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Chronic reserpine treatment on adrenergic neuronal and receptor function in the isolated perfused mesenteric blood vessels of the dog
EUROPEAN
JOURNAL
OF PHARMACOLOGY
I2 (1970)
378-381.
NORTH-HOLLAND
PUBLISHING
COMPANY
Short communication
CHRONIC
RESERPINE
RECEPTOR
TREATMENT
FUNCTION
ON ADRENERGIC
IN THE ISOLATED
BLOOD
VESSELS
PERFUSED
NEURONAL
AND
MESENTERIC
OF THE DOG
David E. CLARKE, H. Richard ADAMS and Joseph P. BUCKLEY Department
Received
20 August
of Pharmacology, School of Pharmacy, Pittsburgh, Pennsylvania 15213.
University of Pittsburgh, USA
Accepted
1970
8 September
1970
D.E. CLARKE, H.R. ADAMS and J.P. BUCKLEY, Chronic reserpine treatment on adrenergic neuronaland receptor fiinction in the isolated perfused mesenteric blood vessels of the dog, European J. Pharmacol. 12 (1970) 378-381. Chronic low dosage reserpine treatment significantly depressed the frequency-response curve to sympathetic nerve stimulation in the isolated perfused dog mesenteric and intestinal blood vessels. This neuronal depression was reversed by norepinephrine, indicating that chronic reserpine dosage fails to produce a drastic impairment of adrenergic nerve function. The dose-response curve to injected norepinephrine was not significantly altered, thus supersensitivity to norepinephrine following chronic reserpine treatment is not a common phenomenon in all vascular tissue. The pharmacological interaction of certain adrenergic agents with sympathetic neuronal and receptor mechanisms appeared unaltered by reserpine.
Mesenteric vessels, perfused Chronic reserpine treatment
Sympathetic nerve stimulation Norepinephrine
1. INTRODUCTION In man therapeutic doses of reserpine are almost invariably administered on a chronic basis; however, only a paucity of experimental studies in animals have simulated this chronic schedule of treatment. This present report concerns the effects of treatment for one year with small, daily, oral doses of reserpine on adrenergic neuronal and receptor function in the mesenteric and intestinal blood vessels on mongrel dogs.
2. METHODS Male and female mongrel dogs received 0.137 mg of reserpine (Serpasil) per OStwice daily. After twelve months of treatment, the dogs were immobilized with
Reserpine
sodium pentobarbital, 20 mg/kg, i.v., and additional amounts were titrated until surgical anesthesia was produced. Control dogs received placebo treatment, twice daily, and were anesthetized with sodium pentobarbital, 35 mg/kg. The small intestine and accompanying mesenteric blood vessels were removed and placed in 1 1 of Krebs’ bicarbonate solution at room temperature and vigorously bubbled with 0s 95% and COZ 5%. Individual intestinal loops about 4 to 6 cm in length were perfused through the main mesenteric arterial branch with oxygenated Krebs solution at 37°C by means of a Sigmamotor pump. A Statham transducer was used to record perfusion pressure, the latter being adjusted to give an initial pressure of 25 to 50 mm Hg. Drugs were injected into the perfusion fluid just prior to the perfusion cannula but in front of the side arm leading to the transducer. The perfused mesenteric arterial branch and accom-
319
D.E.CIarke et al., Chronic reserpine treatment
attenuated by chronic reserpine treatment. The responses to norepinephrine were increased on the systemic blood pressure and significantly potentiated on the femoral artery studied in vitro, whereas no significant potentiation was obtained on the isolated superior mesenteric artery. This latter finding is in agreement with the present observations (table 1). It seems, therefore, that not all vascular tissue or beds are uniformly influenced with regard to adrenergic effector-receptor sensitivity following chronic reserpine treatment. However, an increased responsiveness to norepinephrine might have existed at some time period during the year since there is a definite tendency towards an increased effectiveness of this amine in the reserpine-treated preparations. Cocaine enhanced the responses to both injected norepinephrine and nerve stimulation indicating that chronic reserpine treatment, like acute large doses of reserpine, fails to influence the neuronal uptake mechanism for norepinephrine (Iversen, 1967). Also, since
panying sympathetic nerves were placed over bipolar platinum electrodes and the nerves were stimulated using a Grass square-wave stimulator at supramaximal voltages, with a pulse duration of 2 msec over a range of frequencies (1 to 48 shockslsec) for 20 set at 3 to 6 min intervals. In each loop, frequency-response curves to nerve stimulation were obtained before dose-response curves to injected norepinephrine bitartrate. The responses to nerve stimulation were characterized as originating from post-ganglionic sympathetic fibers since they were blocked by bretylium tosylate (2 to 4 mg), acetylcholine iodide (20 pg) and phentolamine mesylate (1 mg) but not by hexamethonium hydrochloride (10 mg) or atropine sulphate (40 gg). Cocaine hydrochloride (100 and 500 pg) increased the effect of nerve stimulation at all frequencies, the increase being dose related. Similarly norepinephrine responses were potentiated by cocaine, and were completely blocked by phentolamine, but were unaffected by atropine.
