Popat N. Patil

Differences in the applicability of the Easson-Stedman hypothesis to the α1- and α2-adrenergic effects of phenethylamines and imidazolines

The enantiomers of 2-(3,4, alpha-trihydroxybenzyl)imidazoline and the corresponding desoxy derivative, 2-(3,4-dihydroxybenzyl)imidazoline, were evaluated at alpha 1- and alpha 2-adrenergic receptors to test the applicability of the Easson-Stedman hypothesis to the imidazoline class of alpha-adrenergic agonists. A series of closely related phenethylamines was included for comparison. The Easson-Stedman hypothesis states that optically active adrenergic agonists possessing an asymmetric hydroxyl-substituted benzylic carbon atom will have the following relative potencies: R(-) greater than S(+) = desoxy. While the phenethylamines were found to adhere to the Easson-Stedman hypothesis at both alpha 1- and alpha 2-adrenergic receptors, the optically active imidazolines did not. These findings further support our previous observations that the phenethylamines and imidazolines may interact differently with alpha-adrenergic receptors.

The isomers of cocaine and tropacocaine: Effect on 3H-catecholamine uptake by rat brain synaptosomes

Abstract Compared to (+)- pseudo cocaine, (−)-cocaine was 20 times more potent in inhibiting uptake of 3 H-norepinephrine ( 3 HNE) by cortical synaptosomes and 66 times more potent with respect to 3 H-dopamine ( 3 HDA) uptake by striatal synaptosomes. Although the tropacocaine isomers were equipotent as inhibitors of 3 HNE uptake in the cortex, tropacocaine was 3.9 times more potent as an inhibitor of 3 HDa uptake in the striatum than pseudo tropococaine. A major known cocaine metabolite, benzoylecgonine failed to inhibit the accumulation of 3 HNE and 3 HDA by synaptosomes from the cortex and striatum, respectively. The implications of these findings in relation to the motor stimulation seen with (−)-cocaine, (+)- pseudo cocaine and benzoylecgonine in rats are discussed.

Comparison of post-junctional alpha-adrenoceptors in iris dilator muscle of humans, and albino and pigmented rabbits

The relative potency of α-adrenoceptor agonists and the dissociation constants of competitive antagonists were studied to characterize the post junctional α-adrenoceptor of the human iris dilator muscle. The data obtained from human iris dilator tissue was compared to that from rabbit. The iris dilator muscle was mounted in an organ bath and tension changes were recorded. (−)-Norepinephrine, (−)-phenylephrine (PE), oxymetazoline and p-aminoclonidine caused contractile responses in albino rabbit, pigmented rabbit and human iris dilator muscle in a concentration-dependent manner. The imidazoline molecules were partial agonists. In rabbit iris dilator, desensitization occurred to repeated oxymetazoline application at an interval of 1 h but recovery to the agonist activity was complete in about 3 h. Exposure to cocaine (10 μmol/l), hydrocortisone (100 μmol/l) and U-0521, a catechol-O-methyltransferase inhibitor (100 pmol/l), significantly potentiated the response to norepinephrine by 92-, 32- and 7 fold in iris dilator tissue of albino rabbit, pigmented rabbit and human, respectively. After block of “uptake1” and “uptake2”, the EC50 values of norepinephrine in the albino rabbit, pigmented rabbit and human iris dilator did not differ and ranged from 99 to 195 nmol/l. Small but significant potentiation by uptake blockers was also observed in the responses to PE in the albino rabbit or pigmented rabbit iris dilator. The average maximum tension induced by 100 μmol/l PE was 96 ± 11 mg (n = 10), 197 ± 11 mg (n = 11), 45 ± 5 mg (n = 27) in albino rabbit, pigmented rabbit and human iris dilator, respectively.In human iris dilator, the responses to PE were competitively antagonized by prazosin, 5-methylurapidil and phentolamine with apparent pKB values of 7.3, 6.6 and 7.5, respectively. The pKB values of the prazosin-PE interaction in iris dilator of albino and pigmented rabbit were 8.6 and 6.4, respectively.These results suggest that the post-junctional α-adrenoceptors in iris dilator may be similar to that in pigmented rabbit iris. The α-adrenoceptor of the human or pigmented rabbit iris dilator may be characterized as α1L-adrenoceptor subtype. The α-adrenoceptor of albino rabbit iris dilator appears to be a high affinity subtype.Furthermore, albino rabbit may not be the best strain for the drug research which is relevant to human ocular therapeutics.

Further observations on the interaction of prostaglandin F2α with cholinergic mechanisms in canine salivary glands

Abstract The mechanism by which prostaglandin F2α (PGF2α) produces salivation has been further characterized. Intraarterial injection of PGF2α (0.05–0.5 μg) or acetylcholine (10–100 μg) produced dose-related salivation. Treatment with atropine (30 μg, i.a.) shifted the dose-response curve for acetylcholine to the right in parallel fashion, but shifted the response curve for PGF2α to the right in non-parallel fashion. Tetrodotoxin (12 μg, i.a.) antagonized salivation induced by chorda tympani nerve stimulation and by PGF2α, while the response to exogenous acetylcholine remained unaltered. PGF2β produced salivation but was considerably less potent than PGF2α. 15-epi PGF2α did not produce salivation. Chronic decentralization of the chorda shifted the dose-response curve for PGF2α to the left. Saliva produced by stimulation of the chorda or by infusion of PGF2α (4 μg/kg/min) was slightly alkaline, rich in sodium, and contained low concentrations of potassium and total protein. These results are consistent with the previous suggestion that PGF2α produces salivation by liberation of endogenous acetylcholine.

