blocking agents, may display a low degree of specificity." 2 Other alpha adrenergic

Transcription

1 EFFECT OF PHENOXYBENZAMINE ON NEURAL AND HUMORAL CONTROL OF SWEAT GLANDS*,t BY DAVID P. C. LLOYD THE ROCKEFELLER UNIVERSITY Communicated January 17, 1968 It is evident that the role of phenoxybenzamine as an alpha adrenergic blocking agent is suspect in the sense that it, in concert with some other alpha adrenergic blocking agents, may display a low degree of specificity." 2 Other alpha adrenergic blocking agents the specificity of which has been questioned include bretylium and guanethidine. They have been described as exerting, in doses of from 10 to 30 times that for adrenergic nerve blockade, a curarelike (which is to say, an antinicotinic) action in cholinergic systems.3' 4 These latter two agents, considered by some as seemingly less controversial than phenoxybenzamine, will be discussed in a subsequent paper.' In the meanwhile, and for present purposes, it is well to bear in mind that controversy exists concerning phenoxybenzamine. The reasons for using alpha adrenergic blocking agents in connection with study- of transmission to, and chemical action on, sweat glands are simple. Cholinergic innervation of the sweat glands is established beyond peradventure.6 And yet the glands respond by secretion to injected noradrenaline, the action not being blocked by atropine, the anticholinergic (muscarinic) blocking agent that is so effective in blocking action by sudomotor impulses, injected acetylcholine, and injected pilocarpine.7 8 An account of events leading to the foregoing statement is given in reference 9. Granting, as one must, that noradrenaline acts, the problem is to attempt evaluation of its role, if any, in neuroglandular transmission. It is to this end that alpha adrenergic blocking agents have been employed. In commenting on their study of the cholinergic blocking action of antiadrenergic agents, Boyd et al."0 stress their view that "the use of autonomic drugs as analytical tools is not reliable unless combined with both histochemical examination of the preparation and assay of the substances released upon stimulation of the nerves." This is more easily said than done. In fact, the last-mentioned condition for reliability, at least insofar as the sweat glands are concerned, seemingly is a priori impossible, for inextricably intermeshed with the sweat glands are other structures, adrenergically innervated, that could with equal justification be designated the origins of released adrenergic transmitter, supposing this were to be recovered for assay. Such a problem did not face Dale and Feldberg in establishing acetylcholine as the/a transmitter to sweat glands.6 If, then, one accepts the strictures of Boyd et al.,'0 it follows that the observations in this and subsequent papers on adrenergic blocking agents cannot be construed as constituting irrefutable evidence for an adrenergic step in the sudomotor innervation of sweat glands. Nevertheless, the question of such a step in sweat gland innervation is raised and must be considered a possible, even possibly a probable, aspect of the largely confused general picture of autonomic transmission and 816

2 VOL. 59, 1968 PHYSIOLOGY: D. P. C. LLOYD 817 transmitters. Presumably the strictures would be less rigorous, but the interpretation still equivocal, had not the role of cholinergy been demonstrated. The preparations were cats anesthetized by the use of intraperitoneal chloralose (40 mg/kg) and urethane (0.8 mg/kg). Zinc-zinc sulphate electrodes were placed one upon the surface of the central foot pad, the other subcutaneously at a short distance from the first. These led to a d-c amplifier and recorder. For sudomotor stimulation the plantar nerves were isolated, centrally severed, fitted with stimulating electrodes, and submerged in a mineral oil bath. Phenoxybenzamine was slowly infused intravenously with the aid of a Harvard infusion pump. Total dosage varied between 10 and 22 mg/kg delivered over a period extending to 45 minutes or more. Admittedly the higher doses might have been too high to allow one to discount the possibility of cholinergic blockade. However, the effect of phenoxybenzamine was established, although not completed, by the time that from 3.5 to 7.5 mg/kg had been infused. In addition, as the ensuing results show, the phenoxybenzamine blockade, at least with respect to considerations of adrenergy, is not located at the same site as is the established cholinergic blockade by atropine. There is, then, circumstantial evidence of an adrenergic step in transmission to sweat glands which is, naturally, subject to the uncertainty that always befogs circumstantial evidence. Action on Transmission.-Figure 1 illustrates the experimental procedure and the influence of phenoxybenzamine. The upward deflections are "galvanic skin responses" elicited by single stimuli to the plantar nerves iterated at intervals of 25 seconds, stimulation having begun up to an hour before recording to stabilize the level of response. Infusion began at the beginning of the top record. There is a considerable latent period for the onset of phenoxybenzamine effect occasioned in part presumably by the slow rate of infusion, although delayed onset of action also must be considered, due presumably to the necessity for in vivo formation of active intermediates." The latter cause could be of great significance in the present type of experiment. Figure 1 is presented principally to illustrate the nature of the experiment and of the raw data obtained. Figure 2 illustrates to better advantage the course of phenoxybenzamine blockade of sudomotor transmission. In it are plotted, as ordinates, amplitudes of the galvanic skin responses against, on the abscissae, time in minutes from the onset of phenoxybenzamine infusion at the rate of ca ml/minute (total dose 10.4 mg/kg). Not only is the onset of effect delayed due to the aforementioned causes, but the course of developing blockade is smoothly progressive and complete only after approximately 105 minutes. In some experiments blockade was incomplete. Duration of phenoxybenzamine blockade is prolonged in this situation as in others that have been studied. Effect upon Action of Acetylcholine.-Intravenous injection of acetylcholine causes sweat formation that may be measured in any one of a variety of ways. For the present purpose use has been made of the fact that acetylcholine gives rise to a secretory potential of positive sign at the footpad electrode relative to the distant electrode.12 Since potential changes across the footpad were employed to gauge the action of phenoxybenzamine on transmission (Figs. 1 and

