(M-CPP) Induced Intracavernous Pressure Responses in Anaesthetized Rats

Metachlorophenylpiperazine (m-CPP) induced intracavernous pressure responses in anaesthetized rats

ES Hayes1* and PG Adaikan1

1Department of Obstetrics and Gynaecology, the National University of Singapore, Singapore

Here we have recorded the effects of metachlorophenylpiperazine (m-CPP) on intracavernous pressure (ICP) in anesthetized rats pretreated with various pharmacological agents in an attempt to determine the mechanism and relevance of the m-CPP induced ICP response to other models of erection. m-CPP elicited consistent and significantly greater increases in ICP (71.5 Æ 6.6 mmHg) compared with the mixed 5-HT2a=2c agonists trifluoromethylphenylpiperazine (3.4 Æ 1.3 mmHg) and quipazine (10.9 Æ 1.8 mmHg). Blockade of 5-HT2a receptors with ketanserin failed to unmask any stimulatory effect of quipazine (7.2 Æ 1.0 mmHg). m-CPP induced ICP responses (71 Æ 7.0 mmHg) were unaffected in the presence of mianserin (63 Æ 5 mmHg) and ketanserin (51 Æ 12 mmHg). Spiperone significantly reduced the m-CPP induced increase in ICP (8.0 Æ 1.0 mmHg). Naloxone, yohimbine and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OHDPAT) failed to elicit increases in ICP on their own. All three drugs significantly reduced the latency to the first m-CPP induced ICP response compared to saline. Yohimbine increased the duration of m- CPP induced ICP responses whereas 8-OHDPAT increased the mean number of m-CPP induced ICP responses compared to saline. The effects of m-CPP on ICP in anesthetized rats may not be mediated by 5-HT2c receptors and appears to be similar to erection in copula, but not erection elicited by other drugs or penile sheath retraction. International Journal of Impotence Research (2002) 14, 287–294. doi:10.1038=sj.ijir.3900888

Keywords: intracavernous pressure; cavernous nerve; erection; serotonin; agonist; antagonist

