Androgenic maintenance of the erectile response in the rat

Steroids 64 (1999) 605– 609

Androgenic maintenance of the erectile response in the rat Thomas M. Millsa,b,*, Yutian Daib, Vivienne S. Stoppera, Ronald W. Lewisb a

Department of Physiology and Endocrinology, Urology Section Medical College of Georgia, Augusta, Georgia 30912-3000, USA b Department of Surgery, Urology Section Medical College of Georgia, Augusta, Georgia 30912-3000, USA

Abstract 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. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Penile erection; Testosterone; Nitric oxide; Norepinephrine; Blood flow

1. Introduction The erectile response of the penis is based on hemodynamic changes in the corpus cavernosum, which result from arteriolar dilation and relaxation of cavernosal smooth muscle. Under the driving force of the mean arterial pressure, the cavernosal sinuses fill with blood, and as they expand, the veno-occlusive mechanism is activated, which limits the rate of blood outflow from the sinuses. The combination of increased inflow and decreased outflow results in the high intracavernosal pressure characteristic of penile erection. Essential to the erectile response is the relaxation of the smooth muscle cells that line the cavernous sinuses and the smooth muscle cells in the tunica media of the cavernosal arterioles [1]. Studies from several laboratories have elucidated a variety of neurotransmitters and second messenger pathways that regulate the state of contraction/relaxation of the arteriolar and cavernosal smooth muscle cells [2,3]. Nitric oxide (NO) is widely accepted to be the principle agent that causes smooth muscle relaxation, although pros* Corresponding author. Tel: ⫹1-706-721-3401; fax: ⫹1-706-7217299. E-mail address: [email protected] (T.M. Mills)

taglandin E1 and vaso-active intestinal polypeptide may also play a role. Contraction of the cavernosal smooth muscle is caused by norepinephrine and possibly by endothelin-1. Less well understood is the extent to which androgens regulate the erectile response, and how these steroid hormones may influence the actions of neurotransmitters on smooth muscle cells. Over the past several years, studies from this laboratory have investigated the effects of castration and testosterone replacement on the rat’s erectile response based on the working hypothesis that androgens act at multiple sites in the erectile response [4 –11]. This presentation will summarize many of our findings.

2. Materials and methods 2.1. Castration and testosterone replacement In all experiments, rats were castrated and immediately implanted with a pellet of testosterone (TESTO) or with a pellet of cholesterol (CASTRATE). In some experiments, nonoperated rats (INTACT) were employed for comparison. TESTO rats had circulating levels of testosterone of ⬃1 ng/ml, whereas values in CASTRATE rats were 20 –50

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T.M. Mills et al. / Steroids 64 (1999) 605– 609

Fig. 1. The erectile response (CCP/MAP) in INTACT, TESTO, and CASTRATE rats. Brackets represent SEM; asterisks indicate significant difference from TESTO values (n ⫽ 5– 8). Reprinted from [5] with permission.

pg/ml. In the INTACT animals, blood testosterone levels were 2–3 ng/ml [5]. 2.2. Measuring the erectile response Under general anesthesia (ketamine/xylazine) rats were fitted with several cannulae, including a carotid cannula, for continuous monitoring of mean arterial pressure (MAP); an intracavernosal cannula, for continuous monitoring of the intracavernosal pressure (CCP); and a second intracavernosal cannula, for the injection of drugs directly into the erectile tissue. Once all cannulae were in place, electrodes were positioned on the major pelvic ganglion (MPG), the autonomic ganglion that regulates the intracavernosal blood flow, for stimulation at 1–5 V, 12 Hz [7]. In some studies [11], intracavernosal blood flow (CCF) was monitored with a laser Doppler flow meter (FC302, PeriMed Stockholm, Sweden). The detection probe for this flow meter was positioned at the base of the penis to minimize movement artifacts caused by changes in the size of the penis during erection.

