Intracavernous administration of SIN-1 þ VIP in an in vivo rabbit model for erectile function

(2002) 14, 44 49 ß 2002 Nature Publishing Group All rights reserved 0955-9930/02 $25.00 www.nature.com/ijir Intracavernous administration of SIN-1 þ VIP in an in vivo rabbit model for erectile function O Sazova 1, A Kadioğlu 2,LGürkan 2, Z Kayaarasi 2, S Bross 1, M Manning 1 and KP Jünemann 1 * 1 Department of Urology, Mannheim Medical Faculty, University of Heidelberg, Germany; and 2 Department of Urology, Istanbul Faculty of Medicine, Istanbul University, Turkey In accordance with the data reporting the identification of nitric oxide synthase (NOS) and vasoactive intestinal polypeptide (VIP) positive nerve fibres in the trabecular meshwork of the corpus cavernosum, we suggest that nitric oxide (NO) and VIP may serve complementary physiological roles in penile erection. Therefore SIN-1 and VIP were administered alone and in combination in an in vivo rabbit model. All rabbits revealed basal pressure values of 5 8 cm H 2 O intracavernously. In the rabbits intracavernously (i.c.) injected with SIN-1 alone and VIP alone, no adequate erectile responses were observed. Whereas, in the group intracavernously injected with the combination of SIN-1 þ VIP, erectile responses with mean maximal intracavernous pressure (max. ICP) 52.8 ( 13.2) cm H 2 O were noted. These pressure elevations do not statistically diverge (P > 0.05) than the ones obtained in the control group administered i.c. injections of the combination of papaverine=phentolamine (mean max. ICP 51 ( 14.73) cm H 2 O). Referring to our results, we conclude that the combined use of SIN-1 þ VIP could play an important role in the physiological treatment of erectile dysfunction. (2002) 14, 44 49. DOI: 10.1038=sj=ijir=3900813 Keywords: nitric oxide; vasoactive intestinal polypeptide; SIN-1; erectile dysfunction; cgmp; camp Introduction Penile erection is a neurovascular phenomenon consisting of arterial dilatation, venous compression and sinusoidal relaxation. Subsequent to neurological relaxation of the smooth muscle cells of the cavernous sinusoidal spaces and penile arterial vessel walls, the sinusoidal spaces of both corpora cavernosa are filled with blood. The expansion of these sinusoidal spaces rapidly leads to elongation and expansion of the penis. The indistensible character of tunica albuginea causes compression of the subtunical veins between the sinusoidal spaces and the tunica albuginea. In contrast, the subtunical arterioles and nerves are protected from compression by the surrounding loose areolar tissue. 1 Therefore the arterial blood flow during erection is maintained. Moreover, the compressed emissary veins easily become occluded, thus impeding the reflux of blood from the penis. 2 Relaxation of the sinusoidal smooth muscle cells is probably mediated through the intracellular cyclic *Correspondence: KP Jünemann, Klinik für Urologie, Universitaetsklinikum Kiel, Christian-Albrechts-Universität zu Kiel, Arnold-Heller-Str. 7, D-24105 Kiel, Germany. E-mail: kjunemann@urology.uni-kiel.de Received December 1998; accepted June 1999 nucleotide pathway. Here cyclic guanosine monophosphate (cgmp), generated by guanylate cyclase and cyclic adenosine monophosphate (camp), generated by adenylate cyclase, play major roles. 3 The main mechanism controlling smooth muscle relaxation in penile erection is the autonomic nervous system. It is now believed that the chemical nature of the main postganglionic neurotransmitter within the corpus cavernosum that mediates erection is not cholinergic, but nonadrenergic-noncholinergic (NANC). It is assumed that the latter plays a crucial role in penile erection. 4 In the past, it was thought that numerous neurotransmitters, such as, NO, VIP, calcitonin gene related peptide, neuropeptide Y, tyrosine hydroxylase and substance P were the main action neurotransmitters in the relaxation of trabecular smooth muscle. Today, nitric oxide (NO) is considered to be the major neurotransmitter. However, it is still uncertain whether NO is the only transmitter and this remains a subject of debate. In our previous study on human erectile tissue, we reported the identification of NOS and VIP positive nerve fibres in the trabecular meshwork of the corpus cavernosum ( > 90%) and surrounding the helicine arteries ( > 50%). 5 According to these results we planned to use the NO releasing chemical compound SIN-1 and VIP in combination in an