Introduction actions at 5-HT1b receptors within the spinal cord. However, the receptor that mediates the effects of m- CPP on CN and ICP responses in rats remains Metachlorophenylpiperazine (m-CPP) is a non-se- undetermined. lective serotonin (5-HT) agonist that induces erec- Previously we demonstrated that the novel 5- tion in conscious rats in a manner that is dependent HT2c agonist, RSD992, also elicits erection in upon functional supraspinal 5-HT receptors.1,2 2c conscious rats in a manner dependent upon 5-HT2c Other non-selective 5-HT2a=2c agonists also induce receptors.6 However, unlike m-CPP RSD992 failed to erection in conscious rats when 5-HT2a receptors are induce CN and ICP responses in anesthetized rats.7 masked by pretreatment with selective 5-HT 2a Furthermore, the selective 5-HT1a and 5-HT1b 3,4 5 antagonists. Steers and de Groat found that m- agonists 8-OHDPAT and CGS12066B enhanced CPP also elicits firing of the cavernous nerve (CN) in and inhibited, respectively the m-CPP induced CN anesthetized rats with concomitant increases in and ICP responses.7 Others have demonstrated that intracavernous pressure (ICP). The same authors 8-OHDPAT and CGS12066B inhibit and have no showed that the effects of m-CPP on the CN and ICP effect on m-CPP induced erections, respectively, in in anesthetized rats were blocked by the non- conscious rats.3 Therefore contradictory evidence selective 5-HT antagonist metergoline, enhanced exists for the effects of m-CPP on erection in rather than inhibited by spinal transection and conscious rats versus its effects on CN and ICP abolished by pelvic nerve section. Based on the responses in anesthetized rats. results of those experiments the authors suggested ICP responses resulting from direct nerve stimu- that the m-CPP induced responses were due to lation and drug administration are often used as an index of penile erection.8,9 Therefore, the following studies were undertaken in an effort to determine if 5-HT receptors are responsible for the effects of m- *Correspondence: ES Hayes, University of Washington, 2c RPRC, 3000 Western Avenue, Seattle, WA 98121, USA. CPP on ICP responses in anesthetized rats, deter- E-mail: [email protected] mine the effects that drugs known to modulate Received 10 September 2001; accepted 25 April 2002 erection and=or sexual behavior in conscious rats m-CPP induced intracavernous pressure responses ES Hayes and PG Adaikan 288 have on m-CPP induced CN and ICP responses in ICP responses was determined by counting the anesthetized rats and, discuss the relevance of the number of obvious and rapid increases in ICP to m-CPP induced ICP response in relation to other pressures equivalent to or above those attained models of erection. during pre-drug nerve stimulation. Latency to the onset of the first m-CPP induced ICP response was calculated from the start of drug administration to Materials and methods the first rapid rise in ICP. The duration of m-CPP induced ICP responses were calculated as the time from the onset of the first response to the time at The National University of Singapore animal use which the ICP returned to pre-drug levels. and care committee approved all experimental The first experiment examined the effects of the procedures. Drugs were obtained from Sigma non-selective 5-HT agonists m-CPP, trifluoromethyl- Chemical Co (m-CPP, naloxone, quipazine, yohim- phenylpiperazine (TFMPP) and quipazine on CN bine) or Research Biochemicals Inc. (8-OHDPAT, and ICP responses in anesthetized rats. The second ketanserin, mianserin, spiperone, trifluoromethyl- experiment examined the effects of a series of 5- phenylpiperazine) and prepared according to HT2a=2c antagonists on the m-CPP induced CN and manufacturers instructions. ICP responses in anesthetized rats. The third Male Sprague-Dawley (SD) rats were anesthetized experiment examined the effects of drugs known to with sodium pentobarbitone (65 mg=kg; i.p.) and modulate erection and sexual behavior in conscious prepared for drug administration and blood pressure rats on the m-CPP induced CN and ICP responses in recording through cannulation of the external anesthetized rats. Table 1 provides a description of jugular vein and internal carotid artery, respec- the pharmacological and binding profiles of the test tively. The trachea was cannulated for assisted drugs at selected receptors. respiration at a volume of 10 ml=kg and a rate of Data are expressed as the mean Æ standard error of 60 breaths=min. the mean (s.e.m.) for n ¼ 5 animals per test group. A median incision was made in the lower pelvic Differences between drug and saline treated groups region and the cavernous nerve was isolated from for m-CPP induced ICP variables were determined the prostatic pelvic plexus by blunt dissection and using ANOVA followed by Dunnett’s test for multi- suspended on platinum hook electrodes. The pelvic ple comparisons and P < 0.05 was taken as a cavity was filled with warm mineral oil to prevent significant result in all cases. dehydration. Blunt dissection of the ischiocaver- nous musculature was used to isolate the right penile crus. ICP responses elicited by direct stimu- Results lation of the cavernous nerve and drug administration were recorded on computer using polyethylene Agonist induced intracavernous pressure responses tubing connected to a 27-gauge needle that was ICP responses resulting from direct stimulation of placed into the crus. the CN (20 Hz, 2.5 V, 1 ms pulse width) were similar Experiments were initiated by stimulation of the cavernous nerve and recording of ICP to determine viability of nerve responses. Fifteen minutes after Table 1 Selected pharmacological and binding profiles of the initial nerve stimulation saline or test drugs were drugs used in the present study

