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Effect of Guanethidine and Other Sympatholytic Drugs*
EFFECT OF GUANETHIDINE AND OTHER SYMPATHOLYTIC DRUGS* U P O N T H E PUPILLARY RESPONSE TO ELECTRICAL S T I M U L A T I O N OF T H E S Y M P A T H E T I C SYSTEM I N RABBITS L U C I A N O B O N O M I , M.D.,
AND P I E T R O D I
COMITÉ,
M.D.
Bari, Italy Several sympatholytic drugs have been shown to lower the intraocular pressure, whether administered locally or systemically. Recently ophthlamologists have been in troduced to the adrenergic neurone blocking agents, which act by blocking release of the chemical transmitter from the sympathetic nerve endings without impairing the respon siveness of the sympathetic receptors.
mydriatic response to electrical stimulation of the ipsilateral sympathetic chain in rabbit was chosen. T h e following solutions were used* :
One of these drugs, Guanethidine, is known to produce significant lowering of the intraocular pressure, both in rabbit and human eyes (normal or glaucomatous), by markedly decreasing aqueous production. Similar effects have been obtained 1 " 3 with the local use of Reserpine, Bretylium and B.W. 467 C 60. Since the role of the autonomie nervous system in the regulation of intraocular dy namics is poorly understood, interest in these drugs lies not only in the hope that they may be of practical use in the therapy of glaucoma but also in their possible value in the experimental study of this subject. In order to use these substances appropri ately and to interpret their action correctly, it seemed essential, as a first step, to investi gate the following factors : 1. T h e effectiveness of these drugs in blocking the ocular sympathetic nerves when applied locally into the conjunctival sac. 2. The degree and duration of the sympa thetic blockade. 3. T h e dosage of each drug needed to ob tain the effect.
O n e drop of the solution under investiga tion was instilled into the conjunctival sac of the animals' right eyes three times at fiveminute intervals. T h e left eyes were untreat ed controls. At various times after treat ment the pupillary behavior was checked. T h e sympathetic nerve was isolated under general anesthesia (Kemital 10%, 0.5-0.8 ml/kg, intravenously) at both sides of the neck; fine stainless steel electrodes were ap plied in front of the nerve's entrance into the superior cervical ganglion; electrical square wave stimulations were applied from a Palmer electric stimulator (intensity, 2 to 6 v ; frequency, 5 to 20 c / s ; duration of each impulse, 1.5 m s ) . T h e pupillary size was observed under constant lighting and measured with finetipped calipers.
Guanethidine 1%, 2.5%, 5%, 10% Reserpine 0.25%, 1% Bretylium 1%, 5%, 10% B.W. 467 C 60 1%, 2.5% Xylocholine 2.5%, 10%
M A T E R I A L S AND M E T H O D S
A s an index of the function of the sympa thetically innervated ocular structures, the * From the Department of Ophthalmology, Uni versity of Bari (Director: Prof. F. D'Ermo). Pres ent address : Clinica Oculistica, University of Padova. 544
RESULTS
T h e effects of electrical stimulation of the sympathetic system upon the pupillary re sponse are shown in Table 1. Stimulation of the ipsilateral sympathetic chain constantly produced in the untreated eyes a moderate mydriasis when low intensity and frequency * Guanethidine was supplied by the Ciba Industria Chimica of Milan; Reserpine by the Boehringer Prodotti Chimici Farmaceutici of Flor ence; Bretylium and B.W. 467 C 60 by the Wellcome Research Laboratories of Beckenham and Xylocholine by the Smith, Kline and French Lab oratories of Philadelphia.