3. RESULTS AND DISCUSSION The effect of reserpine on the frequency-response curve to nerve stimulation is shown in fig. 1, and some of the mean individual data, obtained for each experimental animal, is shown in table 1. Whereas reserpine treatment caused a significant reduction in the responses to nerve stimulation it failed to significantly increase the responses to exogenously administered norepinephrine. The reserpine-induced neuronal defect appears to be mediated through a noncompetitive type of antagonism similar to that reported with nerve stimulation to the cat nictitating membrane (Trendelenburg et al., 1962; Haefely et al., 1964). It should be noted that the preparations obtained from dogs Rl and R2 (table 1) suffered severe impairment of adrenergic nerve function, whereas the preparations obtained from dogs R3 and R4 suffered a lesser depression. Thus at fherapeutic dose levels the effects of reserpine appear to be approximately dose related. These findings on sympathetic neuronal function are consistent with in vivo studies made on dogs in another aspect of this study (Jandhyala et al., unpublished). The response to tilt, bilateral carotid occlusion and hexamethonium were significantly
L
I
I
Y
I
3 LOG
6 FREOUENCY
9
I2
24
46
(SHOCKI/SLC.)
Fig. 1. The effect of chronic reserpine treatment for 1 year on the frequency-response curve to sympathetic nerve stimulation to the isolated perfused mesenteric and intestinal blood vessels of dogs. o-o control; l -m reserpine treated. *p
380
D. E. Clarke et al., Chronic reserpine treatment
Table 1 The effect of chronic reserpine treatment for 1 year on the responses to injected norepinephrine (NE) and nerve stimulation (NS) in the isolated perfused mesenteric and Intestinal blood vessels of dogs. _ Reserpine Mean response (mm Hg) to NE Mean response (mm Hg) to NS Control Reserpine dose n dogs dogs 1.0 2.0 4.0 8.0 6 9 12 24 bdkdday) (shocks/set)
01g) Rl R2 R3 R4
34.0 * 25.0 26.7 21.5
4 3 2 4
Mean+ SE.
3
Mean f S.E.
10
39.8 31.2 85.0 60.0
58.1 48.7 125.0 88.3
54.0 f 12.0 ____~_ 25.6 f 3.0
80.0 + 17.2 41.1 + 4.9
82.3 78.1 151.0 105.3
110.6 106.2 175.0 131.6
104.3 + 130.9 f 16.1 15.7 _ 71.9 + 103.1 f. 6.0 6.3
1.25 11.6 20.0 17.5
1.25 14.2 22.0 26.9
3.7 27.5 30.0 33.1
5.6 16.0 30.0 38.7
12.6 f 4.2
16.2 + 5.6
23.6 + 6.7
22.6 + 7.3
36.4 rt 11.0
61.2 + 9.2
90.1 + 6.3
107.2 + 3.9
* The dose of reserpine was reduced to 17 pg/kg/day after 8 months (see text). The mean responses to norepinephrine in reserp ine-treated preparations are not significantly greater @ = 0.05) than those obtained from control preparations.
