The Relaxation Effect and Mechanism of Action of Higenamine in the Rat Corpus Cavernosum

ORIGINAL ARTICLE The relaxation effect and mechanism of action of higenamine in the rat corpus cavernosum

SC Kam1,JMDo1, JH Choi1, BT Jeon2, GS Roh2, KC Chang3 and JS Hyun1

1Department of Urology, Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju, Korea; 2Department of Anatomy, Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju, Korea and 3Department of Pharmacology, Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju, Korea

Higenamine mediates cardiotonic, vascular relaxation and bronchodilator effects. The relaxation effects and the mechanism of action of higenamine on the rat corpus cavernosum (CC) were assessed to investigate the effect of higenamine on penile erection. Strips of CC and aorta were used in organ baths for isometric tension studies. Tension was measured with isometric force transducers, and muscle relaxation was expressed as the percent decrease in precontraction induced by phenylephrine (PE). The relaxation reactions were investigated in an endothelial-denuded group and groups pretreated with N(G)-nitro-L-arginine methyl ester (NO synthesis inhibitor), þ propranolol (b-receptor blocker), indomethacin (COX inhibitor), glibenclamide (KATP channel inhibitor), 4-aminopyridine (membrane potential-dependent potassium channel inhibitor) and methylene blue (guanylyl cyclase inhibitor) for 30 min. Intracavernous pressure (ICP) was assessed in rats after the intravenous administration of higenamine, and changes in guanosine 30,50-cyclic monophosphate and adenosine 30,50-cyclic monophosphate (cAMP) concentrations were measured on the basis of the higenamine concentration. Also, the combined reaction of higenamine and the phosphodiesterase type-5 (PDE-5) inhibitors was assessed. Higenamine induced relaxation of the CC and the aortic strips precontracted with PE in a dose-dependent manner. The CC was significantly more relaxed than the aortic rings in response to the same higenamine concentration (Po0.05). The CC relaxation reaction was suppressed by the b-receptor blocker propranolol. The cAMP concentration increased gradually with increased higenamine concentration (Po0.05). The ICP also increased with increased higenamine concentration in vivo (Po0.05). In the group pretreated 7 with 10 M higenamine, the relaxation reaction of CC induced by the PDE-5 inhibitor increased significantly, compared with CC exposed to the PDE-5 inhibitor but not pretreated with higenamine (Po0.05). In conclusion, higenamine induced relaxation of the rat CC in a dose-dependent manner. The effect may be mediated through b-adrenoceptors. The results suggest that higenamine may be valuable as a new lead compound for treating erectile dysfunction. International Journal of Impotence Research (2012) 24, 77–83; doi:10.1038/ijir.2011.48; published online 29 September 2011