3 81818~~~PHYSIOLOGY: D. P. C. LLOYD PO.N PROC. N. A..S. i I A I 11 jj i --j - a I. I i i - i i L.L " L. 111i1I-Ui iiliilu[lull11111lii11 I i LI I i I I. I I1 I I 1 i- I i I i I I 1 Li. L I a a a I I I FIG. 1.-"Galvanic skin responses" or action potentials of the sweat glands of the cat's foot pad (negativity of the foot-pad surface upwards) elicited by a series of single shocks repeated at 25-sec intervals. Beginning of top row coincided with onset of slow intravenous infusion of phenoxybenzamine. Block of response begins after considerable latent period and, as the subsequent rows illustrate, is smoothly progressive. Time bar below third record: 5 min. 2), this method was that of choice for observing responses to injected acetyicholine (and vide infra noradrenaline). Figure 3 exemplifies the result of intravenous injection of acetyicholine (0.08 mg/kg) prior to phenoxybenzamine infusion (upper record) and subsequent to complete transmission blockade by phenoxybenzamine (lower record). There is in the former record a typical positive secretory potential indicative of sweat formation. In the latter there is complete absence of any response to an otherwise equipotent dose of acetylcholine. Therefore, phenoxybenzamine blocks the action upon sweat glands of acetylcholine. Effect on Action of Noradrenacline.-The experiment illustrated by Figure 4 is similar in all respects to that of Figure 3 save that noradrenaline (40 uig/kg) was injected intravenously in place of acetylcholine. One sees in the upper record the secretory potential consequent upon noradrenaline injection. Following phenoxybenzamine transmission block there is no response to noradrenaline administered in the same dosage. Thus phenoxybenzamine blocks the action upon sweat glands of noradrenaline.

4 VOL. 59, 1968 PHYSIOLOGY: D. P. C. LLOYD k 0 Q~ v 20 E I0 U minutes FIG. 2.-Plotted time course of phenoxybenzamine block. Ordinate: amplitude of galvanic skin responses. Abscissae: time from onset of intravenous phenoxybenzamine infusion. 01%j 4.--~~~~~~~~j,_ I FIG. 3.-Action potential and secretory potential (positivity at foot-pad surface downwards) consequent upon intravenous injection (0.08 mg/kg) acetylcholine chloride (upper record). Between taking of the first and second recordings, transmission had been blocked by use of phenoxybenzamine. Lower recording shows absence of response to acetylchloline after phenoxybenzamine transmission block. Time bar: 5 min. FIG. 4.-Action and secretory potential response of sweat glands to noradrenaline (40 pg/kg intravenously) prior to phenoxybenzamine (upper record) and absence of such response subsequent to phenoxybenzamine transmission block (lower record). Time bar: 5 mi.