EFFECT OF SYMPATHOLYTIC DRUGS
545
TABLE 1 PUPILLARY RESPONSES TO ELECTRICAL STIMULATION OF THE IPSILATERAL CERVICAL SYMPATHETIC CHAIN AFTER INSTILLATION OF VARIOUS SYMPATHOLYTIC DRUGS
Hours 6
12
24
++ ++ +-
+++-
+++++
++-
++
++-
1
Guanethidine 1% Guanethidine 2 . 5 % Guanethidine 5% Guanethidine 10% Reserpine 0.25% Reserpine 1% Bretylium 1% Bretylium 5% Bretylium 10% B.W.467C60 1% B.W.467C60 2.5% Xylocholine2.5% Xylocholine 10%
4
+-
stimuli were applied (2 v ; 5 c/s and a maximum mydriasis when higher intensity and frequency were used (6 v ; IS c/s). This response is indicated in Table 1 by + + . The notation H— indicates that the response was less than in the control eyes and that a more powerful stimulation was needed to produce clear results (at least 6 v ; 15 c/s). The notation — indicates a com plete lack of pupillary response even after maximum stimulation. As shown in Table 1, Guanethidine, Bre tylium and B.W. 467 C 60 produced com plete and long-lasting sympathetic blockade, while Xylocholine and Reserpine merely depressed the pupillary response. Higher concentrations of Reserpine were not used, since the animals treated with the 1% solu tion already showed marked systemic effects (diarrhea, sedation and miosis of the fellow eye). In previous experiments,4 however, we demonstrated that the concentrations used were sufficient, when instilled into the con junctival sac of rabbits, to produce an al most complete depletion of the uveal stores of norepinephrine. In addition to investigating the effect of electrical stimulation of the sympathetic upon the pupillary response, the direct effects of the drugs upon the pupillary size prior to any stimulation were also recorded. Guanethidine, Bretylium and B.W. 467 C
48
++-
++ ++ +++
+-
+-
60 produced an early and transient mydri asis followed by a slight miosis which lasted as long as the response to electrical stimula tion was blocked. Reserpine produced no direct pupillary change during the first two hours, after this a slight miosis was seen. Xylocholine, however, caused a clear and early miosis which lasted many hours. These results are summarized in Table 2 which, for clarity of interpretation, refers only to the most effective dosage and to ob servations made at the first and at the 12th hour after treatment. DISCUSSION
Our results demonstrate that all the drugs tested have a definite sympatholytic effect upon the ocular structures of the rabbit when instilled into the conjunctival sac. In order to obtain complete blockage of the sympathetic impulses, however, B.W. 467 C TABLE 2 D I R E C T PUPILLARY EFFECT OF INSTILLATION OF VARIOUS SYMPATHOLYTIC DRUGS IN RABBIT
Hours
Guanethidine 10% Reserpine 0.25% Bretylium 10% B.W.467 C 60 2.5% Xylocholine 2.5%
1
12
Mydriasis No effect Mydriasis Mydriasis Miosis
Slight miosis Slight miosis Slight miosis Slight miosis Miosis
546
LUCIANO BONOMI AND PIETRO DI COMITE
60 ( 1 % to 2.5%), Guanethidine (2.5% to 10% or Bretylium ( 5 % to 10%) should be used. Guanethidine 1%, Bretylium 1%, Reserpine or Xylocholine significantly re duce the pupillary response to sympathetic stimulation but fail to produce complete blockage. Perhaps our results can be better under stood by using the theoretical model postu lated by Brodie to explain the intrinsic mechanism of function of the sympathetic nerve ending and the mode of action of the drugs thereon.5·6 The distinctive features of the pupillary effects of Guanethidine, Bretylium and B.W. 467 C 60 are: (1) rapid onset of com plete sympathetic blockage and (2) the pres ence of an initial transient sympathomimetic effect which, during the first hour, paradoxi cally coexists with an unresponsiveness of the sympathetic structures to electrical stim ulation. These features can be explained by the fact that these drugs directly affect the physiologic mechanism by which the nerve impulse releases norepinephrine from its storage site at the nerve ending. The Reserpine blockage is incomplete and delayed, however, producing no noticeable sympathomimetic effect. Indeed, Reserpine acts by depleting the stores of norepi nephrine at the nerve endings. The depletion of the transmitter takes place slowly4 so that the effect becomes obvious only after a cer tain delay. Furthermore, since the release mechanism remains unimpaired, the mini mal amounts of the transmitter still present at the storage site can be released under maximum stimulation and diffuse onto the receptors, a fact which explains the incom pleteness of the blockage. A similar result is produced by Xylocho line, which is believed to decrease the stored norepinephrine by impairing its synthesis.7 Unlike Reserpine, Xylocholine also exerts a strong muscarinic effect, which accounts for its marked miotic action. With the exception of Xylocholine, all the