Keywords: corpus cavernosum; erection; higenamine

Introduction important previously have appeared. Among them, male erectile dysfunction (ED) is a disease that With the development of medicines and improve- greatly affects the quality of life. ments in general hygiene, the average lifespan The incidence of ED in males older than 40 is 52% has been prolonged and several problems concern- according to the Massachusetts Male Study.1 ing the quality of life that were not considered Additionally, the incidence of ED increases with age. That is, many males past middle age have ED. Recently developed phosphodiesterase type-5 (PDE-5) inhibitors have been widely used as first- Correspondence: Dr J-S Hyun, Department of Urology, line therapeutics for treating ED. Although large Institute of Health Sciences, Gyeongsang National Uni- multicenter clinical trials have shown the efficacy versity School of Medicine, 90 Chilamdong, Jinju 660-702, South Korea. and tolerability of these drugs in ED with various E-mail: [email protected] etiologies and a broad range of severity, 30–35% of Received 12 January 2011; revised 5 July 2011; accepted patients fail to respond. The reported 62% prescrip- 17 August 2011; published online 29 September 2011 tion renewal rate at 3-4 months of follow-up, which Mechanism of action of higenamine on penile erection SC Kam et al 78 drops to approximately 30% by 6-12 months, condition was restored by washing the preparation suggests that patients stop taking the drug for with KH solution three more times. Such mani- reasons other than treatment failure.2,3 The use of pulation was repeated, and contracture within PDE-5 inhibitors may result in side effects, such as 100±10% of the previous contraction was defined visual disturbance, headache, facial flushing, rhini- as the ideal resting optimal isometric tension. tis and indigestion. Other treatments for ED include Although maintaining the resting optimal isometric injection therapy within the penis or penile tension, tissues were contracted by pretreatment 6 implants. However, such methods are invasive and with PE (5 10 M) within the bath, higenamine in 4 7 5 irreversible, and have not been used widely. Thus, five-fold dilutions from 10 to 10 M was added, there is a continuing need for the development and the relaxation level was assessed by observing of new non-invasive and effective therapies for the change in the tension curve. patients with ED. The relaxation reactions were investigated in Kosuge et al.5 purified a substance from the endothelial-intact or endothelial-denuded groups perennial Aconiti tuber plant belonging to to examine the mechanism of higenamine-induced Ranunculaceae, which has an active cardiotonic relaxation of the CC. Endothelial-intact or -denuded component, characterized its chemical structure, tissues from the CC were incubated for 30 min in KH and named it ‘higenamine.’ Higenamine mediates solution containing various drugs. After incubation, 5,6 7 8 6 cardiotonic, vascular relaxation, bronchodilator the tissues were contracted with PE (5 10 M), and anti-thrombotic effects,9,10 and reduces apopto- during which period the plateau state contraction tic cell death in vivo,11 suggesting that higenamine was attained. The relaxation reaction following the has various actions on the cardiovascular system addition of increasing concentrations of higenamine and that it may have an effect on penile erection. was then observed. Drug probes included N(G)- 5 In this study, we investigated the effect of nitro-L-arginine methyl ester (L-NAME; 10 M,NO 5 higenamine on relaxation of the corpus cavernosum synthesis inhibitor), propranolol (10 M, b-receptor 5 (CC) in rats. The mutual action of higenamine and blocker), indomethacin (3 10 M, COX inhibitor), 5 þ PDE-5 inhibitors was also assessed. glibenclamide (10 M,KATP channel inhibitor), 5 4-aminopyridine (10 M, membrane potential- dependent potassium channel inhibitor) and methy- 5 12–14 lene blue (10 M, guanyl cyclase inhibitor). Materials and methods Eight sections were used for each group. CC sections were pretreated with 3 ml 0.3% 3-[(3-cholamide Organ-bath study of the CC propyl)]-1-propane sulfonate (CHAPS) for 20 s to We sectioned the CC of male Sprague Dawley rats, remove vascular endothelial cells. The sections 9–11 weeks old, weighing 280–320 g; eight animals were rubbed lightly using the thumb and index per group were used. The rats were killed by finger for 20 s, washed with KH solution, and the cervical dislocation, the penis was dissected cells were removed by rolling gently on dry paper. immediately, and 2 2 6-mm sections of the CC The removal of vascular endothelial cells was were prepared. The thoracic aorta was extracted, confirmed by the failure of the loss of tissue 5 connective tissues were removed, and aortic rings of contraction induced by acetylcholine (10 M)in 6 3 mm in length were prepared. CC sections and response to PE (5 10 M). aortic rings were transferred to an organ bath; one end was connected to a muscle fixation ring and the other end was connected to an isometric tension transducer (SG-10). The signals were relayed to a Measurement of guanosine 30,50-cyclic physiography instrument (PowerLab ADI Instru- monophosphate (cGMP) and adenosine 30,50-cyclic ments, Sydney, Australia) and measured. Chart 5 monophosphate (cAMP) in the rat CC software (ADI Instruments) was used for real-time Contraction of the smooth muscle sections was monitoring of tension. Krebs–Hanseleit (KH) solu- equalized for 1 h in KH solution, induced with tion was used for the organ bath at 37 1C and pH 7.4, PE (5 106 M), and the tissues were exposed and gassed continuously with 95% O2/5% CO2. to higenamine for 10 min. Then, the tissues were When the initial tension of each section was frozen rapidly in liquid nitrogen and homogenized maintained at approximately 2 g, the KH solution in ice-cold 6% trichloroacetic acid. The homoge- was changed approximately every 30 min (total nized tissues were centrifuged for 15 min, and equilibration period: 2 h) and allowed to reach cellular proteins in the supernatant were separated a stable condition. Once a stable condition was by conventional methods. Samples were divided reached, phenylephrine (PE) was added and into a normal CC group and groups of 107,106 and 5 the contraction level was observed. Each section 10 M according to the concentration of higen- was progressively stretched to the optimal point amine, and the concentrations of released cGMP on its length-tension curve as determined by the (n ¼ 8/group) and cAMP (n ¼ 8/group) were active tension developed to PE. Afterwards, a stable measured by immunoassay. The immunoassay was

