The Relaxation Effects Of Tetrandrine On The Corpus Cavernosum Tissue Of Rabbit In Vitro

Objective: In the course of our studies on the development of naturally occurring agents for the treatment of ED, we demonstrated that the five extracts, tetrandrine, kakonein, scutellarin, ginsenoside Rg1 and ginsenoside Rb1, from traditional Chinese medicine can relaxation of corpus cavernosum smooth muscle. Moreover, we have identified tetrandrine was more potent than the other four extracts. In the present study, the relaxation mechanisms of tetrandrine on the corpus cavernosum smooth muscle were investigated.Methods: â‘  The fluctuation of the dose-response relaxation curves for the concentrations of KCl induced by tetrandrine were observed with isolated rabbit corpus cavernosum tissues. The effects of tetrandrine were examined on isolated muscle stripes precontracted with phenylephine (PE) alone, in the presence of indomethacin (cyclooxygenase inhiditor), N^w-nitro-L-arginine (LNNA, a nitric oxide synthase inhibitor), 1-H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ, a guanylyl cyclase inhibitor), tetraethylammonium (TEA, Ca²⁺-activated K⁺ channel blocker), 4-aminopiridine (4-AP, voltage dependent K⁺ channel blocker) and glibenclamide (ATP sensitive K⁺channel blocker). â‘¡ The corpus cavernosum smooth muscle cells from New Zealand White rabbits were cultured in vitro. [Ca²⁺]i was measured by Fluorescence Ion Digital Imaging System (FIDIS), using Fluo-2/AM as a Ca²⁺-sensitive fluorescenct indicator. â‘¢ The effects of tetrandrine on the concentration of cyclic adenosine monomphsopate (cAMP) and cyclic guanosine monomphsopate (cGMP) in rabbit corpus carvemosum tissues were recorded by means of ¹²⁵I radioimmunoassay.Results: â‘  The dose-response curves of KCl were shifted to the right nonparallelly, and the maximal responses were depressed to 73.0±3.8% and 41.5±3.4%, respectively, in the presence of tetrandrine (10μmol/L, 30μmol/L). Corpus cavernosum stripes showed relaxation in response to tetrandrine (10^-8～10^-3mol/L) in a concentration-dependent manner with an IC₅₀ of 3.73×10^-5mol/L. However they were not affected by indomethathacin, LNNA, ODQ and K⁺-channel blockers. â‘¡ Tetrandrine (1μmol/L, 10μmol/L, 100μmol/L) had no effect on the resting [Ca²⁺]_i (P>0.05). In the presence of extracellular Ca²⁺(2.5 mmol/L), tetrandrine (1μmol/L, 10μmol/L,100μmol/L) inhibited [Ca²⁺]_i elevation induced by high K⁺ and phenylephrine (PE) in a concentration-dependent manner (P<0.05). In calcium free solution containing egtaic acid (EGTA), tetrandrine (1μmol/L and 10μmol/L) had no inhibitory effects on [Ca²⁺]_i elevation induced by PE (P>0.05). However, tetrandrine (100μmol/L) inhibited [Ca²⁺]_i elevation induced by PE (P<0.05). â‘¢ The basal concentration of cAMP in rabbits corpus carvemosum tissues is 5.67±0.97 pmol/mg. tetrandrine increased cAMP concentration directly in a concentration-dependent manner (P<0.05), but this effect couldn't be inhibited by adenylate cyclase inhibitor (MDL-12, 330A) (P>0.05). The accumulation of cAMP to PGE₁ (a stimulator of cAMP) can also be augmented by tetrandrine in a concentration-dependent manner (P<0.05).The basal concentration of cGMP in rabbits corpus carvemosum tissues is 0.44±0.09 pmol/mg. Tetrandrine didn't affect any concentration of cGMP in corpus carvemosum tissues in the presence or in the absence of a guanylate cyclase inhibitor (ODQ) (P>0.05). Nor did it enhance SNP (a stimulator of cGMP)-induced cGMP production (P>0.05).Conclusion: The results of the present study suggest that tetrandrine possesses a relaxant effect on rabbit corpus cavemosum tissues. The mechanism is manifested by the fact that tetrandrine reduces the intracellar free Ca²⁺ level of corpus carvemosum smooth muscle cells by means of the following three ways:(1) the inhibition of extracellular Ca²⁺ infux from the extracellular site via voltage-activated Ca²⁺ channel and α₁-adrenoceptor-operated Ca²⁺ channel, (2) the inhibition of release of intracellular stored Ca²⁺, and (3) the inhibition of activities of phosphodiesterase and then enhancement of the concentration of cAMP in rabbit corpus carvemosum tissue. But it is not mediated by the release of nitric oxide, prostaglandins or by the activation of potassium channels.