injected and CN and ICP responses were recorded Drug Receptor Agonist (pEC50) Antagonist (pKB or pA2) for a further 15 min. After administration of saline or test compounds m-CPP was injected and CN and m-CPP 5-HT1b 6.5 — ICP responses were monitored for an additional 5-HT2c 6.9 — TFMPP 5-HT1b 6.9 — 15 min. To end the experiments the CN was again 5-HT2c 6.8 — stimulated and ICP recorded to conﬁrm responsive- Quipazine 5-HT1b — 6.2 ness to direct stimulation. All drugs were prepared 5-HT2c 6.2 — fresh in saline and injected intravenously in a Ketanserin 5-HT2a — 9.3 5-HT — 6.5 volume of 1.0 ml=kg. 2c Mianserin 5-HT2a — 8.0 Arterial blood pressure (BP) and ICP were 5-HT2c — 8.7 1 recorded on a MacIntosh computer using Ma- Spiperone 5-HT2a — 9.1 1 1 cLab analog to digital conversion and Chart 5-HT2c — 6.1 software. The CN was stimulated with square-wave 8-OHDPAT 5-HT1a 8.2 — 1 5-HT1b 4.9 — pulses delivered from the MacLab output at 20 Hz, Yohimbine a2 — 8.5 2.5 V and 1 ms pulse width until a plateau in ICP 5-HT2b — 7.3 was observed (30 – 60 s). Changes in ICP were Naloxone OP3 — 9.1 calculated as the difference between the maximal Data are expressed as pEC50 values (agonists) or, pKB or pA2 response and the mean ICP over 2 min prior to direct values (antagonists) as adopted from Hoyer et al,10 Bonhaus stimulation or drug administration. The number of et al,14 Bigoni et al41 and Paiva et al.42

International Journal of Impotence Research m-CPP induced intracavernous pressure responses ES Hayes and PG Adaikan 289 amongst treatment groups and ranged from number, latency and duration of ICP responses 27 Æ 4 mmHg to 41 Æ 10 mmHg (P > 0.30). Prelimin- between treatment groups. The CN was responsive ary dose-ranging studies using doses of m-CPP (0.1 – to direct stimulation after the administration of test 1.0 mg=kg; n ¼ 3), TFMPP (0.3 – 3.0 mg=kg; n ¼ 3) and drugs. However, ICP responses to direct CN stimula- quipazine (0.3 – 3.0 mg=kg; n ¼ 3) known to induce tion in the TFMPP treated group (15 Æ 3 mmHg) erection in conscious rats3 indicated that m-CPP were significantly lower (P < 0.001) compared with was the only drug to elicit CN and ICP responses in ICP responses in the other drug treatment groups anesthetized rats at doses producing erection in (range 38 Æ 11 mmHg to 55 Æ 4 mmHg). conscious rats (data not shown). Therefore, further experiments were conducted using equivalent doses of the three test drugs at the lowest m-CPP dose Antagonism of the m-CPP induced intracavernous = producing consistent ICP responses (0.3 mg kg). pressure response Saline and ketanserin failed to induce ICP responses greater than pre-drug mean ICP values that ranged between 7 Æ 2 mmHg and 15 Æ 3 mmHg (data not Pre-drug ICP responses resulting from direct stimu- shown). lation of the CN were similar amongst treatment TFMPP, quipazine (QUIP) and quipazine in the groups and ranged from 30 Æ 5 mmHg to presence of ketanserin (QUIP=KET) failed to elicit 38 Æ 4 mmHg (P > 0.50). The 5-HT antagonists ke- ICP responses similar to those observed for m-CPP tanserin, mianserin and spiperone failed to induce (Figure 1). m-CPP produced a maximal increase in increases in ICP above pre-drug mean ICP values ICP of 72 Æ 7 mmHg compared to 3 Æ 1 mmHg, that ranged between 4 Æ 4 mmHg and 17 Æ 5 mmHg 11 Æ 2 mmHg and 7 Æ 1 mmHg for TFMPP, QUIP (P > 0.10). ICP responses resulting from direct and QUIP=KET, respectively. Pre-drug mean ICP stimulation of the CN and pre-drug mean ICP values were not significantly different between responses were not significantly different from those treatment groups and ranged from 4 Æ 1 mmHg to of the saline=m-CPP treatment group from the first 12 Æ 3 mmHg. In the presence of saline m-CPP experiment. Therefore, the saline=m-CPP group elicited 1 Æ 0 ICP responses with a mean latency of from the first experiment (72 Æ 7 mmHg) also served 285 Æ 18 s and mean duration of 135 Æ 24 s. Changes as control for this experiment. in ICP produced by TFMPP, QUIP and QUIP=KET m-CPP induced maximal changes in ICP of simply reflected fluctuations about the baseline. 63 Æ 5 mmHg and 51 Æ 12 mmHg in the presence of Therefore, the inability of the other test drugs to mianserin and ketanserin, respectively. However in elicit ICP responses precluded a comparison of the the presence of spiperone the m-CPP induced ICP

