Calcium channel blocking activity: Screening methods for plant derived compounds.

Calcium channel blockers are a heterogenous group of substances that inhibit influx of Ca(2+) into the cell. Their main therapeutical influence is on the function of heart and blood circulation. A vast number of pure natural compounds with calcium antagonistic activity, mainly coumarins, have been isolated from plants and identified. Screening natural products for calcium channel blockers has been done through classical in vitro aorta or ileum strip assays, but recently in vitro screens using animal cell lines also have been established. The number of in vivo assays on natural compounds is quite limited so far. Much more research is needed to clarify the basic pharmacology and to determine the possible clinical use of pure compounds discovered from plants.

Comparison of enzymatic hydrolysis of pilocarpine prodrugs in human plasma, rabbit cornea, and butyrylcholinesterase solutions.

Various bispilocarpic acid diesters (double prodrugs of pilocarpine) were synthesized, and their in vitro esterase catalyzed hydrolysis was evaluated in diluted human plasma, rabbit cornea homogenate, and specific butyrylcholinesterase solution. The structural changes greatly affected the rate of enzymatic hydrolysis of the prodrugs. Bispilocarpic acid with 2 cyclopropane substituents was the most stable derivative, whereas bispilocarpic acid with 2 cyclobutane substituents was the most labile derivative. The charged bispilocarpic acid diester hydrolyzed more slowly than the unchanged form. Comparison of the results obtained from different plasma and cornea homogenate batches is difficult because of the variety of the enzyme systems involved. This variety also makes comparing the results between different laboratories difficult.

Relaxation of the bovine retractor penis muscle by a small K+ excess and the role of K+ in its neurogenic relaxation.

The mechanism of the relaxation of the bovine retractor penis muscle induced by 6.7 mM K+ as well as the role of K+ in the neurogenic relaxation of this muscle induced by nicotine, acetylcholine or electrical field stimulation, was studied. The relaxation induced by 6.7 mM K+ was, contrary to that induced by nicotine or electrical field stimulation, abolished by 10(-7) M ouabain. 15 min exposure to 10(-5) M NG-nitro-L-arginine, 3.2 x 10(-6) M tetrodotoxin, 5.0 x 10(-4) M hexamethonium, 5.3 x 10(-4) M methylene blue or hypoxia, all known to inhibit the neurogenic relaxation, did not affect the relaxation induced by 6.7 mM K+, which was also unaffected by 10(-5) M apamin, 3 x 10(-3) M 4-aminopyridine, 2.6 x 10(-2) M tetraethylammonium and 7.3 x 10(-4) M Ba2+. Exposure to K(+)-free solution reversibly abolished the neurogenic relaxations. The relaxations caused by 5.0 x 10(-7) M cromakalim and 2.0 x 10(-6) M pinacidil were totally blocked by 10(-5) M glibenclamide. Glibenclamide and apamin did not affect the tone of the muscle or its neurogenic relaxations. 4-Aminopyridine 4.0 x 10(-5) to 3.0 x 10(-3) M and tetraethylammonium 10(-4) to 2.6 x 10(-2) M raised the tone and enhanced the relaxations elicited by electrical field stimulation. The results indicate that the relaxation induced by 6.7 mM K+ is partly mediated by activation of Na(+)-K(+) ATPase and that its mechanism is thoroughly different from that of the neurogenic relaxations.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of the bovine retractor penis muscle for the assessment of ganglion-blocking activity of neuromuscular blocking and other drugs.

Inhibition of relaxation of strips of the bovine retractor penis muscle induced by a standard dose of nicotine (30 microM) was used for quantitative assessment of the ganglion-blocking activity of eight neuromuscular blocking agents that are in clinical use. The order of potency of the drugs studied was (+)-tubocurarine >> alcuronium > vecuronium > metocurine >> pancuronium > atracurium >>> suxamethonium > gallamine. The results have been compared to those obtained with other methods. On the basis of the present results, it is concluded that inhibition of the nicotine-induced relaxation of the bovine retractor penis muscle can be used as an alternative, sensitive in vitro method for the assessment of the ganglion-blocking activity of a neuromuscular blocking agent relative to that of, for example, (+)-tubocurarine. Earlier results have showed that this method is useful also for the assessment of the ganglion-blocking activity of other drugs, because it has yielded comparable and reproducible results at the quantitation of this property of actual ganglion-blocking and various antimuscarinic agents. In addition, this method may be useful for rapid screening of ganglion-blocking activity.

Inhibition of nicotine-induced relaxation of the bovine retractor penis muscle by beta-adrenoceptor antagonists.