drugs used in the present experiment were tested in previous studies of their effects upon the intraocular pressure in rabbits.1'3·8 Bretylium was tested only in man.2 Inter estingly, lowering of the intraocular pres sure and blockage of the sympathetic are obtained with the same dosages and the du ration of the two phenomena corresponds, suggesting a definite cause-and-effect rela tionship between peripheral sympathetic blockage and lowering of the intraocular pressure. SUMMARY
Appropriate concentrations of Guanethi dine, Bretylium and B.W. 467 C 60, instilled into the conjunctival sac of rabbits, produce a complete and long-lasting blockage of the mydriatic response normally elicited by the electrical stimulation of the ipsilateral sym pathetic chain. Reserpine and Xylocholine markedly reduce the mydriatic response but fail to produce complete blockage. Clinica Oculistica Dell 'Università REFERENCES
1. Bonomi, L. : Effetti della instillazione locale di Reserpina sul tono intraoculare del coniglio. Atti XLVII Cong. Soc. Oftal. Ital., 1963, v. 21. 2. Bonomi, L., and Di Comité, P. : Azione del bretilio sulla pressione endoculare e sulla dinamica dell'umor acqueo nel glaucoma ad angolo aperto. Boll. Ocul., 42:719, 1963. 3. Di Comité, P. : Comportamento della tensione oculare e del diametro pupillare nel coniglio dopo somministrazione locale di un farmaco simpaticolitico (B.W.467 C 60). Boll. Ocul., 43:333, 1964. 4. Bonomi, L. : Spectrophotofiuorimetric research on catecholamines in ocular tissues. Boll. Ocul., 41 : 562, 1962. 5. Brodie, B. B. : Recent views on mechanisms for lowering sympathetic tone. Circulation, 28 :970, 1963. 6. Brodie, B. B., and Costa, E. : Concept of the neurochemical transducer as an organized molecu lar unit at sympathetic nerve endings. Activ. nerv. sup. (Praha), 5 :264, 1963. 7. Bain, W. A. : Interference with the release of transmitter in response to nerve stimulation. In Adrenergic Mechanisms. London, Churchill, 1960, p. 131. 8. Oosterhuis, J. A.: Guanethidine (Ismelin) in ophthalmology: I. Observations in rabbits. Arch. Ophth., 67:592, 1962.
AND P I E T R O D I
COMITÉ,
M.D.
Bari, Italy Several sympatholytic drugs have been shown to lower the intraocular pressure, whether administered locally or systemically. Recently ophthlamologists have been in troduced to the adrenergic neurone blocking agents, which act by blocking release of the chemical transmitter from the sympathetic nerve endings without impairing the respon siveness of the sympathetic receptors.
mydriatic response to electrical stimulation of the ipsilateral sympathetic chain in rabbit was chosen. T h e following solutions were used* :
One of these drugs, Guanethidine, is known to produce significant lowering of the intraocular pressure, both in rabbit and human eyes (normal or glaucomatous), by markedly decreasing aqueous production. Similar effects have been obtained 1 " 3 with the local use of Reserpine, Bretylium and B.W. 467 C 60. Since the role of the autonomie nervous system in the regulation of intraocular dy namics is poorly understood, interest in these drugs lies not only in the hope that they may be of practical use in the therapy of glaucoma but also in their possible value in the experimental study of this subject. In order to use these substances appropri ately and to interpret their action correctly, it seemed essential, as a first step, to investi gate the following factors : 1. T h e effectiveness of these drugs in blocking the ocular sympathetic nerves when applied locally into the conjunctival sac. 2. The degree and duration of the sympa thetic blockade. 3. T h e dosage of each drug needed to ob tain the effect.
O n e drop of the solution under investiga tion was instilled into the conjunctival sac of the animals' right eyes three times at fiveminute intervals. T h e left eyes were untreat ed controls. At various times after treat ment the pupillary behavior was checked. T h e sympathetic nerve was isolated under general anesthesia (Kemital 10%, 0.5-0.8 ml/kg, intravenously) at both sides of the neck; fine stainless steel electrodes were ap plied in front of the nerve's entrance into the superior cervical ganglion; electrical square wave stimulations were applied from a Palmer electric stimulator (intensity, 2 to 6 v ; frequency, 5 to 20 c / s ; duration of each impulse, 1.5 m s ) . T h e pupillary size was observed under constant lighting and measured with finetipped calipers.