Analysis of the effects of apomorphine and bulbocapnine in relation to the proposed dopamine receptor

On rat isolated vas deferens, apomorphine was found to be l/5th as active as dopamine. Reserpine or cocaine pretreatment failed to reduce the response to apomorphine. Low concentrations of apomorphine which do not cause contraction of the tissue, antagonize the effects of dopamine. The effects of dopamine were antagonized equally by phentolamine or apomorphine. On rabbit aortic strips 4 × 10−4M apomorphine repeated at 45 min intervals induces pronounced tachyphylaxis. During the tachyphylactic period dopamine was not inhibited to a significantly greater extent than was phenylephrine. Bulbocapnine tested against dopamine and phenylephrine yielded identical pA2 values of 6. Only at 10−5M was bulbocapnine observed to produce a preferentially greater blockade of dopamine. Dopamine or isoprenaline‐induced relaxations of aortic strips were not blocked by bulbocapnine. Dopamine and 3 times 10−6M bulbocapnine increased the chronotropic effects in atria. Apomorphine and higher doses of bulbocapnine produced rate decelerating effects. On atria low doses of apomorphine were equally effective in reducing the effects of dopamine or histamine. These results are discussed in light of the proposal that the effects of apomorphine and bulbocapnine involve dopamine receptor interactions. In all three tissues there was no clear cut evidence of specific dopamine receptors.

Some factors which affect the ocular drug responses

Abstract Although the cornea represents a major barrier for the transport of drugs into the aqueous humor, the metabolizing enzymes, such as esterases, monoamine oxidase and catechol-O-methyl transferase can limit the duration of drug action in the eye. In addition, the drug binding by ocular melanins can limit the early initaition of the ocular response. Where a drug exhibits large differences in the duration of the ocular responses in lightly pigmented and heavily pigmented irides, the drug binding by melanins appears to be the most important factor that contributes to the observed changes. This capacity of melanins to bind drugs far exceeds that of any other known biopolymer isolated from living tissues. The drug bound by melanins can be subsequently released to prolong the duration of response. Thus, 8–10 days duration of the mydriatic effect of atropine, for example, is related to the binding to the pigment cell melanins. The bound atropine is slowly released onto muscarinic receptors.

Influence of group selective reagents in tissues containing α- and β-adrenoceptors☆

N-ethylmaleimide which is known to react irreversibly with free -SH groups of protein when incubated with rabbit aorta (alpha-adrenoreceptor) or atria (beta-adrenoreceptor) markedly depressed the dose-response curves of norepinephrine isomers in the aorta but not in atria. Dithiothreitol, a reagent which reduces S-S linkages caused a parallel shift of the dose-response curves of both isomers to an equal degree in aorta. However, in atria the reagent drastically and selectively reduced the maximal effects of (+)-norepinephrine and dopamine. This selective alteration of the (+)-isomer by the reagent probably reflects the conformational changes at the beta-adrenoreceptor. The concentrations of N-ethylmaleimide and dithiothreitol required to alter the activity of norepinephrine isomers was higher for atria than for aorta. It may be that alpha-adrenoreceptors are more susceptible than the beta-adrenoreceptors on the -SH group selective reagents. In the highest concentration tested, two other reagents 2,4-dimethoxybenzylamine and N-acetylimidazole, did not influence the activity of the steroisomers on aorta. In the presence of dithiothreitol, the pharmacologic effects of a nonadrenoreceptor stimulant drug, histamine was potentiated on aorta (H1) and not on atria (H2). Results are discussed in the light of similarity and dissimilarity of adrenoreceptors as reflected by the group selective reagents and the stereoisomers.

Binding of 14C-imipramine by pigmented and non-pigmented tissues

When pigmented and non-pigmented rabbit irides were incubated with various concentrations of 14C-imipramine at equilibrium (120 min), the accumulation of the drug by the pigmented iris was 1.5 times as great as that by the non-pigmented iris. The accumulated drug is lost from both types of irides in a complex fashion. However, even after 120 min of washing, the differences in accumulation remain nearly constant. When accumulation of the drug in the non-pigmented iris was analyzed by discontinuous sucrose density gradient, it was observed that the drug was bound mainly by the low density sucrose fractions where the synaptosomes separate. On the contrary, in the pigmented iris approximately 70% of the drug was found in the melanin-containing fraction. The homogenate from the substantia nigra accumulated 1.5 times more than that from the human brain cortex. The affinity of the drug for bovine iris melanin granules and the synthetic L-dopa melanin was 9.9 X 10(5) M-1 and 3.8 X 10(3) M-1, respectively. On the rabbit iris sphincter muscles, imipramine was evaluated for antimuscarinic effects. The apparent dissociation constants, KB values, for the antagonist in the non-pigmented and pigmented iris were 1.7 X 10(-7) M and 3.8 X 10(-6) M, respectively. The low antimuscarinic activity in the pigmented iris is attributed to the loss of the drug to the pigment. On this basis, relevancy of the drug binding by pigmented tissues to the effects of this tricyclic drug is discussed.