bretylium, acetylcholine and phentolamine all blocked the responses to nerve stimulation in reserpine treated preparations it is evident that other adrenergic mechanisms had not been materially altered over the one-year period of reserpine treatment. The acetylcholine-induced block of adrenergic neurotransmission occurred without any concomitant reduction in the response to injected norepinephrine and could be fully prevented by atropine. Thus, these observations extend the recent reports of Malik and Ling (1969) and Lbffelholz and Muscholl (1969) that muscarinic receptors are present on postganglionic sympathetic neurones. The depressant effect of reserpine on adrenergic neurotransmission was significantly restored by injections of norepinephrine. Such a reversal of adrenergic function is not usually possible if extensive damage to intraneuronal vesicular storage and uptake mechanisms is present; as is the case after large doses of reserpine (Haggendal and Malmfors, 1969; Malmfors, 1969). This observation indicates that small daily doses of reserpine do not summate to produce pharmacological effects equivalent to large acute doses of this agent. A similar conclusion has been made from studies on the cardiovascular sympathetic tone of rats given small daily doses of reserpine (Clarke, 1967). Dog Rl was the smallest dog in the study, and
therefore received the largest mg/kg dose of reserpine (table 1). After 8 months of treatment, this dog exhibited a marked Parkinsonism-like syndrome with pronounced depression and loss of body weight. During the last four months of treatment the reserpine dose was reduced by 50% but the dog continued to exhibit extensive depression and tremors although body weight was regained. In spite of this dose reduction, the preparations from this dog consistently exhibited the most severe depression of adrenergic nerve function. It is possible therefore that the initial regimen of chronic reserpine dosage may modify the intensity of effect obtained from subsequent changes in dose level. Evidence for such an effect has been obtained in rats (Clarke, 1967), although in this species small daily doses of reserpine appeared to antagonize the effects of a subsequently administered large dose.
ACKNOWLEDGEMENTS This work was supported by U.S. Public Health Service grant number GM 15587 from the National Institutes of General Medical Sciences. Reserpine (Serpasil) was kindly donated by Dr. Plummer, Ciba Pharmaceutical Company, Summit, NJ., U.S.A.
D.E.Clarke et al., Chronic reserpine treatment
REFERENCES Clarke, D.E., 1967, The effect of differing reserpine pretreatments on the cardiovascular response to tyramine, J. Pharm. Pharmacol. 19,474. Haefely, W., A. Hurliian and H. Thoenen, 1964, A quantitative study of the effect of cocaine on the response of the cat nictitating membrane to nerve stimulation and to injected noradrenaline, Brit. J. Pharmacol. 22,s. Haggendal, J. and T. Mahnfors, 1969, The effect of nerve stimulation on catecholamines taken up in adrenergic nerves after reserpine pretreatment, Acta Physiol. &and. 75,33. Iversen, L.L., 1967, The Uptake and Storage of Noradrenaline in Sympathetic Nerves (Cambridge University Press, Cambridge).
381
Mffelholz, K. and E. Muscholl, 1969, A muscarinic inhibition of the noradrenaline release evoked by postganglionic sympathetic nerve stimulation, Arch. Exptl. Pathol. Pharmakol. 265, 1. Malik, K.U. and G.M. Ling, 1969, Modification by acetycholine of the response of rat mesenteric arteries to sympathetic stimulation, Circulation Res. 25, 1. Malmfors, T., 1969, Histochemical studies on the effect of nerve impulses on exogenous catecholamines taken up into the adrenergic nerves of reserpine-pretreated animals, Pharmacology 2, 193. Trendelenburg, U., A. Muskus, W.W. Fleming and Gomez Alonso De La Sierra, 1962, Modification by reserpine of the action of sympathomimetic amines in spinal cats; a classification of sympathomimetic amines, J. Pharmacol. Exptl. Therap. 138, 70.
JOURNAL
OF PHARMACOLOGY
I2 (1970)
378-381.
NORTH-HOLLAND
PUBLISHING
COMPANY
Short communication
CHRONIC
RESERPINE
RECEPTOR
TREATMENT
FUNCTION
ON ADRENERGIC
IN THE ISOLATED
BLOOD
VESSELS
PERFUSED
NEURONAL
AND
MESENTERIC
OF THE DOG
David E. CLARKE, H. Richard ADAMS and Joseph P. BUCKLEY Department
Received
20 August
of Pharmacology, School of Pharmacy, Pittsburgh, Pennsylvania 15213.