5 820 PHYSIOLOGY: D. P. C. LLOYD PROC. N. A. S. Comment.--In view of the prevailing climate of opinion concerning the action(s) of phenoxybenzamine, it would be folly to state baldly that the foregoing results demonstrate an adrenergic step in sudomotor transmission. Still, noradrenaline does activate sweat glands, and phenoxybenzamine, whatever else it may be, is an adrenergic blocking agent, and it does prevent the action on sweat glands of noradrenaline (and of nerve impulses and acetylcholine). The concept of an adrenergic step thus is worth consideration and continued study despite the ambiguities and inability to comply fully with all the experimental desiderata for establishing, as distinct from postulating, a given transmitter substance. On a number of occasions a dual transmitter mechanism has been postulated, with acetylcholine the agent in one stage, or indeed in both stages.'3-'5 In these instances quoted it has been the curare-sensitive nicotinic action of acetylcholine that is, or at least may be, involved. The plantar nerve-sweat gland system, assuming the postulation of duality of transmitters, is unique among hypothetical dual transmitter systems in that action of the cholinergic step is muscarinocholinergic rather than nicotinocholinergic. In consideration of the foregoing it is noteworthy that a clean-cut block of sympathetic action, with the parasympathetic action unaffected, has been reported in situations involving the muscarinic action of the transmitter substance."6 The case for adrenergy in sudomotor innervation may not be as weak as to some it might appear at first glance. Two observed facts remain to be considered and commented upon. The first of these is that the antimuscarinocholinergic agent, atropine, rapidly blocks sudomotor transmission and the action upon sweat glands of injected acetylcholine, but does not interfere with the effect of injected noradrenaline.7 8 The second is that the antiadrenergic agent phenoxybenzamine blocks all three modes of activation: by nerve impulses, by acetylcholine, and by noradrenaline. These facts constitute a demonstration, in classical terms, that the two blocking agents act upon different receptor sites. This is so whether-or -not one adopts a dual-transmitter hypothesis. However, since one might reasonably suppose- an antiadrenergic agent in high dosage, by virtue of low specificity, to block cholinoceptive receptor sites, so one should at the same time equally reasonably expect the fact that different sites are involved to mean that the adrenergic blocking agent is not effective by reason of an anticholinergic (in this case antimuscarinocholinergic) action. Adopting pro tem a aual hypothesis, it follows from the observations on blockade that the action of acetylcholine and of noradrenaline would have to be located serially rather than in parallel, and that the cholinergic step would have to antecede the adrenergic step. I thank the Research and D)evelopment division, Smith Mline & French Laboratories (Philadelphia), for a supply of phenoxybenzamine (dibenzyline hydrochloride). * The investigation was supported by a U.S. Public Health Service research grant NB from the National Institute of Neurological Diseases and Blindness. t Presented in part at the July meeting of the Physiological Society, 1963; Lloyd, D. P. C., J. Physiol., 169, P (1963).

6 VOL. 59, 1968 PHYSIOLOGY: D. P. C. LLOYD Ariens, E. J., "The structure-activity relationships of beta adrenergic drugs and beta adrenergic blocking drugs," Ann. N.Y. Acad. Sci., 139, (1967). 2 Benfey, B. G., and S. A.. Grillo, "Antagonism of acetylcholine by adrenaline antagonists," Brit. J. Pharmacol., 20, (1963). 3 Boura, A. L. A., and A. F. Green, "The actions of bretylium: adrenergic neurone blocking and other effects," Brit. J. Pharmacol., 14, (1959). 4Dixit, B. N., 0. D. Gulati, and S. D. Gokhale, "Action of bretylium and guanethidine at the neuromuscular junction," Brit. J. Pharmacol., 17, (1961). 5 Lloyd, D. P. C., "Effect of bretylium and guanethidine on tranmission to sweat glands in the cat," in preparation. Preliminary note, Lloyd, D. P. C., J. Physiol., 175, P (1964). 6 Dale, H. H., and W. Feldberg, "The chemical transmission of secretory impulses to the sweat glands of the cat," J. Physiol., 82, (1934). 7 Nakamura, Y., and K. Hatanaka, "Effect of denervation of the cat's sweat glands on their responsiveness to adrenaline, nicotine and mecholyl," Tohoku J. Exptl. Med., 68, (1958). 8 Lloyd, D. P. C., "Response of cholinergically innervated sweat glands to adrenaline and noradrenaline," Nature, 184, (1959). 9 Lloyd, D. P. C., "Cholinergy and adrenergy in the neural control of sweat glands," in Studies in Physiology (Berlin, Heidelberg, and New York: Springer-Verlag, 1965), pp Boyd, H., G. Burnstock, G. Campbell, A. Jowett, J. O'Shea, and M. Wood, "The cholinergic blocking action of adrenergic blocking agents in the pharmacological analysis of autonomic innervation," Brit. J. Pharmacol., 20, (1963). 11 Nickerson, M., "The pharmacology of adrenergic blockade," Pharmacol. Rev., 1, (1949). 12 Lloyd, D. P. C., "Action and secretory potential of sweat glands," these PROCEEDINGS, 47, (1961); Lloyd, D. P. C., "Secretion and reabsorption in eccrine sweat glands," in Advances in Biology of Skin (Oxford: Pergamon Press, 1962), vol. 3, pp Burn, J. H., and M. J. Rand, "Sympathetic postganglionic mechanism," Nature, 184, (1959). 14 Burn, J. H., "A new view of adrenergic nerve fibres, explaining the action of reserpine, bretylium and quanethidine," Brit. Med. J., 1, (1961). 15 Koelle, G. B., "A new general concept of the neuro-humoral functions of acetylcholine and acetylcholinesterease," J. Pharm. Pharmacol., 14, (1962). 16 Boyd, G., J. S. Gillespie, and B. R. Mackenna, "Origin of the cholinergic response of the rabbit intestine to stimulation of its sympathetic nerves after exposure to sympathetic blocking agents," Brit. J. Pharmacol., 19, (1962).