Muscarinic receptors of rabbit irides

SummaryAfter occlusion of a fraction of the receptors with the irreversible blocker dibenamine, dissociation constants (KA) of several muscarinic stimulants like carbachol, muscarine, bethanechol and oxotremorine are determined in four types of irides (presence or absence of pigment and/or plasma atropinesterase).(1)The negative log molar ED50 and KA values of carbachol in the iris sphincter obtained from the pigmented atropinesterase negative rabbits were 5.92 and 4.50, respectively. The corresponding values for irides obtained from albino atropinesterase positive and negative animals, were ED50 5.66, KA 4.48 and ED50 5.69, KA 4.44, respectively.(2)The negative log molar KA of dissociation constants for carbachol, oxotremorine, bethanechol and muscarine, obtained from albino atropinesterase positive animals, were 4.48, 5.03, 3.88 and 5.29, respectively. For each agonist similar values were obtained in the sphincter obtained from albino esterase negative animals.(3)The ED50 of acetylcholine in the sphincters obtained from albino and pigmented irides were not significantly different.(4)Pilocarpine caused a small contraction of the iris sphincter. The drug, however, behaves as a competitive antagonist at the muscarinic receptor of the iris. The pA2 value of pilocarpine vs. carbachol obtained from the Schild plot in the albino iris of atropinesterase positive rabbit was 5.4. Based on the dose-ratios obtained at 100 μM of pilocarpine in the pigmented and albino iris, the drug appears to be 2.6 times more effective antagonist in the latter type. Since the ED50 (or the dissociation constant) of carbachol was similar in the two irides, the apparently low receptor blockade by pilocarpine in the pigmented irides must be inversely related to the binding of pilocarpine by the pigment.(5)Although both in the pigmented and albino irides, a muscarinic receptor blocker, 3H-quinuclidinyl benzylate binds with the apparently similar dissociation constants, KD, and capacity, Bmax, the pigmented iris possesses an additional high capacity non-receptor binding which in all probability is related to the binding of the drug to the pigment.(6)The inhibition of the muscarinic receptor-related binding of 3H-quinuclidinyl benzylate by low concentrations of atropine was similar in albino and pigmented iris. At the higher concentrations of atropine, as compared to that observed in the albino iris, the inhibition was significantly less in the pigmented iris. Thus, the pharmacological activity of many muscarinic stimulants like carbachol, oxotremorine, bethanechol and muscarine in irides is not influenced by the presence or absence of plasma atropinesterase or the melanin pigment. The dissociation constants obtained from the pharmacological (KA) and biochemical (KD) studies indicates that unequal effects of certain drugs in irides may be largely related to the drug binding to the pigment. Hence, the contribution of the atypical muscarinic receptor, if any, to explain the unequal pharmacological effects in different types of irides must be small.

Adrenoceptor-mediated effects of optically active catecholimidazolines in pithed rat.

SummaryAdrenoceptor-mediated effects of the enantiomers of the optically active imidazoline, 2-(3,4,α-trihydroxybenzyl)imidazoline, and the corresponding desoxy derivative, 2-(3,4-dihydroxybenzyl)imidazoline, have been investigated in the pithed rat. The enantiomers and desoxy derivative were potent pressor agents with a direct action mediated predominantly via postsynaptic vascular α2-adrenoceptor. These compounds were significantly less potent at presynaptic α2-adrenoceptor in rat heart. The rank order of potency for the two enantiomers and desoxy derivative at postsynaptic vascular α1- and presynaptic cardiac α2-adrenoceptor in pithed rat were: desoxy≥R(−) >(+), consistent with our previous findings in vitro. This order of potency is not in agreement with the rank order of R(−)>S(+)=desoxy which is predicted by the Easson-Stedman Hypothesis. The β-adrenoceptor-mediated chronotropic and β-adrenoceptor-mediated vasodepressor effects of these imidazolines were also investigated in pithed rat and found to be weaker than either the α1- or α2-adrenoceptor-mediated effects. However, the rank order of potency of the enantiomers and corresponding desoxy derivative for β1- and β2-adrenoceptor-mediated effects was found to be similar to that order predicted by the Easson-Stedman Hypothesis. Studies with these optically active imidazoline enantiomers and corresponding desoxy derivativative indicate that quantitative as well as qualitative differences exist in the stereochemical requirements of α- and β-adrenoceptor. The results also support our previous observations which suggest that phenethylamines and imidazolines may interact differently with α-adrenoceptor since the former adhere strictly to the Easson-Stedman Hypothesis whereas the latter do not.