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 rho-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.

Receptor-specific influence of endothelin-1 in the erectile response of the rat.

Specific receptor antagonists were used to examine the role of endothelin-1 (ET-1) in the erectile response of the rat. In these studies, intact rats were cannulated to permit the continuous recording of mean arterial pressure (MAP) and intracavernosal pressure (CCP). Erection was induced by electrical stimulation of the autonomic ganglion, which regulates blood flow to the penis. The animals were subjected to intracavernosal injection with vehicle only (Cont) or with an antagonist to the endothelin-A receptor (ET(A)) or to the endothelin-B receptor (ET(B)). Blockade of the ET(A) or the ET(B) had no effect on the erectile response (CCP/MAP) during maximal ganglionic stimulation. When ET-1 was injected into Cont rats, there was a marked vasoconstriction with a sharp rise in MAP and a decline in CCP as the cavernosal arterioles constricted and limited inflow. The injection of the ET(A) antagonist prevented the vasoconstriction after ET-1 injection into Cont rats, whereas blockade of the ET(B) had no effect on the vasoconstrictive effect to ET-1. Similar results were obtained during submaximal ganglionic stimulation. With minimal levels of ganglionic stimulation, ET-1 injection led to a moderated degree of vasodilation in the presence of the ET(A) antagonist. The ET(B) antagonist failed to alter the CCP response during minimal stimulation, but it did have a marked effect on the MAP response to ET-1 injection. The results of these studies confirm that cavernosal tissue of the rat penis is highly responsive to ET-1. However, the failure of the ET-1 antagonists to affect penile erection in response to ganglionic stimulation reflects a minimal role of ET-1 in the erectile response in the rat.

Androgenic maintenance of the erectile response in the rat.

Ongoing studies in this laboratory have used the castrated rat, with and without testosterone replacement, to investigate how androgens maintain the erectile response. The high intracavernosal pressures during erection depend on both an increase in the rate at which blood flows into the sinuses of the corpus cavernosum and a decrease in the rate at which blood flows out (veno-occlusion). Accordingly, our studies investigated androgenic regulation of the arterioles that regulate inflow and of the intracavernosal muscle that regulates the veno-occlusive mechanism controlling outflow. The results of these studies show that castration causes a decline in the rate of inflow and that androgen replacement reverses this decline. The decline in inflow in the castrated rats is also reversed by the administration of a nitric oxide donor drug, suggesting that the androgen may regulate inflow by increasing the synthesis of nitric oxide. Testosterone also appears to regulate outflow by controlling the sensitivity of the erectile mechanisms to norepinephrine, considered to be the principle vaso-constrictor neurotransmitter in the erectile response. Taken together, the results of these studies suggest that androgens control the erectile response by altering the synthesis and action of the neurotransmitters that normally alter the state of contraction and relaxation of smooth muscle in the erectile tissue.

Androgenic maintenance of inflow and veno-occlusion during erection in the rat.

Ongoing studies in this laboratory are designed to determine the role of androgens in the maintenance of the erectile response in the rat. Testosterone-treated castrated rats (TESTO) and untreated castrated rats (CASTRATE) were used for measurement of the rate at which blood flows into the cavernous sinuses by timed collections of blood after partial amputation of the penis. A laser Doppler flow meter was employed to determine whether androgens also regulate the veno-occlusive mechanism that controls the rate of blood flow out of the sinuses. Erection was induced by direct electrical stimulation of the autonomic ganglion that controls cavernosal blood flow in the erectile response. The results of these studies showed that blood flow into the sinuses was approximately twice as great in the TESTO animals as the CASTRATE rats. Furthermore, during ganglionic stimulation, veno-occlusion occurred in the TESTO rats but failed to occur in the CASTRATE rats. The dependence of these responses on nitric oxide (NO) was demonstrated by showing that injection of sodium nitroprusside (SNP) enhances the intracavernosal pressure response in TESTO rats but not CASTRATE animals. However, when SNP injection was combined with ganglionic stimulation, veno-occlusion did occur in the CASTRATE animals. Taken together, these studies show that both the rate of blood flow into the cavernous sinuses and the blood flow out are under androgenic regulation and may involve the actions of NO.

The loss of alpha-adrenergic effect during the erectile response in the long-term diabetic rat.

