Inhibitory effect of mitragynine, an analgesic alkaloid from Thai herbal medicine, on neurogenic contraction of the vas deferens.

The effect of an indole-alkaloid mitragynine isolated from the Thai medicinal herb kratom (Mitragyna speciosa) on neurogenic contraction of smooth muscle was studied in guinea-pig vas deferens. Mitragynine inhibited the contraction of the vas deferens produced by electrical transmural stimulation. On the other hand, mitragynine failed to affect the responses to norepinephrine and ATP. Mitragynine did not reduce KCl-induced contraction in the presence of tetrodotoxin, prazosin and alpha,beta-methylene ATP. Mitragynine inhibited nicotine- or tyramine-induced contraction. By using the patch-clamp technique, mitragynine was found to block T- and L-type Ca2+ channel currents in N1E-115 neuroblastoma cells. In the Ca2+ measurement by a fluorescent dye method, mitragynine reduced KCl-induced Ca2+ influx in neuroblastoma cells. The present results suggest that mitragynine inhibits the vas deferens contraction elicited by nerve stimulation, probably through its blockade of neuronal Ca2+ channels.

[Analysis of the contractions evoked by sympathetic nerve stimulation, and thermal effect on the guinea-pig vas deferens--study as a model for the thermotherapy of benign prostatic hyperplasia].

BACKGROUND: The present study was designed to investigate the mechanism of thermotherapy on benign prostatic hyperplasia (BPH), using the guinea-pig vas deferens as a model for BPH. The components of contractions elicited by electrical field stimulation and nicotine were analyzed, and the thermal effect on the vas deferens was examined. METHODS: The vas deferens was dissected, suspended vertically through two silver ring electrodes, and attached to an isometric transducer. The electrical stimulation of 10 constant current pulses (10 mA) with 0.3 msec in duration of 5, 10, and 40 Hz was achieved under air-gap condition. Drugs were added directly to a 5 ml Magnus tube containing Tyrode solution (36 degrees C) gassed with a 95% O2-5% CO2 mixture. The components of contractions evoked by electrical stimulation and nicotine were investigated by tetrodotoxin (TTX), and blocking agents of alpha 1-adrenoceptors and/or purinoceptors. Thermal effect on electrically evoked contractions was examined at incubation temperature of 25 degrees C (control), 43 degrees C, 45 degrees C, 46 degrees C and 47 degrees C for 1 hour. RESULTS: Nicotine (200 microM) elicited biphasic contractions, which were triggered by corelease of noradrenaline (NA) and ATP (N-ATP) from sympathetic nerve terminals by activation of prejunctional nicotine receptors. NA and N-ATP caused the corresponding contractions, alpha 1 and N-ATP components, respectively. Combined application of prazosin (1 microM) and suramin (50 microM) abolished these contractions. Activation of post-junctional alpha 1-adrenoceptors by NA caused release of ATP from muscle cells to produce the contraction (alpha 1-ATP component), which was sensitive to both suramin and prazosin. N-ATP and alpha 1 components attributed to fast and slow part of the contraction, respectively. Electrical field stimulation caused biphasic contractions which consisted of both neurogenic (TTX-sensitive) and non-neurogenic (TTX-insensitive) components. An increase in stimulation frequency (5 to 40 Hz) increased the neurogenic components, which contained alpha 1 and N-ATP components, as well as the case of nicotine. The non-neurogenic components consisted of alpha 1-ATP, muscle-derived ATP (m-ATP) and unknown substance 'X' components. Nifedipine (10 microM). L-type Ca2+ channel blocker, markedly reduced the contractions induced by bath applied phenylephrine (alpha 1-agonist, 100 microM) but only partially blocked the contractions produced by bath applied ATP (500 microM). The contractile force in amplitude and neurogenic components induced by electrical field stimulation did not change at 43 degrees C, but both declined significantly above 45 degrees C. The neurogenic components at 45 degrees C and 46 degrees C were suppressed to 22 +/- 6% and 14 +/- 3% (mean +/- SD) of control, respectively. All the contractile responses were abolished at 47 degrees C. CONCLUSION: The contractions of the guinea-pig vas deferens evoked by electrical field stimulation consisted of alpha 1, N-ATP, alpha 1-ATP, m-ATP and X components. Sympathetic nerve fibers in the muscles were completely inactivated by thermal exposure at 47 degrees C for 1 hour. The results suggest that the minimal temperature for thermotherapy of BPH should be 47 degrees C.

Changes in sensitivity of the isolated guinea-pig vas deferens induced by a lyophilized Phoradendron latifolium leaf infusion.