in vivo rabbit model as a combination of these substances seemed adequate for pharmacological treatment of erectile dysfunction. Materials and methods Male Chinchilla rabbits (b.w. 3.5 4.0 kg) were divided into four groups. Each rabbit was intramuscularly sedated with Ketanest (ketamine, 15 mg=kg) and anaesthesia was induced by intravenous infusion of Ketanest (0.15 ml=min) and Rompun (xylazin, 0.075 ml=min). The animals were placed in supine position and after removal of the muscle and connective tissue surrounding the penis, a 24-gauge needle was inserted into one of the corpora cavernosa for pressure recording. A WIEST 6000 pressure transducer was utilised for monitoring and recording the intracavernous pressure. The arterial blood pressure was also recorded in one of the femoral arteries. In previous reports and in our trials, 5 IU heparin were administered every 2 h to prevent blood clotting. The heparin dosage was kept low to avoid changes in penile hemodynamics. The first group, consisting of seven rabbits was intracavernously injected with 0.15 mg=0.15 ml SIN- 1. The six rabbits in the second group were administered i.c. injections of 2 mg=0.2 ml VIP. These doses of SIN-1 and VIP are similar to the doses administered in previous rabbit studies with these substances. SIN-1 þ VIP were administered to the eight rabbits in the third group in the same amounts used in previous groups. SIN-1 was dissolved in distilled water and freshly prepared for each single injection. VIP was dissolved in 0.05 M acetic acid. Acetic acid is not expected to show any effect on the intracavernosal pressure. The fourth group served as a control group and the rabbits in this group (n ¼ 7) were administered i.c. papaverine=phentolamine (3 mg and 0.1 mg respectively in 0.2 ml of distilled water). This represents the most commonly used and effective dose of pharmacological substances in in vivo animal models relating to penile erection. The pressure was recorded for at least 30 min after each injection. If there was a pressure rise over 25 cmh 2 O, we continued the pressure recording for at least 2 h as this condition was considered to be an erectile response. The basal value of intracavernous pressure before injections was 5 8 cmh 2 O. The maximal pressure recorded after the injection was named maximal intracavernous pressure (max. ICP) and the time interval between the half of the maximal pressure during increase and the half of the maximal pressure during decrease of the blood pressure was named duration of the erection. Statistical evaluation of the results was carried out in accordance with the Student s t-test, comparing the mean max. ICPs. Results In seven of the eight rabbits injected with IC SIN-1 (group 1), no erectile response was noticed. In one of these eight rabbits, the max. ICP increased from 8 to 40 cmh 2 O and this erection lasted for 70 min (Table 1). The mean value of max. ICPs was 18.63 ( 9.32) cmh 2 O. A typical response to IC injection of SIN-1 alone can be seen in Figure 1. VIP alone was injected in six rabbits (group 2) and no significant increase in the ICP was detected (Table 2). The mean value of the max. ICPs in this group was 17.5 ( 3.93) cmh 2 O. Figure 2 shows the results of one of the pressure recordings after injection of VIP alone. Erectile response was noticed in six of the eight rabbits injected with the combination of the same amounts of SIN-1 and VIP (group 3). The mean max. ICP of these six erectile responses was 52.8 ( 13.2) cmh 2 O. In two of these six rabbits the erection lasted for more than 2 h, the mean duration in the other four rabbits was 45 ( 16.99) min. In the last two rabbits of this group, no significant increase in Table 1 Rabbits in group 1 with i.c. injections of SIN-1 1 16 no erectile response 2 12 no erectile response 3 16 no erectile response 4 19 no erectile response 5 10 no erectile response 6 40 70 7 15 no erectile response 8 21 no erectile response Mean max. ICP of the rabbits in group 1: 18.63( 9.32) cmh 2 O. Figure 1 A typical response to SIN-1. The line shows the injection of 0.15 mg SIN-1 in 0.15 ml distilled water. Table 2 Rabbits in group 2 with i.c. injections of VIP 1 12 no erectile response 2 16 no erectile response 3 20 no erectile response 4 19 no erectile response 5 23 no erectile response 6 15 no erectile response Mean max. ICP of the rabbits in group 2: 17.5 ( 3.93) cmh 2 O. 45

46 Figure 2 A typical response to VIP. The line shows the injection of 2 mg VIP dissolved in 0.2 ml 0.05 M acetic acid. the ICP was noticed. Taken on average, the ICP values of the eight rabbits in this group, the mean max. ICP was 45.5 ( 17.6) cmh 2 O (Table 3). Figure 3 shows the results of a pressure recording after the combined use of SIN-1 þ VIP. In the last group consisting of seven rabbits injected with papaverine=phentolamine (group 4), all rabbits responded with erections with mean max. ICP 51 ( 14.73) cmh 2 O. Except in one rabbit with an erection lasting for more than 2 h, the average duration was 48 ( 31.87) min (Table 4). A typical response after IC injection of papaverine=phentolamine is depicted in Figure 4. There were statistically significant differences between the maximal intracavernous pressures of groups 1 and 3, as well as between groups 2 and 