3. Results and discussion 3.1. The effect of androgens on the erectile response In Fig. 1, the erectile response, expressed as the ratio of intracavernosal pressure to mean arterial pressure (CCP/ MAP), is shown for INTACT, CASTRATE, and TESTO rats [5]. This method of expressing the results was used because stimulating the MPG leads to a transitory decline in MAP in some animals. Fig. 1 shows that the response is significantly lower in the CASTRATE animals compared to the INTACT and TESTO rats. We have previously reported that the response is measurably lower by 24 h after castration, with only a moderate further decline by the seventh

Fig. 2. The rate of blood flow into the corpora cavernosa of TESTO and CASTRATE rats. Each point is the mean of seven measurements with brackets representing SEM. Asterisks indicate significant differences between TESTO and CASTRATE at the same voltage. From [11] and used with permission.

week of castration [5]. However, even in these long-term, castrated animals, a portion of the erectile response remains, demonstrating that there is an androgen-dependent and an androgen-independent component to the erectile response in rats. Fig. 1 also points to the fact that the circulating level of testosterone may be in excess of that needed to maintain erection. Even though there is approximately twice as much testosterone in the blood of the INTACT rats compared to the TESTO animals, the magnitude of the erectile responses is not different. Fig. 2 shows the results of the direct measurement of the rate at which blood flows into the cavernous sinus during stimulation of the MPG to induce erection [11]. To accomplish this measurement, the distal end of the penis was amputated just proximal to the os penis while the penis was in the nonerect state. At the cut end, the dorsal vein and corpus spongiosum were ligated with microclips so that only blood from the corpus cavernosum emerged from the remaining portions of the shaft. By timing the interval required to collect 100 ␮l of blood into a capillary tube, the flow rate in ml/min could be determined. The results of these measurements clearly demonstrate that at 3, 4, and 5 V, the rate of blood flow into the erectile tissue was significantly greater in the TESTO than in the CASTRATE animals. From this observation, it can be suggested that there is either more cavernosal smooth muscle in the TESTO rats or the degree of relaxation of the existing smooth muscle is greater when testosterone is present. 3.2. The androgenic regulation of NO synthesis in the erectile response In previous studies, we used N-nitro-L-arginine (LNNA), a competitive inhibitor that blocks the binding of substrate L-arginine to nitric oxide synthase (NOS) and thereby blocks the synthesis of NO. When this inhibitor was infused

T.M. Mills et al. / Steroids 64 (1999) 605– 609

Fig. 3. The erectile response (CCP/MAP) in CASTRATE and TESTO rats before any treatment (STIM), 10 min after LNNA injection into the erectile tissue (200 ␮g/kg body weight), and after injection of a 100-fold excess of L-arginine (L-ARG). Each bar represents the mean of five animals; brackets equal SEM. Means with different superscripts are significantly different from one another. Reprinted from [9] with permission.

systemically (jugular vein), there was a marked reduction in the magnitude of the erectile response, suggesting that NO was mediating a portion of the erectile response in rats. Systemic infusion of LNNA also significantly increased mean arterial blood pressure [4]. Fig. 3 demonstrates that a single, intracavernosal injection of 200 ␮g of LNNA/kg inhibited the erectile response in both CASTRATE and TESTO rats, but did not alter MAP. To confirm that LNNA is a competitive inhibitor of NOS in the erectile tissue, administration of a 100-fold excess of L-arginine partially reversed the effect of LNNA and increased CCP in both CASTRATE and TESTO animals [4]. Based on the demonstration that inhibition of NOS activity reduced the magnitude of the erectile response, we hypothesized that androgens act primarily to regulate NO availability during erection. It would then follow that in castrated rats with no source of androgen, there would be a deficiency of NO and that exogenous NO should enhance the response in the castrated rats more than the response in the TESTO animals. To test this hypothesis, CASTRATE and TESTO rats were treated with an intracavernous injection of sodium nitroprusside (SNP), a drug that releases NO; measurements were made with and without ganglionic stimulation [9]. Figure 4 shows that, during stimulation only (STIM), the expected difference in the response of CASTRATE and TESTO was apparent. Following injection of SNP only into the intracavernosal sinuses (SNP), there was a similar rise in the CCP/MAP ratio in both treatment groups. Ganglionic stimulation following SNP administration (STIM ⫹ SNP) failed to significantly increase the response in TESTO rats; however, in the CASTRATE group, the supplemental NO (i.e., STIM ⫹ SNP) yielded an additional effect, with the magnitude of the CCP/MAP rise greater than in either STIM or SNP. These observations support the hypothesis that androgens regulate the availabil-