Figure 1 The effects of metachlorophenylpiperazine (m-CPP; 0.3 mg=kg; solid), triﬂuoromethylphenylpiperazine (TFMPP; 0.3 mg=kg; cross-hatch), quipazine (QUIP; 0.3 mg=kg; diagonal stripe) and quipazine plus ketanserin (QUIP=KET; 0.3 mg=kg=0.03 mg=kg; horizontal stripe) on intracavernous pressure (ICP) in anesthetized rats. The vertical axis indicates ICP in mmHg. Data are expressed the mean Æ s.e.m. for n ¼ 5 animals per test group. An asterisk (*) indicates a signiﬁcant difference compared with m-CPP treated animals.

International Journal of Impotence Research m-CPP induced intracavernous pressure responses ES Hayes and PG Adaikan 290 response was significantly reduced to 8 Æ 1 mmHg Modulation of the m-CPP induced intracavernous (Figure 2). Mean ICP values prior to m-CPP admin- pressure response istration (4 Æ 1 mmHg to 11 Æ 2 mmHg) were not significantly different between treatment groups in which m-CPP induced ICP responses. However, this Direct stimulation of the CN resulted in mean value was significantly higher in the spiperone=m- increases in ICP ranging from 28 Æ 4to39Æ 7 mmHg CPP treatment group as compared to the saline=m- 7 mmHg (P > 0.50) from pre-stimulation mean va- CPP and ketanserin=m-CPP treatment groups lues ranging between 4 Æ 2 mmHg and 13 Æ 7 mmHg (P < 0.05). In the presence of mianserin and ketan- (P > 0.60). Administration of saline or test drugs serin m-CPP induced 1 Æ 0 ICP responses, a value (Naloxone, Yohimbine and 8-OHDPAT) failed to not significantly different from the 1 Æ 0 m-CPP directly influence ICP (Panel A in Figures 3 and 4). induced ICP responses in the presence of saline. Baseline ICP values and maximal changes in ICP However, mianserin and ketanserin did significantly after saline or test drug administration were not reduce the latency to the first ICP response to significantly different between treatment groups 183 Æ 12 s and 155 Æ 20 s, respectively, from a la- (data not shown). tency of 285 Æ 18 s in saline pre-treated rats. Mean In the presence of naloxone, yohimbine and 8- duration of the m-CPP induced ICP responses were OHDPAT m-CPP elicited consistent ICP responses 168 Æ 14 s and 189 Æ 29 s in mianserin and ketanser- above those obtained during direct stimulation of in pretreated rats, respectively and were not sig- the cavernous nerve in the first and second set of nificantly different from a mean duration of experiments (Panel B in Figures 3 and 4). m-CPP 135 Æ 24 s in saline pre-treated animals (P > 0.30). All test groups responded to direct stimulation of the cavernous nerve after m-CPP administration with mean increases in ICP ranging from 31 Æ 3 mmHg to 48 Æ 5 mmHg (P > 0.60).

Figure 2 The effects of saline (SAL; 1.0 ml=kg; solid), mianserin (MIA; 0.03 mg=kg; cross-hatch), ketanserin (KET; 0.03 mg=kg; diagonal stripe) and spiperone (SPIP; 0.03 mg=kg; horizontal Figure 3 The effects of saline (1.0 ml=kg) alone (A) and m-CPP stripe) pretreatment on m-CPP (0.3 mg=kg) induced increases in (0.3 mg=kg) in the presence of saline (B) on blood pressure (BP) intracavernous pressure (ICP) in anesthetized rats. The vertical and intracavernous pressure (ICP) in anesthetized rats. Horizontal axis indicates ICP in mmHg. Data are expressed the mean Æ s.e.m. axes indicate time in minutes (15 min) whereas vertical axes for n ¼ 5 animals per test group. An asterisk (*) indicates a indicate pressures in mmHg. Arrows indicate the administration signiﬁcant difference compared to saline treated animals. of saline (A) or m-CPP (B).