The effect of a lyophilized mistletoe infusion (LMI) was studied on isolated guinea-pig vas deferens. LMI caused a contraction which was partially blocked by phentolamine but not by atropine. LMI caused a shift to the left of the norepinephrine concentration-effect curve (CEC), an effect which appeared to be blocked by atropine and was absent in animals previously treated with reserpine and alpha-methyl-para-tyrosine. The increase of the norepinephrine maximal response induced by LMI was not blocked by atropine or pharmacological denervation. LMI caused a shift to the right of the acetylcholine CEC and had no effect on the acetylcholine maximal response. These results suggest that the effects seem to be due mainly to the presence of potassium ion in the LMI; however, the participation of muscarinic agonist(s) of reduced intrinsic activity or some tyramine-like substance could not be ruled out.

Study of the effects of nootropic agents on the adrenergic neurotransmission in smooth muscles of young and old animals.

The effects of compounds from the group of nootropic agents on the peripheral adrenergic neurotransmission in animals of different ages were studied in in vitro experiments. The experiments were performed on smooth muscle preparations isolated from young sexually mature (4-5-month-old) and old (22 month-old) rats treated twice daily for 7 days with 50 mg/kg of the nootropic agents piracetam, its structural analogues p-H and p-Cl, aniracetam and Ginseng extract. The animals were decapitated on the 7th day, the changes in the contractile responses of the smooth muscles to noradrenaline (NA) or electrical stimulation (ES) were studied in isolated preparations from vas deferens, aorta and anococcygeal muscle. The results show that the compounds studied can modulate the contractile effects of NA and ES, their action being most often potentiating and more pronounced in the smooth muscles manifesting weaker responses. In some cases the reactivity of the smooth muscle preparations to the stimuli applied was reduced after the nootropic agents. These effects can be partly explained with changes in the sensitivity of the pre- and postsynaptic alpha-adrenergic receptors. The possible variabilities in the influence on the two receptor populations in the different smooth muscles could explain the differences in the observed modulatory effects of the nootropic agents. The data obtained show that in prolonged therapeutic application of nootropic agents it is necessary to bear also in mind their possible influence on the peripheral neurotransmitter systems.

Radioenzymatic assay measurement of yohimbine's influence on adrenergic neurotransmission of rat vas deferens: unique consequence of using normetanephrine as an uptake2-blocking agent.

The effect of the alpha 2-receptor antagonist, yohimbine on norepinephrine overflow was studied in the transmural-stimulated isolated rat vas deferens. A radioenzymatic assay was used to measure the endogenous norepinephrine overflow. In initial studies conducted in the presence of uptake1 blocker desipramine (10(-6) M) and uptake 2 blocker, normetanephrine (10(-5) M) there was an apparent uncoupling of the influence of yohimbine on nerve-stimulated contractile response from norepinephrine overflow. These results were found to be due to the electrolytic O-demethylation of normetanephrine with the resultant generation of large quantities of norepinephrine obscuring the influence of yohimbine on nerve-stimulated norepinephrine overflow from the vas deferens. These findings serve as a warming to the use of normetanephrine as an uptake1 blocker when radioenzymatic assay is used to measure norepinephrine overflow from transmural-stimulated isolated tissue preparations. Yohimbine (10(-6) M), in the absence of uptake blockade, causes a 3-fold enhancement of nerve-stimulated norepinephrine overflow at 1 Hz and a 2-fold enhancement at 10 Hz. This report demonstrates utilization of a radioenzymatic assay to study endogenous norepinephrine overflow from rat vas deferens. Results for yohimbine are complementary to others using measurement of 3H-label overflow from [3H]norepinephrine prelabeled isolated tissue.

Progress in microfluorometric identification of monoamine fluorophores.

Techniques for the microfluorometric identification of monoamine fluorophores have been put forward along two different lines: (1) extension of the excitation range into the far UV and (2) computer-assisted on-line scanning recordings in combination with an automatic microfluorometric identification of fluorophores. By an extension of the excitation range to 250 nm it is possible to distinguish fluorophores of DOPA from those of dopamine, fluorophores of noradrenaline from those of adrenaline, and fluorophores of 5-OH-tryptophan from those of 5-OH-tryptamine. In a computer-assisted on-line scanning procedure using a high-aperture inverted illuminating microscope equipped with UV transmitting optics the specific formaldehyde-induced catecholamine fluorescence is characterized microfluorometrically by corrected ratios of excitation maxima (370:320 nm and 320:275 nm). Excitation data are plotted out with corresponding coordinates in the x- and in the y-axis. Furthermore the digitized values of fluorescence intensities obtained with the scanning procedure are processed according to principles of automatic image-analysis procedures.