International Journal of Impotence Research Mechanism of action of higenamine on penile erection SC Kam et al 79 based on the competitive binding technique in Combined effects of higenamine and the PDE-5 which cGMP and cAMP present in a sample inhibitors on the CC compete with a fixed amount of horseradish perox- The relaxation effects of the PDE5 inhibitor on 6 idase-labeled cGMP and cAMP for sites on a precontracted CC strips with PE (5 10 M) were rabbit polyclonal antibody. During the incubation, assessed for any synergistic effect of combined PDE5 the polyclonal antibody becomes bound to the goat inhibitor and higenamine treatment. The PDE5 anti-rabbit antibody coated on the microplate. inhibitor and combinations diluted five-fold from 5 4 Following a wash to remove excess conjugate and 10 to 5 10 M were added and relaxation was unbound sample, a substrate solution is added to assessed. The minimal concentration of higenamine the wells to determine the bound enzyme activity. inducing relaxation of the CC was obtained from the The color development is stopped and the absor- above experiment. The CC sections were contracted bance is read at 450 nm. The intensity of the color is by pretreatment with higenamine for 20 min at inversely proportional to the concentration of cGMP the minimal concentration obtained from the above and cAMP in the sample. experiment, the PDE-5 inhibitor was added and the relaxation level was observed.

Induction of an erection by electrical stimulation The 32 Sprague-Dawley male rats were divided into Drugs and solutions four groups (n ¼ 8 each): group 1, normal control; 1 PE, L-NAME, glibenclamide, 4-aminopiridine, in- group 2, intravenously injected 0.0005 mg kg domethacin, methylene blue, propranolol and higenamine; group 3, intravenously injected CHAPS were purchased from Sigma (St Louis, MO, 0.001 mg kg1 higenamine; and group 4, intrave- 1 USA). The cGMP immunoassay kit (KGE003) and nously injected 0.002 mg kg higenamine. cAMP immunoassay kit (KGE002) were purchased Experimental rats were anesthetized with keta- 1 from R&D Systems (Minneapolis, MN, USA). mine (50 mg kg ) injected into the peritoneal cavity. The PDE5 inhibitor (udenaphyl) was supplied by Animals were placed in the supine position on an Dong-A Pharmaceuticals (Seoul, South Korea). The 1 operation table, a catheter (polyethylene-50 tube) composition of the KH solution was (mM l ) NaCl, was inserted into the carotid, and blood pressure 118.1; NaHCO , 25; KCl, 4.6; KH PO , 1.2; CaCl , (BP) was monitored. The prostate was exposed with 3 2 4 2 2.5; MgSO4, 1.2 l; glucose, 11.0; and pH 7.4. a midline incision of the abdomen. Additionally, the Higenamine was synthesized and purified as de- pelvic ganglion located in the posterolateral side of scribed in Chang et al.16,17 the prostate was assessed, and the pertinent pelvic nerves and cavernosal nerves were assessed and dissected without injuring the nerves. After intravenous injection of higenamine through the internal Data analysis jugular vein, the cavernosal nerves were stimulated Statistical significance was analyzed by Student’s using an electric stimulator and platinum electrodes t-test and one-way analysis of variance with after about 10–15 min. To assess an erection in Scheffe’s F-test using SPSS software (version 14.0; response to electrical stimulation of the nerves, the SPSS, Chicago, IL, USA). Results are expressed penis was dissected up to the penile crura, the as the mean±s.d. A P-value o0.05 was deemed to tunica albuginea of the penis was assessed by indicate statistical significance. dissecting the ischial CC muscles surrounding the penile crura and a 25-gauge needle was installed (polyethylene-50 tube pretreated with 250 U ml1 Results heparin) into the CC.15 The BP and the intracavernosal pressure (ICP) Effect of higenamine on aortic rings and CC were measured using a BP manometer transducer As the concentration of higenamine increased from 7 and recorder (PowerLab, ADI Instruments). Chart 5 10 M, the aortic rings and CC pre-contracted with software (ADI Instruments) was used for real-time PE relaxed. The first relaxation reaction occurred at 7 BP monitoring. Electrical stimulation was per- a concentration of 10 M and increased according to formed at a voltage of 5 V and duration of 60 s. the increase in the higenamine concentration. When 5 To examine the hemodynamic reaction in re- the concentration of higenamine was 10 M, relaxa- sponse to autonomic nerve stimulation, pressure tion of the aortic ring and CC was approximately was measured prior to nerve stimulation, during 35.3±2.6 and 92.5±6.9%, respectively. stimulation and after stimulation. Additionally, to Higenamine induced relaxation of the CC, and the examine the reactions induced by nerve stimulation, aortic rings contracted in response to PE in a dose- the percentage of the maximal ICP/mean systemic dependent manner (Po0.05). The CC group (n ¼ 8) arterial BP (max ICP/MAP 100) was measured by showed significantly more relaxation than the stimulating the CC nerves, and differences among aortic-ring group (n ¼ 8) in response to the same the groups were compared. concentration of higenamine (Po0.05; Figure 1).