The present study was designed to investigate the effect of long-term, streptozotocin-induced diabetes on the erectile response in the laboratory rat. Mean arterial blood pressure (MAP) and intracavernosal blood pressure within the erectile tissue (CCP) were continuously monitored during erection elicited by stimulation of the autonomic innervation of the penis. MAP and CCP were also measured during administration of two drugs: nitroglycerin, a nitric oxide donor drug and phenylephrine, an alpha-adrenergic agonist. The results of these studies show that during graded electrical stimulation of the ganglion, the overall magnitude of the erectile response was greater in the diabetic rats than in untreated control animals. Neither diabetic nor control animals responded significantly to infusion of nitroglycerin. However, diabetic rats and control rats responded very differently to administration of phenylephrine; in the control rats, this alpha agonist caused a sharp decline in CCP as the cavernosal vessels constricted in response to the drug. The same dose of phenylephrine had no discernible effect on CCP in the diabetic animals. This loss of alpha responsiveness may be confined to the penile circulation because MAP was elevated to approximately the same extent in both groups. Taken together, these results show that long-term diabetes leads to a failure of alpha-adrenergic responsiveness in the cavernosal circulation. The greater erectile response to ganglionic stimulation in the diabetic animals is likely due to the loss of response to endogenous norepinephrine.

Androgenic regulation of NO availability in rat penile erection.

Prior studies from this laboratory, using untreated castrated (CASTRATE) rats and testosterone-treated castrated (TESTO) rats, have shown that the magnitude of the intracavernosal pressure increase during erection is androgen dependent. Studies from this and other laboratories have also presented evidence suggesting that penile erection is mediated principally by nitric oxide (NO). The present report was designed to confirm that androgens maintain the availability of cavernosal NO and to determine if this androgenic action is exerted at the genomic level modulating the expression of the neuronal form of the nitric oxide synthase gene (nNOS). The results showed that administration of supplemental L-arginine failed to augment the erectile response in either group, suggesting that substrate availability is not a cause of the reduced response in CASTRATE animals. Inhibition of NO synthesis with a nitro-arginine competitive inhibitor of nitric oxide synthase enzyme protein (NOS) resulted in strong inhibition of erection in both TESTO and CASTRATE rats. When given in conjunction with ganglionic stimulation to induce erection, the NO releasing drug, sodium nitro-prusside (SNP), increased intracavernosal pressure in CASTRATE rats but not in TESTO rats, suggesting a deficiency of the available NO in CASTRATE-animals. Finally, reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that mRNA levels for the enzyme nNOS in the penis were greater in TESTO animals than in CASTRATE rats. These results support the hypothesis that androgens mediate the erectile response in the rat penis by stimulating the expression of the neuronal isoform of nitric oxide synthase, thus maintaining an adequate supply of NO.

Androgens modulate the alpha-adrenergic responsiveness of vascular smooth muscle in the corpus cavernosum.

Rat penile erection is an androgen-dependent process with castration leading to a loss of potency. The present study was designed to determine if one of the mechanisms by which androgens maintain the erectile response is the regulation of the alpha-adrenergic responsiveness of cavernosal smooth muscle. Electrical stimulation of the major pelvic ganglion (MPG) was used to elicit erection in untreated, castrated rats (CASTRATE) or castrated rats given testosterone replacement (TESTO). The effects of phenylephrine (an alpha 1-adrenergic agonist) and prazosin (an alpha 1-adrenergic antagonist) on the erectile response were investigated. Phenylephrine, when administered to both TESTO and CASTRATE animals during erection, resulted in a dose-dependent decrease in the intracavernosal pressure (CCP) with an ED50 value of 1.8 +/- 0.48 micrograms/kg BW for TESTO rats; in the CASTRATE animals, the ED50 was significantly reduced to 0.29 +/- 0.08 microgram/kg BW. The increases in mean arterial pressure (MAP) resulting from phenylephrine injection in TESTO and CASTRATE animals were of similar magnitude and were not significantly different. Prazosin administration resulted in an enhancement of the erectile response in CASTRATE but not in TESTO animals. Taken together these results demonstrate that the cavernosal vasculature in CASTRATE animals possesses increased reactivity to alpha-adrenergic stimulation as compared to the sensitivity in TESTO rats. Based on these findings, we conclude that one of the mechanisms by which androgens maintain erectile function is by regulating the alpha 1-adrenergic responsiveness of the cavernosal smooth muscle.

Androgenic maintenance of the rat erectile response via a non-nitric-oxide-dependent pathway.