The relative potency in inhibiting nicotine-induced relaxation of the bovine retractor penis muscle (BRP) was estimated for the racemates of seven beta-adrenoceptor antagonists, both of the optical isomers of propranolol, and lidocaine. The order of potency of the drugs studied was (+)-propranolol greater than (-)-propranolol greater than propranolol greater than alprenolol greater than metoprolol greater than lidocaine greater than acebutolol greater than pindolol greater than sotalol greater than atenolol. It is concluded that the inhibition of the relaxation was not due to blockade of beta-adrenoceptors but to the nonspecific effects of the beta-adrenoceptor antagonists. It is also concluded that the neurotransmitter(s) which was (were) released from the non-adrenergic non-cholinergic inhibitory nerves in the BRP did not relax the muscle by activating the beta-adrenoceptors. It is suggested that the beta-adrenoceptor antagonists inhibited the release of the inhibitory neurotransmitter(s) by a mechanism which is significantly correlated to their lipophilicity.

Inhibition of nicotine-induced relaxation of the bovine retractor penis muscle by compounds known to have ganglion-blocking properties.

1. The relative potency in blocking the nicotine-induced relaxation of the bovine retractor penis muscle (BRP) was estimated for 12 drugs known to have ganglion-blocking properties. 2. The order of potency of the drugs studied was mecamylamine greater than chlorisondamine greater than pentolinium greater than propantheline greater than (+)-tubocurarine greater than hexamethonium greater than emepronium greater than tetraethylammonium greater than glycopyrrolate greater than decamethonium greater than butylscopolamine greater than scopolamine. 3. The results conform well to those obtained with other pharmacological methods used for the estimation of ganglion-blocking activity. 4. It is concluded that blockade of the nicotinic relaxation of the BRP can be used as an alternative method for quantitative assessment of ganglion-blocking activity. 5. Advantages of this technique are that it discriminates well between antinicotinic and antimuscarinic activity and that it satisfies most or all ethical and economical demands. 6. It is also possible that this method has certain value in predicting whether a drug has enough ganglion-blocking activity to be likely to cause impotence.

Histochemical demonstration of nerve cell bodies in the retractor penis muscle and the penile artery of the bull.

The presence of single nerve cell bodies and small ganglia in the retractor penis muscle and the penile artery of the bull was demonstrated by using antisera to neurofilament protein and neuron specific enolase. In the retractor penis muscle the findings were confirmed by staining for acetylcholinesterase. It was also shown that relaxation of strips of the retractor penis muscle induced by 70 microM acetylcholine was totally blocked by a 2.0 microM concentration of the ganglionic blocking drug chlorisondamine. The hypothesis is presented that the relaxation of the bovine retractor penis muscle and the bovine penile artery induced by nicotinic ganglionic stimulating drugs is at least in part mediated via receptors located on the nerve cell bodies described in this study.

Pharmacological analysis of nicotinic relaxation of bovine retractor penis muscle.

Relaxations of the isolated bovine retractor penis muscle elicited by nicotine and the three other nicotinic agonists acetylcholine, carbachol, and dimethylphenylpiperazinium were studied. Nicotine (10-45 microM) induced dose-dependent relaxations that closely resembled those evoked by transmural stimulation of inhibitory nerves. The relaxations induced by this dose level of nicotine were abruptly abolished by hypoxia and totally blocked by 1.2 microM mecamylamine, 45 microM lidocaine, and 13 microM methylene blue. They were reduced 50-90% by 0.16 microM tetrodotoxin, but they were unaffected by 17 microM scopolamine. Provided that sufficient concentrations of scopolamine and eserine were present, the relaxations caused by acetylcholine (30-140 microM) were exactly like those evoked by nicotine, and they were identically affected by hypoxia and the blocking drugs. Also carbachol and dimethylphenylpiperazinium induced relaxations qualitatively identical to those effected by the above-mentioned doses of nicotine. Relaxations induced by larger doses of nicotine were less susceptible to hypoxia and the blocking drugs. The results suggest that nicotine concentrations ranging from about 10-45 microM relax the bovine retractor penis muscle by a rather selective activation of inhibitory nerves, whereas higher concentrations may additionally activate some other less specific inhibitory mechanism. They further strongly suggest the presence of nerve cell bodies in this muscle. It is suggested that one physiological role of acetylcholine in the development of penile erection is nicotinic activation of inhibitory nerves. Moreover, nicotinic activation of these nerves of the bovine retractor penis muscle can be used as a model for further characterization of mammalian erectile nerves.