Guanethidine 1%, 2.5%, 5%, 10% Reserpine 0.25%, 1% Bretylium 1%, 5%, 10% B.W. 467 C 60 1%, 2.5% Xylocholine 2.5%, 10%
M A T E R I A L S AND M E T H O D S
A s an index of the function of the sympa thetically innervated ocular structures, the * From the Department of Ophthalmology, Uni versity of Bari (Director: Prof. F. D'Ermo). Pres ent address : Clinica Oculistica, University of Padova. 544
RESULTS
T h e effects of electrical stimulation of the sympathetic system upon the pupillary re sponse are shown in Table 1. Stimulation of the ipsilateral sympathetic chain constantly produced in the untreated eyes a moderate mydriasis when low intensity and frequency * Guanethidine was supplied by the Ciba Industria Chimica of Milan; Reserpine by the Boehringer Prodotti Chimici Farmaceutici of Flor ence; Bretylium and B.W. 467 C 60 by the Wellcome Research Laboratories of Beckenham and Xylocholine by the Smith, Kline and French Lab oratories of Philadelphia.
EFFECT OF SYMPATHOLYTIC DRUGS
545
TABLE 1 PUPILLARY RESPONSES TO ELECTRICAL STIMULATION OF THE IPSILATERAL CERVICAL SYMPATHETIC CHAIN AFTER INSTILLATION OF VARIOUS SYMPATHOLYTIC DRUGS
Hours 6
12
24
++ ++ +-
+++-
+++++
++-
++
++-
1
Guanethidine 1% Guanethidine 2 . 5 % Guanethidine 5% Guanethidine 10% Reserpine 0.25% Reserpine 1% Bretylium 1% Bretylium 5% Bretylium 10% B.W.467C60 1% B.W.467C60 2.5% Xylocholine2.5% Xylocholine 10%
4
+-
stimuli were applied (2 v ; 5 c/s and a maximum mydriasis when higher intensity and frequency were used (6 v ; IS c/s). This response is indicated in Table 1 by + + . The notation H— indicates that the response was less than in the control eyes and that a more powerful stimulation was needed to produce clear results (at least 6 v ; 15 c/s). The notation — indicates a com plete lack of pupillary response even after maximum stimulation. As shown in Table 1, Guanethidine, Bre tylium and B.W. 467 C 60 produced com plete and long-lasting sympathetic blockade, while Xylocholine and Reserpine merely depressed the pupillary response. Higher concentrations of Reserpine were not used, since the animals treated with the 1% solu tion already showed marked systemic effects (diarrhea, sedation and miosis of the fellow eye). In previous experiments,4 however, we demonstrated that the concentrations used were sufficient, when instilled into the con junctival sac of rabbits, to produce an al most complete depletion of the uveal stores of norepinephrine. In addition to investigating the effect of electrical stimulation of the sympathetic upon the pupillary response, the direct effects of the drugs upon the pupillary size prior to any stimulation were also recorded. Guanethidine, Bretylium and B.W. 467 C
48
++-
++ ++ +++
+-
+-
60 produced an early and transient mydri asis followed by a slight miosis which lasted as long as the response to electrical stimula tion was blocked. Reserpine produced no direct pupillary change during the first two hours, after this a slight miosis was seen. Xylocholine, however, caused a clear and early miosis which lasted many hours. These results are summarized in Table 2 which, for clarity of interpretation, refers only to the most effective dosage and to ob servations made at the first and at the 12th hour after treatment. DISCUSSION
Our results demonstrate that all the drugs tested have a definite sympatholytic effect upon the ocular structures of the rabbit when instilled into the conjunctival sac. In order to obtain complete blockage of the sympathetic impulses, however, B.W. 467 C TABLE 2 D I R E C T PUPILLARY EFFECT OF INSTILLATION OF VARIOUS SYMPATHOLYTIC DRUGS IN RABBIT
Hours
Guanethidine 10% Reserpine 0.25% Bretylium 10% B.W.467 C 60 2.5% Xylocholine 2.5%
1
12
Mydriasis No effect Mydriasis Mydriasis Miosis
Slight miosis Slight miosis Slight miosis Slight miosis Miosis
546
LUCIANO BONOMI AND PIETRO DI COMITE