International Journal of Impotence Research m-CPP induced intracavernous pressure responses ES Hayes and PG Adaikan 291 rats, respectively. However, maximal changes in ICP were not significantly different between treatment groups (0.30 < P < 0.60) despite a trend for larger maximal changes in ICP for rats pre-treated with test drugs compared to saline (Table 2). The test drugs did increase the number of ICP responses induced by m-CPP administration. In rats pre-treated with 8- OHDPAT m-CPP elicited 4.0 Æ 0.8 ICP responses compared to 1 Æ 0 ICP responses in saline pre- treated rats (P < 0.01; Figures 3 and 4; Table 2). Naloxone and yohimbine also increased the mean number of m-CPP induced ICP responses to 2.4 Æ 0.2 and 1.2 Æ 0.2 responses, respectively, but this effect was not significant. Latency to the first m-CPP induced ICP response was significantly reduced from 220 Æ 46 s in saline pre-treated rats to 76 Æ 12 s, 102 Æ 17 s and 43 Æ 9 s in naloxone, yohimbine and 8-OHDPAT pre-treated rats, respectively (Table 2). The duration of ICP responses was prolonged from 149 Æ 38 s in saline pre-treated rats to 321 Æ 112 s 696 Æ 130 s and 492 Æ 147 s in rats pre-treated with naloxone, yohimbine and 8-OHDPAT, respectively, but this effect was only significant in the yohimbine pre-treated group (Table 2).

Discussion

The results of the present study demonstrate that: (1) at equivalent doses m-CPP produces increases in ICP in anesthetized rats whereas its structural analogs TFMPP and quipazine do not; (2) at Figure 4 The effects of 8-OHDPAT (0.64 mg=kg) alone (A) and m- equivalent doses the 5-HT2 antagonists spiperone, CPP (0.3 mg=kg) in the presence of 8-OHDPAT (B) on blood but not mianserin and ketanserin, antagonize the m- pressure (BP) and intracavernous pressure (ICP) in anesthetized rats. Horizontal axes indicate time in minutes (15 min) whereas CPP induced increases in ICP; and (3) naloxone, vertical axes indicate pressures in mmHg. Arrows indicate the yohimbine and 8-OHDPAT reduce the latency to the administration of 8-OHDPAT (A) or m-CPP (B). onset of the ﬁrst m-CPP induced ICP response and, tend to increase the number and duration of m-CPP induced ICP responses. induced maximal changes in ICP of 59 Æ 8 mmHg, Previously we described the erection inducing 132 Æ 42 mmHg, 82 Æ 19 mmHg and 99 Æ 31 mmHg effects of the novel compound RSD992 and sug- from baseline ICP values of 7 Æ 2 mmHg, gested that this response was dependent upon 6 11 Æ 4 mmHg, 4 Æ 4 mmHg and 9 Æ 2 mmHg in saline, agonist action at 5-HT2c receptors. m-CPP has also naloxone, yohimbine and 8-OHDPAT pre-treated been shown to elicit erection in conscious rats in a

Table 2 Effects of m-CPP on intracavernous pressure (ICP) in anesthetized rats pretreated with various drugs known to modulate erection and=or sexual behavior in conscious rats

Treatment group

Saline (1.0 ml=kg) Naloxone (4.0 mg=kg) Yohimbine (2.0 mg=kg) 8-OHDPAT (0.64 mg=kg)

ICP maxa 59 Æ 8 132 Æ 42 82 Æ 19 104 Æ 35 ICP responsesb 1 Æ 0 2.4 Æ 0.2 1.2 Æ 0.2 4.0 Æ 0.8* ICP latencyc 220 Æ 46 76 Æ 12* 102 Æ 17* 43 Æ 9* ICP durationd 149 Æ 38 321 Æ 112 696 Æ 130* 492 Æ 147

The effects of saline (1.0 ml=kg), naloxone (4.0 mg=kg), yohimbine (2.0 mg=kg) and 8-OHDPAT (0.64 mg=kg) on maximal changes in ICP (a, mmHg), number of ICP responses (b) and latency (c, seconds) and duration (d, seconds) of ICP responses produced by m-CPP (0.3 mg=kg). Values represent the mean Æ s.e.m. for ﬁve animals. Values in brackets indicate volume in ml=kg for saline or dose in mg=kg for test drugs. An asterisk (*) indicates a signiﬁcant difference compared to the saline pre-treated group (P < 0.05).