3 Table 3 Rabbits in group 3 with i.c. injections of SIN-1 þ VIP 1 68 70 2 48 33 3 69 > 120 4 24 no erectile response 5 38 35 6 53 42 7 23 no erectile response 8 41 > 120 Mean max. ICP of all rabbits in group 3: 45.5 ( 17.6) cmh 2 O. Mean max. ICP of only the erectile responses in group 3: 52.8 ( 13.2) cmh 2 O. Mean duration of the erectile responses in group 3: 45 ( 16.99) min. (for both P < 0.01). Using the Student s t-test, no significant statistical difference in maximal intracavernous pressure between groups 3 and 4 (P > 0.05) was observed (Table 5). As the mean max. ICP of group 3, both values mentioned above (mean value of six erectile responses and mean value of all eight measurements in this group) were taken as a comparison to group 4. The first comparison was the one between the erectile responses of both groups, the second comparison was the one of both groups in general. During all these recordings, the systemic blood pressure was between 75 and 90 mmhg, and this can be considered normal under ketamine and xylazin anaesthesia. Discussion The trabecular smooth muscle relaxation within the cavernosal bodies of the penis is believed to be mediated through the intracellular cyclic nucleotide pathway. The cyclic nucleotides camp and cgmp Table 4 Rabbits in group 4 with i.c. injections of papaverine= phentolamine 1 34 29 2 56 29 3 48 60 4 76 19 5 34 45 6 59 > 120 7 50 106 Mean max. ICP of the rabbits in group 4: 51 ( 14.73) cmh 2 O. Mean duration of the erectile responses in group 4: 48 ( 31.87) min. Figure 4 A typical response to papaverine=phentolamine. The line shows the injection of papaverine=phentolamine (3 mg and 0.1 mg, respectively in 0.2 ml of distilled water). Table 5 Comparisons with the Student s t-test Comparison P-value Figure 3 A typical response to SIN-1 þ VIP. The first line shows the injection of 2 mg VIP in 0.2 ml 0.05 M acetic acid, the second line represents the injection of 0.15 mg SIN-1 in 0.15 ml distilled water. Group 1 Group 3 P < 0.01 Group 2 Group 3 P < 0.01 Group 4 Group 3 P > 0.5 Group 4 Group 3 (only erectile responses considered) P > 0.5

are important intracellular second messengers in mediating the relaxation of smooth muscle cells in various organs. When guanylate cyclase is activated, it causes intracellular accumulation of cgmp. cgmp mainly activates protein kinase G and also protein kinase A. 3 camp on the other hand, which is generated by the stimulation of adenylate cyclase, activates mainly protein kinase A and to a lesser extent also protein kinase G. These two activated protein kinases then stimulate the Ca 2þ pump in the membrane of the sarcoplasmic reticulum. Smooth muscle relaxation follows as the consequence of reduced cytoplasmic Ca 2þ levels. In the smooth muscle, cgmp and camp do not only show their effect through Ca 2þ, several other mechanisms have also been suggested. 6 As mentioned in the introduction, the principal neurotransmitter mediating these alterations for trabecular smooth muscle relaxation in the corpora cavernosa is characterized as a NANC neurotransmitter. This is supported by in vivo and in vitro experiments. 4,7 Many investigators have suggested that the 28 aminoacid peptide VIP is one of the NANC neurotransmitter(s) in the trabecular smooth muscle of the corpus cavernosum. 8,9 In support of this, several observations revealed that VIP immunoreactive fibres densely innervate the trabecular smooth muscle and that VIP elicits relaxation of the trabecular smooth muscle. 10 The results of some in vivo studies demonstrated that VIP can induce an increase in ICP in various species. 11 Jünemann et al concluded that the physiological dose of VIP cannot induce a full erection in dogs and that the major VIP induced hemodynamic change during penile erection is more likely to be found in the venous drainage system. 11 This conclusion became evident after analysing the venous studies of the same group and the data revealed that venous outflow restriction took place after minimal doses of i.c. VIP injection. These results have also been supported by other authors. 12 In 1990 Ignarro et al demonstrated that NANCmediated relaxation in rabbit corpus cavernosum causes the release of NO and the accumulation of cgmp. 13 It has been proven that NO is the neurotransmitter responsible for the NANC activity in the peripheral nervous system of various organs. 14 The localisation of NOS in the peripheral autonomic nerves innervating vascular and nonvascular smooth muscle was further confirmation of the NANC activity of NO. 15 Electrical stimulation of human corpus cavernosum tissue with disrupted endothelium and in the presence of muscarinic adrenergic blockade results in NANC neurogenic relaxation. Some studies have shown that substances that interfere with the synthesis or the effects of NO significantly reduce or block this response. These experiments, together with the existence of NOS in the cavernosal tissue, contribute strong evidence towards the assumption that NO is the principal neurotransmitter that initiates the relaxation of corpus cavernosum trabecular smooth muscle. 