60 ( 1 % to 2.5%), Guanethidine (2.5% to 10% or Bretylium ( 5 % to 10%) should be used. Guanethidine 1%, Bretylium 1%, Reserpine or Xylocholine significantly re duce the pupillary response to sympathetic stimulation but fail to produce complete blockage. Perhaps our results can be better under stood by using the theoretical model postu lated by Brodie to explain the intrinsic mechanism of function of the sympathetic nerve ending and the mode of action of the drugs thereon.5·6 The distinctive features of the pupillary effects of Guanethidine, Bretylium and B.W. 467 C 60 are: (1) rapid onset of com plete sympathetic blockage and (2) the pres ence of an initial transient sympathomimetic effect which, during the first hour, paradoxi cally coexists with an unresponsiveness of the sympathetic structures to electrical stim ulation. These features can be explained by the fact that these drugs directly affect the physiologic mechanism by which the nerve impulse releases norepinephrine from its storage site at the nerve ending. The Reserpine blockage is incomplete and delayed, however, producing no noticeable sympathomimetic effect. Indeed, Reserpine acts by depleting the stores of norepi nephrine at the nerve endings. The depletion of the transmitter takes place slowly4 so that the effect becomes obvious only after a cer tain delay. Furthermore, since the release mechanism remains unimpaired, the mini mal amounts of the transmitter still present at the storage site can be released under maximum stimulation and diffuse onto the receptors, a fact which explains the incom pleteness of the blockage. A similar result is produced by Xylocho line, which is believed to decrease the stored norepinephrine by impairing its synthesis.7 Unlike Reserpine, Xylocholine also exerts a strong muscarinic effect, which accounts for its marked miotic action. With the exception of Xylocholine, all the
drugs used in the present experiment were tested in previous studies of their effects upon the intraocular pressure in rabbits.1'3·8 Bretylium was tested only in man.2 Inter estingly, lowering of the intraocular pres sure and blockage of the sympathetic are obtained with the same dosages and the du ration of the two phenomena corresponds, suggesting a definite cause-and-effect rela tionship between peripheral sympathetic blockage and lowering of the intraocular pressure. SUMMARY
Appropriate concentrations of Guanethi dine, Bretylium and B.W. 467 C 60, instilled into the conjunctival sac of rabbits, produce a complete and long-lasting blockage of the mydriatic response normally elicited by the electrical stimulation of the ipsilateral sym pathetic chain. Reserpine and Xylocholine markedly reduce the mydriatic response but fail to produce complete blockage. Clinica Oculistica Dell 'Università REFERENCES
1. Bonomi, L. : Effetti della instillazione locale di Reserpina sul tono intraoculare del coniglio. Atti XLVII Cong. Soc. Oftal. Ital., 1963, v. 21. 2. Bonomi, L., and Di Comité, P. : Azione del bretilio sulla pressione endoculare e sulla dinamica dell'umor acqueo nel glaucoma ad angolo aperto. Boll. Ocul., 42:719, 1963. 3. Di Comité, P. : Comportamento della tensione oculare e del diametro pupillare nel coniglio dopo somministrazione locale di un farmaco simpaticolitico (B.W.467 C 60). Boll. Ocul., 43:333, 1964. 4. Bonomi, L. : Spectrophotofiuorimetric research on catecholamines in ocular tissues. Boll. Ocul., 41 : 562, 1962. 5. Brodie, B. B. : Recent views on mechanisms for lowering sympathetic tone. Circulation, 28 :970, 1963. 6. Brodie, B. B., and Costa, E. : Concept of the neurochemical transducer as an organized molecu lar unit at sympathetic nerve endings. Activ. nerv. sup. (Praha), 5 :264, 1963. 7. Bain, W. A. : Interference with the release of transmitter in response to nerve stimulation. In Adrenergic Mechanisms. London, Churchill, 1960, p. 131. 8. Oosterhuis, J. A.: Guanethidine (Ismelin) in ophthalmology: I. Observations in rabbits. Arch. Ophth., 67:592, 1962.