International Journal of Impotence Research m-CPP induced intracavernous pressure responses ES Hayes and PG Adaikan 292 1 manner dependent upon 5-HT2c receptors. How- the effects of m-CPP on ICP. The possibility that co- ever, when both compounds were tested for their activation of 5-HT2a receptors might mask a 5-HT2c ability to elicit excitation of the CN and concomitant dependent increase in ICP was excluded by the increases in ICP m-CPP was effective whereas observed lack of ICP responses during quipazine 7 RSD992 was ineffective, thus indicating that the administration in the presence of 5-HT2a blockade effects of m-CPP on erections in conscious rats and with ketanserin. The effect of quipazine plus ICP responses in anesthetized rats could possibly be ketanserin on ICP in anesthetized rats in the present mediated by different mechanisms. study is directly opposite to that produced by the Steers and de Groat5 demonstrated that the m- same drug combination on erection in conscious 3,4 CPP induced effects on CN activity and ICP were rats. It is also unlikely that 5-HT2a receptors are mediated by a 5-HT receptor sensitive to block by responsible for the effects of m-CPP on ICP as metergoline and transection of the spinal cord. TFMPP and m-CPP display similar potency and Although metergoline exhibits a greater affinity for efficacy as partial agonists at 5-HT2a receptors in 10 10,13 5-HT2c vs all other 5-HT receptor subtypes, the vivo and in vitro. Furthermore, quipazine, a dose used by Steers and de Groat5 was high enough more efficacious but equally potent partial agonist at 11 to eliminate any selectivity in antagonist activity. 5-HT2a receptors compared to m-CPP and 13,15 Therefore, although 5-HT2c receptors may be im- TFMPP, failed to elicit ICP responses on its portant for the m-CPP induced erectile responses own. mediated through supraspinal mechanisms in con- Results obtained in the present study with three scious rats, other receptors may be responsible for 5-HT2 antagonists support the idea that 5-HT2a and m-CPP induced CN activity and increases in ICP in 5-HT2c receptors may not be responsible for the m- anesthetized rats. CPP induced ICP response. The m-CPP responses in In an elegant series of experiments Berendsen the presence of molar doses of antagonists (70 – 3 et al, demonstrated a strong correlation (r ¼ 0.98) 100 nmol=kg) known to block 5-HT2 receptors in 11 between 5-HT2c=5-HT2a receptor selectivity ratio, as vivo and in vitro were similar except for spiperone. determined from binding studies, and maximal Mianserin, ketanserin and spiperone antagonize 5- erectogenic effects in vivo for a group of 1-arylpiper- HT2a receptor mediated phospholipase C turnover azine 5-HT2c agonists including m-CPP, TFMPP and in frontal cortex with Kb values of 12, 58 and 6 nM, 16 quipazine. Furthermore ID50 doses for antagonism of respectively and block 5-HT2a mediated responses m-CPP-induced erections in conscious rats were in vivo over dose ranges (4 – 26 mg=kg)17 – 19 encom- also correlated (r ¼ 7 0.89) with 5-HT2c receptor passing the doses used in the present study. There- 3 selectivity of a range of 5-HT2 antagonists. Com- fore, if the m-CPP induced ICP responses were pounds exhibiting selectivity for 5-HT2a receptors mediated by a 5-HT2a receptor mechanism all of the also demonstrate a strong relationship (r > 0.80) antagonists might have been expected to diminish, if between binding affinity at 5-HT2a sites and doses not abolish, the response at the roughly equimolar inhibiting DOI mediated head-twitch response in doses used in this study. Furthermore, mianserin 12 rats (an in vivo bioassay for 5-HT2a antagonists). exhibits similar affinity and potency as an antago- TFMPP, quipazine and m-CPP display similar nist at 5-HT2a a and 5-HT2c receptors whereas affinity for 5-HT2c receptors and, similar potency ketanserin and spiperone exhibit approximately and efficacy in stimulating IP3 turnover in rat 100 and 1000-fold less affinity and potency at 5- 16,20 choroid plexus in vitro (a bioassay for 5-HT2c HT2c receptors. Therefore mianserin, but not agonists).13 We have conducted an analysis of data spiperone, might have been expected to block a 5- 10 14 presented by Hoyer et al and Bonhaus et al and HT2c mediated m-CPP effect on ICP. However, in the have determined a strong correlation for the in vivo present study spiperone completely abolished the and in vitro potency of agonists and antagonists at 5- m-CPP induced ICP response whereas mianserin 3 HT2a=5-HT2c receptors and binding affinities at was without effect. Berendsen et al, have demon- native and cloned human and rat 5-HT2a=5-HT2c strated that, in contrast to the present study, the m- receptors (r ¼ 0.96-0.98; n ¼ 7; t ¼ 7.8 – 15.3, CPP induced erection in conscious rats (5-HT2c)is t(0.05,5) ¼ 2.571; P < 0.001). Therefore, although com- blocked by mianserin and ketanserin with ID50 plete dose – response curves were not examined in values of 0.06 and 2.0 mg=kg, respectively, but not this study, doses were selected based on known in by spiperone (0.1 – 1.0 mg=kg). Taken together these vivo and in vitro potency and selectivity at the results suggest that 5-HT2a and 5-HT2c receptors may various receptor subtypes and, preliminary experi- not be responsible for the effects of m-CPP on ICP. ments. Spiperone is an a-adrenoceptor (ADR) antagonist Preliminary dose – response experiments and sin- with a pA2 for a-adrenoceptor antagonism approxi- gle equivalent dose experiments both indicated that mately 100-fold lower than prazosin21,22 and, ap- TFMPP and quipazine failed to initiate increases in proximately 100-fold lower than its pA2 for 10 ICP over dose ranges known to produce erection in antagonist action at 5-HT2a receptors. Spiperone 3 22,23 conscious rats (0.3 – 3.0 mg=kg), thus supporting the exhibits selectivity for the a1a ADR subtype. idea that 5-HT2c receptors may not responsible for Prazosin, a non-selective a1 – 2 antagonist, is often