Prior studies have demonstrated that the erectile response in the rat penis is androgen dependent and is mediated by nitric oxide (NO), the neurotransmitter synthesized by the enzyme nitric oxide synthase (NOS). The present studies used L-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, to determine if androgens also regulate alternative pathways leading to the erectile response but not mediated by NO. Castrated rats that were treated with L-NAME (L-NAME CASTRATE) exhibited little or no increase in intracavernosal pressure in response to stimulation of the major pelvic ganglion. This ganglion controls blood flow into the penis and, when stimulated, normally leads to erection. However, when castrated animals were treated with testosterone along with L-NAME (L-NAME TESTO), the animals responded to the ganglionic stimulation with increased intracavernosal pressure. This finding suggests that there are other androgen-dependent pathways that lead to penile erection but are not mediated by NO. Erection occurred in both L-NAME CASTRATE and L-NAME TESTO rats in response to intracavernosal injection of sodium nitroprusside (an NO donor drug), proving that the NO responsive mechanisms were unaffected by the inhibition of NOS activity. To investigate further the nature of this NO independent pathway, L-NAME CASTRATE and L-NAME TESTO rats were treated with either zaprinast (a specific phosphodiesterase 5 inhibitor), which would block the breakdown of cGMP to 5'GMP, or methylene blue (an inhibitor of guanylate cyclase) to prevent the synthesis of cGMP. Zaprinast treatment led to increased erectile response in L-NAME TESTO rats but not in L-NAME CASTRATE rats, demonstrating that androgen-sensitive alternative pathways increased guanylate cyclase activity. Methylene blue inhibited the erectile response in all treatment groups, showing that cyclic GMP is critical to the NO-independent pathway as well as the NO-dependent pathway. Taken together, these results support the hypothesis that androgens maintain the erectile response by alternate pathways, including one that is independent of NO but involves the synthesis of cyclic GMP.

Duration of estrogen exposure prior to follicle-stimulating hormone stimulation is critical to granulosa cell growth and differentiation in rats.

Estrogens have been reported to exert both stimulatory and inhibitory effects on granulosa cell function. Previous studies from our laboratory showed that 12 h after administration of diethylstilbestrol (DES; a synthetic estrogen), FSH-stimulated granulosa cell proliferation and aromatase activity were increased; however, 48 h after DES, FSH stimulation of both parameters was inhibited. In other experiments, exposure of rats to DES for a period of 26 h blocked ovulation in response to eCG and hCG administration, whereas the same treatment regimen without DES caused ovulation in all treated rats. Thus, DES may in some cases actually interfere with maturation and development of ovulatory follicles. The present study was designed 1) to confirm that the duration of estrogen pre-exposure determines the way granulosa cells respond to FSH and 2) to investigate the underlying mechanisms involved. While DES was used in preliminary experiments, the majority of the studies were conducted with estradiol, a natural estrogen, in order to conform as closely as possible to the normal physiology. In the experimental protocol, immature female rats received injections of DES or implants of estradiol pellets 12 h (short exposure) or 36 h (long exposure) before 36 h of FSH treatment. Rats were killed, ovaries removed, and granulosa cells collected at the end of the FSH treatment period. The results demonstrate that exposure to either of these estrogens for 12 h allowed the subsequent FSH stimulation to produce high cellular proliferation, high aromatase enzyme activity, and large amounts of FSH receptor and aromatase mRNA. Estrogen exposure for 36 h, however, resulted in significantly decreased FSH stimulation of all these parameters. These findings confirm that short exposure to estrogen enhances the response of granulosa cells to FSH while longer exposure makes granulosa cells refractory to FSH. This differential sensitivity of granulosa cells to estrogen exposure could help explain how dominant follicles survive to ovulate while others are lost to atresia during ovarian cycles.

Sites of androgenic regulation of cavernosal blood pressure during penile erection in the rat.

The present studies were designed to determine the role of reactive vascular smooth muscle in the regulation of blood flow into and out of the cavernous sinuses during penile erection in castrated and testosterone-treated animals. While the mean arterial pressure and intracavernosal pressure were continuously monitored, vasoactive drugs were injected into the aorta or into the cavernous sinuses during erection. The results show that both a NO releasing vasodilatory drug and an alpha adrenergic agonist significantly affected both mean arterial pressure and intracavernosal pressure when injected into the aorta. However, when these same drugs were injected into the cavernous sinuses, neither drug exerted a significant influence on the erectile response. Based on these studies, we conclude that the flow of blood into the cavernous sinuses during erection is regulated by reactive vascular smooth muscle but the outflow is not under the regulation of reactive vascular smooth muscle. Furthermore, the relaxation of the smooth muscle which controls the flow of blood into the cavernous sinuses during erection may be under partial androgenic control.