International Journal of Impotence Research Mechanism of action of higenamine on penile erection SC Kam et al 80 Mechanism of the relaxation reaction induced by higenamine The relaxation reaction of the CC was suppressed only by the b-adrenoreceptor blocker propranolol. 5 When the concentration of higenamine was 10 M, maximal relaxation was approximately 92.6±6.9% in the control group, but it was 42.1±7.3% after pretreatment in the propranolol group (Po0.05; Figure 2). However, pretreatment with L-NAME, indomethacin, glibenclamide, 4-aminopyridine and methylene blue for 30 min, as well as the results of endothelial- denuded tissues did not suppress the relaxation of the CC by higenamine (Figure 2).

Figure 1 Effect of higenamine on aortic rings and CC smooth muscle in the rat. Aortic rings and CC tissue relaxed in response to higenamine in a dose-dependent manner. The CC showed Concentration of cGMP and cAMP in the CC significantly more relaxation than the aortic rings in response to the same concentration of higenamine (Po0.05; n ¼ 8 per group). according to the concentration of higenamine w Po0.05. Concentrations of cGMP and cAMP in normal and higenamine-treated CC were measured using immunoassays. The cAMP concentrations were 26.17±5.69 pmol per mg protein in the normal CC without higenamine, 34.48±4.65 pmol per mg 7 protein in the 10 M higenamine group, 43.95± 6 9.01 pmol per mg protein in the 10 M higenamine group and 49.59±6.36 pmol per mg protein in the 5 10 M higenamine group. The cAMP concentration increased gradually as the higenamine concentration increased, and the concentration of cAMP in 6 5 the 10 and 10 M higenamine groups showed statistically significant increases compared with the control group (Po0.05; Figure 3a). The cGMP concentrations were 0.63±0.26 pmol per mg protein in normal CC without higenamine, 1.38±0.59 pmol 7 Figure 2 Effects of various treatments on the relaxation response per mg protein in the 10 M higenamine group, 6 of the rat CC. The relaxation reaction of the CC was suppressed 2.00±1.13 pmol per mg protein in the 10 M only by the b-adrenoreceptor blocker propranolol. However, higenamine group and 3.86±2.07 pmol per mg 5 pretreatment with L-NAME, indomethacin, glibenclamide, protein in the 10 M higenamine cGMP group. 4-aminopyridine and methylene blue for 30 min, as well as the results of endothelial-denuded tissues, did not suppress the The cGMP concentration increased gradually as relaxation of the CC by higenamine (Po0.05; n ¼ 8 per group). the higenamine concentration increased, but this w Po0.05. was not statistically significant (P40.05; Figure 3b).

Figure 3 Effects of higenamine on (a) cAMP and (b) cGMP in the rat CC. The concentrations of cAMP and cGMP in the CC increased in response to higenamine in a dose-dependent manner, but cGMP had no statistical significance. The concentration of cAMP in the 106 5 and 10 M higenamine groups increased significantly, compared with the control group (Po0.05; n ¼ 8 per group). *Po0.05.