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Fig. 4. The erectile response (CCP/MAP) in CASTRATE and TESTO rats resulting from ganglionic stimulation (STIM), intracavernous injection of SNP (8 ␮g/kg body weight), or stimulation following SNP administration (STIM ⫹ SNP). Each bar represents the mean of 10 rats; brackets equal SEM. Means with different superscripts are significantly different from one another. Reprinted from [9] with permission.

ity of NO to the erectile mechanisms. Studies from other laboratories have drawn the same conclusion [12–14]. Additional investigation into the role of androgens and NO in the regulation of the erectile response employed semi-quantitative reverse transcriptase-polymerase chain reaction to measure the abundance of the neuronal form of nitric oxide synthase (nNOS) mRNA [9]. Fig. 5 shows the results of the electrophoretic analysis of the reverse transcriptase-polymerase chain reaction products in CASTRATE and TESTO animals, in which nNOS mRNA is expressed in terms of the expression of the reporter gene, cyclophilin. These results show that in TESTO rats, the amount of nNOS mRNA in cavernosal tissue is significantly greater than that in CASTRATE animals.

Fig. 5. Reverse transcriptase polymerase chain reaction amplification of cyclophilin (216 bp) and the neuronal form of the nitric oxide synthase (nNOS) cDNA in TESTO and CASTRATE penile tissue. The gels were scanned, and the peak areas of the nNOS bands were normalized to the areas of cyclophilin and expressed as arbitrary units. Each bar is the mean ⫾ SEM for observations in six rats. Asterisk indicates that nNOS is more abundant in the tissues from the TESTO rats than from the CASTRATE animals. Reprinted from [9] with permission.

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Fig. 6. Representative tracing of the intracavernosal pressure (CCP) and mean arterial pressure (MAP) during erection. Arrows indicate the intracavernosal injection of phenylephrine (1 ␮g/kg body weight) into the aorta of a TESTO and a CASTRATE rat. Horizontal bars indicate ganglionic stimulation. Reprinted from [10] with permission.

3.3. Androgens and ␣ adrenergic responsiveness in the erectile response The flow of blood into the cavernosal sinuses is dependent on a balance between neurotransmitters, which cause contraction of arteriolar and cavernosal smooth muscle, and factors such as nitric oxide, which cause this smooth muscle to relax. The principal factor that controls cavernosal smooth muscle contraction is the ␣ adrenergic agonist norepinephrine. Experiments were designed to determine whether androgen also regulates the responsiveness of the cavernosal tissue to ␣ adrenergic stimulation. Fig. 6 shows a representative tracing of CCP and MAP response to ganglionic stimulation in a TESTO and a CASTRATE rat. After CCP had reached a maximum, phenylephrine, a potent ␣ adrenergic agonist, was injected directly into the cavernous sinuses [10]. This figure shows that in both treatment groups, phenylephrine injection caused a rapid decline in CCP, but an increase in MAP, although the decline in the CCP/MAP was greater in the CASTRATE (90% decline) than in TESTO (50% decline). To confirm this apparently greater sensitivity to phenylephrine of CASTRATE rats, dose-response studies were completed in CASTRATE and TESTO rats in which doses of phenylephrine (0.001–50 ␮g/kg body weight) were given (Fig. 7). When these results were analyzed statistically, it was found that the ED50 for the phenylephrine-induced decline in CCP in CASTRATE rats was 0.3 ⫾ 0.1 ␮g/kg, which was significantly less than the ED50 for this response in TESTO rats, 1.8 ⫾ 0.5 ␮g/kg [10].