International Journal of Impotence Research m-CPP induced intracavernous pressure responses ES Hayes and PG Adaikan 293 used to induce erection or enhance erection pro- response does not resemble erectile responses duced by other agents.24 However, in anesthetized elicited by retraction of the penile sheath. In this rats and dogs prazosin tends to reduce ICP elicited regard glans erections observed during penile sheath by stimulation of the CN but this effect is not clearly retraction are not attenuated by section of the dose-dependent and, differs between species.9 On cavernous nerve35 whereas m-CPP induced ICP the other hand, the same authors demonstrated that responses in anesthetized rats are abolished by 5 the selective a1a ADR antagonist REC 15=2841 failed cavernous nerve section, thus suggesting differen- to inhibit CN mediated ICP responses in rats or dogs tial mechanisms for these two stimuli as models of at doses 10 – 30 times higher than the dose of erection. spiperone used in the present study. Although we In tests of copulatory performance naloxone has are not able to completely rule out an a-ADR been shown to produce equivocal effects on latency antagonist action for the effect of spiperone on m- to intromission or ejaculation with no effect at low CPP induced ICP in the present study, it is doses31,36 and reduced latency to both variables at interesting to note that ketanserin is comparable to higher doses.37 In contrast, yohimbine reduces both 38 spiperone in terms of affinity and potency as an a1- intromission and ejaculation latency and, 8-OHD- ADR antagonist23,25 but failed to significantly reduce PAT strongly reduces intromission and ejaculation the m-CPP induced ICP response. latencies and increases the number of ejaculations Naloxone, yohimbine and 8-OHDPAT have dif- in copulatory tests.39,40 In the present study all three ferential effects on erection elicited in different compounds reduced the latency to the first m-CPP experimental models and were employed here in induced response and, 8-OHDPAT was particularly order to gain some insight into the relevance of the effective in this respect. Furthermore, 8-OHDPAT m-CPP induced ICP response to other models of significantly increased the number of ICP responses erection. In conscious rats naloxone enhances elicited by m-CPP. The inability of test drugs to spontaneous and apomorphine induced erectile elicit ICP responses on their own combined with activity26,27 whereas 8-OHDPAT fails to enhance their ability to enhance m-CPP induced responses spontaneous erection on its own2 and, attenuates suggests that their effects are stimulus dependent. apomorphine and m-CPP induced erection.28 The strong effect observed for 8-OHDPAT in the Although yohimbine has not been tested for its present study suggests that the m-CPP induced ICP effects on apomorphine or m-CPP induced erections response may resemble erection and ejaculatory in conscious rats, the selective a2 antagonists patterns observed in copula more closely than piperoxan and idazoxan have been shown to lack erectile responses generated by genital reflex testing the ability to attenuate apomorphine-induced erec- and pharmacological stimulation within the CNS. 29 tion. 8-OHDPAT is a potent inhibitor of 5-HT2c mediated erections in rats and is in keeping with Conclusion results regarding physiological antagonism of 5- 12 HT2c receptor-mediated events by 5-HT1a agonists. Therefore, the fact that 8-OHDPAT significantly The results of this study suggest that m-CPP induced enhanced the m-CPP induced ICP response at doses increases in intracavernous pressure may not be known to elicit maximal agonist action at 5-HT1a mediated by 5-HT2a or 5-HT2c receptors. Although receptors12 strongly suggests that the m-CPP in- the receptor responsible for the m-CPP effects on ICP duced ICP response in anesthetized rats differs from was not identified in the present study it is worthy of the m-CPP induced erectile responses in conscious further investigation based on the observed interac- rats. tion with 5-HT1a receptor stimulation. Naloxone, Naloxone has been shown to either have no effect yohimbine and 8-OHDPAT all reduced the latency to on, or reduce, the number of erections and latency to onset of the first m-CPP induced ICP response and ejaculation in tests employing penile sheath retrac- tended to increase the number and duration of such tion to elicit erection.30,31 Yohimbine has been responses. Therefore the m-CPP induced ICP re- shown to inhibit erectile and ejaculation responses sponse appears to a greater degree of similarity to elicited by penile sheath retraction at doses similar erection and ejaculation in copula as compared to to the one used in the present study.32 Similarly, 8- erection elicited through pharmacological means or OHDPAT and other selective 5-HT1a agonists have genital reflex testing. The ability of 8-OHDPAT to been shown to completely abolish erectile and significantly enhance m-CPP induced ICP responses ejaculatory responses mediated by penile sheath may have implications for the pharmacological retraction.33,34 The results of the present study inducement of erection and ejaculation. clearly demonstrated a positive effect of all the compounds with respect to the number and dura- Acknowledgements tion of ICP responses elicited by m-CPP and a reduction in the latency to onset of the first m-CPP induced ICP response. Based on this experimental These studies were supported through a National evidence it is suggested that the m-CPP induced ICP University of Singapore post-graduate scholarship

International Journal of Impotence Research m-CPP induced intracavernous pressure responses ES Hayes and PG Adaikan 294 awarded to ES Hayes. LC Lau and B Srilatha are 19 Valentin JP et al. Use of the pithed rat model to determine the thanked for technical assistance and review of the relative potencies of 5-HT2A=2C receptor antagonists following acute intravenous and oral administration. Methods Find manuscript. Exp Clin Pharmacol 1995; 17: 267 – 271. 20 Bonhaus DW et al. RS-102221: a novel high affinity and selective, 5-HT2c receptor antagonist. Neuropharmacology 1997; 36: 621 – 629. References 21 Pupo AS. Functional effects of castration on alpha1-adrenoceptors in rat vas deferens. Eur J Pharmacol 1998; 351: 217 – 223. 1 Millan MJ et al. 5-HT2c receptors mediate penile erections in 22 Amobi N et al. 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