Effects of castration and androgen replacement on the hemodynamics of penile erection in the rat.

Previous studies from this laboratory have demonstrated that penile erection in the rat is androgen dependent: 1 wk after castration, there was a significant decline in the magnitude of the intracavernosal pressure (CCP) response during erection induced by stimulation of the autonomic ganglion controlling penile blood flow. The response was altered by vasoactive drugs and appeared to involve nitric oxide synthesis. These earlier studies, however, did not identify the site of androgenic action or the mechanism by which the androgens act. The findings reported here show that even in long-term-castrated animals (up to 7 wk), there remains a rise in CCP in response to ganglionic stimulation, demonstrating that there is an androgen-independent as well as an androgen-dependent portion of the erectile response. Other results show a linear relationship between systemic blood pressure and CCP during erection, although in castrated animals without androgen replacement, the CCP responds less to changes in the systemic pressure than in intact or testosterone-treated animals. This finding could signify a reduced blood inflow and/or an increased blood outflow during erection in the castrated rats. Further studies partially explained the lower erectile pressure by demonstrating that the rate of outflow from the cavernosal spaces was greater in castrated rats than in animals with normal androgen levels. Taken together, these findings show that androgens act to maintain both the inflow and the outflow of blood from the cavernous spaces during erection.

Androgen maintenance of erectile function in the rat penis.

Previous research has shown that the frequency and duration of penile erection is diminished after castration and that replacement with testosterone will restore the process. Using rats, the present study was designed to confirm that erection is androgen-dependent and to determine whether castration and androgen replacement affect the penile vascular smooth muscle responsiveness to vasoactive drugs. Blood pressure in the corpus cavernosum was measured directly during erections induced by electrical stimulation of the autonomic innervation of the penis. Maximal cavernosal pressure was markedly reduced after castration but was returned to normal levels if the castrated animals were treated with testosterone. Infusion of nitroglycerin (vasodilator) or phenylephrine (vasoconstrictor) resulted in a decline in cavernosal pressure in androgen-treated animals but not in castrated animals, even though the mean arterial blood pressure was strongly affected in all treatment groups by these drugs. When an inhibitor of nitric oxide synthesis was infused, cavernosal pressure was decreased in all groups, indicating that this substance is involved in penile erection. Taken together, these results show that androgens maintain the erectile process and may act specifically to support the responsiveness of the vascular smooth muscle to vasoactive drugs.

The use of perfused rabbit ovaries to investigate regional ovarian lymphatic flow.

A method is presented for the perfusion of rabbit ovaries in vitro which allows continuous collection of effluent perfusate from the ovarian vein and lymphatic system. The flow of ovarian lymph and the output of progesterone and 20 alpha dihydroprogesterone in lymph and venous effluent from perfused ovaries were measured and the results compared to the same parameters measured in vivo. Rates of flow of lymphatic and venous effluent and lymph/plasma protein ratios measured from perfused ovaries were similar to those measured in vivo, and were not statistically affected by the presence of corpora lutea in the ovaries. The concentrations of progesterone and 20 alpha dihydroprogesterone in ovarian venous blood/perfusate and lymph was increased by the presence of corpora lutea, but the concentration of progesterone was lower in vitro than in vivo. The concentration of these progestins in lymph suggest that only a small proportion of ovarian lymph is derived from corpora lutea in vitro or in vivo, and most is derived from ovarian interstitium.

The intraovarian progesterone modulation of follicle development in the rabbit ovary.

Intraovarian progesterone levels were manipulated by surgically adjusting the number of corpora lutea (CL) present in rabbit ovaries and this model was used to study the local effect of luteal progesterone on growth of follicles. The results show that when a single CL or several CL were present, follicle growth was inhibited. However, when all CL on one ovary were removed, increased numbers of follicles grew even when a single CL was present in the contralateral ovary. These findings show that progesterone inhibits follicle growth and that at least part of its action is local, i.e., exerted within the ovary. Additionally, ovarian blood vessels and periovarian lymph ducts were cannulated, and samples were collected and analyzed for steroid and protein content. The results show that when CL were present, ovarian vein progesterone levels were elevated 10-30-fold over levels in ovaries without CL; this high concentration points to the blood vascular system as the principal carrier of the steroid within the ovary. Analysis of lymph showed that protein content was consistently high and that the progesterone concentration was not significantly altered with the presence of CL; these two findings show that ovarian capillaries are extremely permeable to proteins, but the unexpectedly low concentrations of progesterone in lymph may signal an intraovarian countercurrent mechanism by which it is returned to the blood.