Fig. 7. The effects of increasing doses of phenylephrine on CCP during induced erection. The dose range for phenylephrine was from 1 ng/kg body weight to 50 ␮g/kg body weight. Statistical analysis revealed that these dose-response curves were significantly different. Reprinted from [10] with permission.

of normal erection. To investigate intracavernosal blood flow through the penis during erection in CASTRATE and TESTO rats, a Laser Doppler flow meter was employed. Fig. 8 shows representative tracings of CCP (expressed in relative units) and CCF (expressed in relative units) in CASTRATE and TESTO rats [5]. In full agreement with our previous findings [5], the CCP values for the TESTO rat were greater than the those for the CASTRATE rats. The CCF also shows an important difference between TESTO and CASTRATE animals: in the androgen-treated rat, the rate of flow rose over the first few seconds of stimulation and then declined to levels that were no different from the prestimulation levels, indicating that outflow was sharply impeded and veno-occlusion had occurred. In the

3.4. Effects of androgens on the rate of blood flow out of the cavernosal sinuses In addition to the increased flow of blood into the cavernosal sinuses, an impediment of blood outflow is part of the normal erectile response. Without this veno-occlusion, blood would not be retained in the cavernous sinuses, and the intracavernosal pressure would not rise to the levels characteristic

Fig. 8. Representative tracings of the intracavernosal pressure (CCP) and intracavernosal blood flow (CCF) during erection in a TESTO and a CASTRATE rat. Note that veno-occlusion is indicated by the sharp decline in flow as maximal pressure is achieved in the TESTO rat. In the CASTRATE rat, CCF remains high, indicating that veno-occlusion failed to occur.

T.M. Mills et al. / Steroids 64 (1999) 605– 609

Fig. 9. The ␣ actin content of penile tissue collected from TESTO and CASTRATE rats was determined after electrophoretic separation and specific staining. The gels were scanned, and the density of the peaks were expressed relative to a rat aorta smooth muscle standard. The results of this preliminary experiment suggest that there is little difference in the ␣ actin content of penile tissue in the two treatment groups.

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pects of the erectile response in rats. Our findings, along with reports from other laboratories, show that testosterone regulates the availability of NO. In addition, androgens regulate the sensitivity of the response to ␣ adrenergic stimulation; because ␣ adrenergic agonists favor detumescence, our finding that testosterone lowers the sensitivity is in keeping with the greater erectile response in the TESTO animals. We also report here a difference between CASTRATE and TESTO treatment groups in the activation of the veno-occlusive mechanism. This difference could be due to a change in the physical properties of the tunica albuginea (tissue compliance) or veno-occlusion could be activated by the greater rate of inflow in the TESTO rats (Fig. 2). Studies into the effects of androgens on other physical properties of the corpus cavernosum (collagen content of the tunica albuginea, number of smooth muscle cells, ␣ actin content of cavernosal cells, etc.) are ongoing.

References CASTRATE rat, CCF remained high, indicating high rates of outflow and little or no veno-occlusion. Thus, veno-occlusion, as indicated by the initial rise followed by a sharp decline, appears to be characteristic of the androgen-maintained erectile response. It can also be suggested from this experiment that the lower CCP values during the erectile response in CASTRATE rats are dependent only on inflow, whereas in the TESTO rats, both inflow and veno-occlusion are involved. 3.5. Androgenic effects on cavernosal smooth muscle cells The protein ␣ actin was used as a marker for smooth muscle cells in the cavernosal tissue. To measure the amount of this protein, penises were removed from CASTRATE and TESTO rats, homogenized, and the proteins extracted. The proteins were separated electrophoretically and then immunochemically stained with a specific antibody for ␣ actin, with the results expressed relative to an ␣ actin standard prepared from rat dorsal aorta (Fig. 9). The results of this preliminary Western analysis suggest that there was no difference in the total amount of this protein in the two treatment groups. Once this observation has been confirmed with additional experiments, the findings will suggest that the number of smooth muscle cells in the two treatment groups (i.e. TESTO and CASTRATE) is not different. It would thereby follow that the differences between the erectile response in TESTO and CASTRATE rats are due to biochemical differences in the erectile response and not only to anatomic changes that result from castration.

4. Summary and conclusions When taken together, the results of these studies strongly support our hypothesis that androgens regulate several as-

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