International Journal of Impotence Research Mechanism of action of higenamine on penile erection SC Kam et al 81 Effect of higenamine on ICP The mean systemic arterial BPs and heart rates of the control group and the experimental groups prior to and after electrical stimulation of the cavernosal nerve were not statistically different. Before cavernosal nerve stimulation, the mean baseline ICP/ MAP did not show a statistically significant difference in four groups (group 1: 10.2±2.7%, group 2: 10.0±2.4%, group 3: 9.3±2.1% and group 4: 10.1±1.7%). At the time of cavernosal nerve stimulation, the mean ICP of group 1 (control) was 72.9±11.7 mm Hg, and the max ICP/MAP 100 was 59.2±6.6%. The mean ICP of group 2 (0.0005 mg kg1 higenamine) was 67.3±13.0 mm Hg and the max ICP/MAP 100 was 59.3±10.4%. The 1 Figure 4 ICP changes during cavernosal nerve stimulation. mean ICP of group 3 (0.001 mg kg higenamine) was Graph shows an increase in ICP in response to cavernosal nerve 87.7±14.4 mm Hg and the max ICP/MAP 100 was stimulation in all groups. Maximal ICP/MAP in the 0.002 74.5±8.4%. The mean ICP of group 4 (0.002 mg kg1 and 0.001 mg kg1 higenamine groups increased significantly, compared with that in the control group (Po0.05; n ¼ 8 per higenamine) was 87.8±14.1 mm Hg and the max w ICP/MAP 100 was 76.0±5.1%. The max ICP/ group). Po0.05. MAP 100 of groups 3 and 4 were statistically higher than that of the control group (Po0.05). However, group 2 (0.0005 mg kg1 higenamine) did not show a statistically significant difference from the control group (P ¼ 0.19; Figure 4).

Combined effects of higenamine and PDE-5 inhibitors in the CC The effect of the PDE5 inhibitors based on the PE concentration of precontracted CC strips was examined. As the concentration of the PDE5 inhibitor was 5 increased from 10 M, the CC showed dose-related relaxation effects. When the concentration of the 4 PDE-5 inhibitor was 5 10 M, the mean relaxation was 84.3±13.9%. As the minimal concentration of Figure 5 Relaxation effects of a PDE-5 inhibitor on the rat CC 7 7 higenamine effective for relaxing the CC was 10 M, in the presence of higenamine (10 M). CC tissue relaxed in 7 the strips were pretreated with 10 M higenamine response to the PDE-5 inhibitor in a dose-dependent manner. 7 Pre-treatment with higenamine (10 M) increased PDE-5 inhi- for 20 min. The strips were contracted with PE w 6 bitor induced-relaxation (Po0.05; n ¼ 8 per group). Po0.05. (5 10 M), and the PDE-5 inhibitor was then added. The results showed that in the group treated 4 4 with 10 or 5 10 M PDE-5 inhibitor, the relaxa- induced relaxation of the CC in a dose-dependent tion reaction of the CC increased significantly, manner. Additionally, the CC responded more compared with the PDE-5 inhibitor group without sensitively to the same higenamine concentration higenamine pretreatment (Po0.05; Figure 5). than the aorta, so the relaxation effects were greater. This confirms that higenamine acts preferentially on the CC rather than systemically. Discussion Higenamine acts on the heart, the blood vessels and the bronchus through actions at the adrenergic Higenamine is a benzyltetrahydroisoquinoline-type b-receptor. The CC contracts in response to the drug derived from the Aconiti tuber.5 Higenamine activation of adrenergic a-receptors18,19 and relaxes acts as an adrenergic b-agonist to effectively treat when adrenergic b-receptors are activated.20,21 That heart failure. Thus, most studies of higenamine have is, activation of a-adrenoreceptors is involved in the focused on its chronotropic effects. Studies have flaccid state of the CC and suppression of penile been conducted on the adrenergic b-receptor ex- erection.22 In contrast, activating b-adrenoreceptors pressed in blood vessels and smooth muscle. is involved in an erection. Similarly, the relaxation However, this is the first reported study that has effect of higenamine was efficiently suppressed by assessed the reaction mechanism and relaxation the non-selective adrenergic b-antagonist proprano- effects of higenamine on CC tissue. Higenamine lol in our experiments. However, the nitric oxide