16 18 Although the fact that NO is the principal neurotransmitter of penile erection is common knowledge nowadays, several studies still doubtfully express that it is not the only one. 19 21 These studies demonstrated the existence of alternative pathways for penile erection independent of NO. Ehmke et al identified the presence of NOS and VIP positive nerve fibres in the trabecular meshwork of the corpus cavernosum ( > 90%) and also surrounding the helicine arteries ( > 50%). 5 NOS positive axons contained neither immunoreactivities of substance P nor of tyrosine hydroxylase. In diabetic patients NOS=VIP immunoreactivity was reduced and in patients with lesions of the cavernous nerve, it was completely absent. They concluded that the high degree of coexistence of NOS and VIP immunoreactivity in human cavernous nerves indicates that NO is not the sole neuronal mediator involved in penile erection. This implication correlates with experimental ICP results gained during nerve stimulation and the concurrent administration of a false substrate of either NOS or VIP antibodies. While the blockade of NOS activity by an infusion of arginine analogues can completely inhibit the rise in ICP in response to nerve stimulation, the administration of VIP antibodies severely compromises the maintenance of erection. 11 On the other hand, the initial engorgement of the penis during nerve stimulation could not be prevented by the administration of VIP antibodies. Conversely, the administration of SIN-1, S-nitroso- N-acetylpenicillamine or sodium nitroprusside - spontaneously NO liberating chemical compounds in in vivo models and chemical trials either caused inadequate erectile responses or very high doses of these compounds were necessary for the induction of erectile responses. 22 25 Inadequate erectile responses only were achieved with VIP. 11,12,26 In addition, reports have been made on the coactivation of NO and VIP in other organs. 27,28 These findings have led to the suggestion that NO and VIP may serve complementary roles during various phases of erection. As NO stimulates intracellular guanylate cyclase and causes the accumulation of cgmp in the smooth muscle cells, it probably triggers the relaxation of the corpus cavernosum trabecular meshwork. cgmp serves as a second messenger molecule with the capacity to influence the corporal smooth muscle contractile state by reducing intracellular Ca 2þ. The half-life of the vasoactive effects of NO is 30 s. 29 However, the half-life of the vasoactive effects of VIP is considerably longer (10 min). This fact and the abovementioned findings imply that the primary role of VIP during erection could be to effect the duration of venous restriction by maintaining the relaxant effects of NO in the smooth muscle cells. 9,11,26 The 47

48 effects of VIP are regulated via camp, which is accumulated within the smooth muscle cells through the activation of adenylate cyclase by VIP. As mentioned above, the accumulation of both these cyclic nucleotides within the smooth muscle cells induces relaxation by reducing cytoplasmic Ca 2þ levels. Recent studies have also suggested that VIP may be important to ensure survival and effectiveness of the short-lived NO. 5,30 Kirkeby et al supported the assumption that short interruptions of NO release in the absence of VIP could result in an interruption of erection. 26 They disclosed that VIP dissolved in 0.5 ml volumes did not significantly increase corpus cavernosum pressure compared with equivalent volumes of solvent, but increased the time interval until return of the pressure to steady-state level. The results achieved by us demonstrated the following: In groups 1 and 2 with SIN-1 and VIP alone, we detected no significant erectile response and this, in general, coincides with the results proclaimed in the bibliography. However, in comparison, some authors reported better results than others when using SIN-1 alone. 31,32 In group 3, we administered SIN-1 þ VIP and erectile responses similar to those with papaverine=phentolamine were achieved. These results support our suggestion that, in the treatment of erectile dysfunction, a good effect is attained by the combined use of SIN- 1 þ VIP. Conclusion With the help of our results, we propose the combined administration of SIN-1 and VIP in further investigations. The dosage of SIN-1 and VIP should be adjusted in future trials in order to avoid prolonged erections as observed in two rabbits of group 3. References 1 Lue TF, Tanagho EA. Hemodynamics of erection. In: Tanagho EA, Lue TF, McClure RD (eds). 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