCATED AT 400 WORDS)