International Journal of Impotence Research Mechanism of action of higenamine on penile erection SC Kam et al 82 synthesis inhibitor, L-NAME, the guanylate cyclase increasing cAMP in the CC. Enhanced relaxation inhibitor methylene blue, the prostaglandin inhibi- effects were observed when we examined tor indomethacin, the membrane potential-depen- the combined effects of a PDE-5 inhibitor and þ dant K channel (KV) blocker 4-aminopyridine, the higenamine. Higenamine treatment significantly ATP-dependant K þ channel blocker glibenclamide increased ICP, compared with the control group. and the presence or absence of endothelial cells did According to the results of this study, higenamine not affect relaxation of the CC induced by higen- activated the b-adrenoceptors. Also, it is well amine. After detecting the presence of b-adrenocep- known that b-adrenoceptors increase the cAMP. So tors in the human CC, several animal experiments considering this phenomenon, it is suggested have been performed to examine which subtypes are that b-adrenoceptors activated by higenamine in- involved in the primary reactions. In canine CC creased the cAMP.6,7 We do not have any data tissues, the b2 and b3 subtypes primarily mediate the regarding the results of this study that higenamine 23,24 relaxation reaction. In contrast, mixed b1- and has some PDE5 inhibitor activity. We guess that b2-adrenoceptors mediate relaxation reactions in the action mechanisms of higenamine and PDE-5 in- horse CC.21 Furthermore, atypical b-adrenoceptors hibitor showed synergistic effect have two signal and b2-adrenoceptors mediate the primary relaxa- pathways. One is b-adrenoceptors/cAMP pathway tion reaction in rabbits.25 However, in our study, the by higenamine and the other is guanylate cyclase/ b-adrenoceptor subtype of the CC that higenamine cGMP pathway by PDE-5 inhibitor. But Kim et al.34 mediated the major relaxation reaction could not reported that cAMP upregulated the intracellular be assessed using the non-selective adrenergic levels of cGMP. They suggested several potential b-antagonist propranolol. Wong et al.8 suggested mechanisms for this regulation. Increased cAMP that b-adrenoceptors sensitive to higenamine are synthesis may lead to stimulation of guanylyl primarily located in the endothelial layer of vessels. cyclase or to inhibition of PDE-5. These alterations However, our results showed that the relaxation of cyclase or phosphodiesterase activity could occur effect of higenamine was not related to the presence via direct interaction with cAMP or in an indirect or absence of the CC endothelial layer. manner via cyclic nucleotide-dependent protein Adrenergic nerves are suppressed when the penis kinase modulation of enzyme activity. Such is stimulated by the local reaction of prostaglandins results suggest that higenamine may be a useful and vasoactive intestinal peptide. The penile drug for ED or a supplemental drug with PDE-5 CC smooth muscles are relaxed due to reactions inhibitors. Additional animal and clinical studies mediated by cholinergic or nonadrenergic-noncho- are required. linergic nerves and endothelium-derived relaxing We investigated the relaxation effect of higen- factors released from vascular endothelial cells. amine on the CC and characterized its mechanism Then, the artery expands and blood influx increases, of action. Additionally, this study was conducted to resulting in an elevation of intra-penile pressure. assess the possibility of using higenamine as a The vein between the tunica albuginea and the therapeutic for ED. The results show that higen- expanded sinusoid is compressed, resulting in amine induced more relaxation in the CC than in the reduced blood efflux, so an erection occurs.26–28 aorta in a dose-dependent manner, and it enhanced cGMP and cAMP are second messengers involved in the relaxation reaction of the CC induced by PDE-5 such a vascular smooth muscle relaxation reaction. inhibitors. Furthermore, higenamine significantly Increases in such second messengers within the increased ICP. However, studies to examine whether cells cause the dephosphorylation of myosin light the higenamine concentration in the human CC chains, which induces relaxation of CC smooth could reach a maximal relaxation concentration and muscle.29 PDE-5 inhibitors, currently used as first- whether the higenamine concentration inducing line therapy for ED, suppress the hydrolysis an erection causes other side effects in humans are of intracellular cGMP, raise cGMP concentration required. and thus, enhance an erection.30,31 Nevertheless, numerous patients with ED have experienced treatment failure. In particular, low treatment success rates have been reported for patients with diabetes Conclusions and patients who have undergone surgery for Higenamine induced relaxation of the rat CC in a prostate cancer.32 Thus, the development of drugs dose-dependent manner. The mechanism was a that can compensate is required. In our study, reaction involving b-adrenoreceptors in the CC. higenamine increased the concentration of cGMP Higenamine induced relaxation of the CC by elevat- and cAMP in the CC tissues in a dose-dependent ing intracellular cAMP. Such reactions were more manner. Nonetheless, although intracellular cAMP specific in the CC than the aorta. Furthermore, increased significantly, the cGMP increase was higenamine elevated ICP and significantly augmen- not statistically significant. Similar to the results of ted the relaxation reaction of the CC induced by studies conducted on other blood vessels,33 higen- PDE-5 inhibitors. Thus, higenamine may be valuable amine induced the relaxation reaction, mainly by as a new lead compound for the treatment of ED.

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