University of Pittsburgh, USA
Accepted
1970
8 September
1970
D.E. CLARKE, H.R. ADAMS and J.P. BUCKLEY, Chronic reserpine treatment on adrenergic neuronaland receptor fiinction in the isolated perfused mesenteric blood vessels of the dog, European J. Pharmacol. 12 (1970) 378-381. Chronic low dosage reserpine treatment significantly depressed the frequency-response curve to sympathetic nerve stimulation in the isolated perfused dog mesenteric and intestinal blood vessels. This neuronal depression was reversed by norepinephrine, indicating that chronic reserpine dosage fails to produce a drastic impairment of adrenergic nerve function. The dose-response curve to injected norepinephrine was not significantly altered, thus supersensitivity to norepinephrine following chronic reserpine treatment is not a common phenomenon in all vascular tissue. The pharmacological interaction of certain adrenergic agents with sympathetic neuronal and receptor mechanisms appeared unaltered by reserpine.
Mesenteric vessels, perfused Chronic reserpine treatment
Sympathetic nerve stimulation Norepinephrine
1. INTRODUCTION In man therapeutic doses of reserpine are almost invariably administered on a chronic basis; however, only a paucity of experimental studies in animals have simulated this chronic schedule of treatment. This present report concerns the effects of treatment for one year with small, daily, oral doses of reserpine on adrenergic neuronal and receptor function in the mesenteric and intestinal blood vessels on mongrel dogs.
2. METHODS Male and female mongrel dogs received 0.137 mg of reserpine (Serpasil) per OStwice daily. After twelve months of treatment, the dogs were immobilized with
Reserpine
sodium pentobarbital, 20 mg/kg, i.v., and additional amounts were titrated until surgical anesthesia was produced. Control dogs received placebo treatment, twice daily, and were anesthetized with sodium pentobarbital, 35 mg/kg. The small intestine and accompanying mesenteric blood vessels were removed and placed in 1 1 of Krebs’ bicarbonate solution at room temperature and vigorously bubbled with 0s 95% and COZ 5%. Individual intestinal loops about 4 to 6 cm in length were perfused through the main mesenteric arterial branch with oxygenated Krebs solution at 37°C by means of a Sigmamotor pump. A Statham transducer was used to record perfusion pressure, the latter being adjusted to give an initial pressure of 25 to 50 mm Hg. Drugs were injected into the perfusion fluid just prior to the perfusion cannula but in front of the side arm leading to the transducer. The perfused mesenteric arterial branch and accom-
319
D.E.CIarke et al., Chronic reserpine treatment
attenuated by chronic reserpine treatment. The responses to norepinephrine were increased on the systemic blood pressure and significantly potentiated on the femoral artery studied in vitro, whereas no significant potentiation was obtained on the isolated superior mesenteric artery. This latter finding is in agreement with the present observations (table 1). It seems, therefore, that not all vascular tissue or beds are uniformly influenced with regard to adrenergic effector-receptor sensitivity following chronic reserpine treatment. However, an increased responsiveness to norepinephrine might have existed at some time period during the year since there is a definite tendency towards an increased effectiveness of this amine in the reserpine-treated preparations. Cocaine enhanced the responses to both injected norepinephrine and nerve stimulation indicating that chronic reserpine treatment, like acute large doses of reserpine, fails to influence the neuronal uptake mechanism for norepinephrine (Iversen, 1967). Also, since
panying sympathetic nerves were placed over bipolar platinum electrodes and the nerves were stimulated using a Grass square-wave stimulator at supramaximal voltages, with a pulse duration of 2 msec over a range of frequencies (1 to 48 shockslsec) for 20 set at 3 to 6 min intervals. In each loop, frequency-response curves to nerve stimulation were obtained before dose-response curves to injected norepinephrine bitartrate. The responses to nerve stimulation were characterized as originating from post-ganglionic sympathetic fibers since they were blocked by bretylium tosylate (2 to 4 mg), acetylcholine iodide (20 pg) and phentolamine mesylate (1 mg) but not by hexamethonium hydrochloride (10 mg) or atropine sulphate (40 gg). Cocaine hydrochloride (100 and 500 pg) increased the effect of nerve stimulation at all frequencies, the increase being dose related. Similarly norepinephrine responses were potentiated by cocaine, and were completely blocked by phentolamine, but were unaffected by atropine.
3. RESULTS AND DISCUSSION The effect of reserpine on the frequency-response curve to nerve stimulation is shown in fig. 1, and some of the mean individual data, obtained for each experimental animal, is shown in table 1. Whereas reserpine treatment caused a significant reduction in the responses to nerve stimulation it failed to significantly increase the responses to exogenously administered norepinephrine. The reserpine-induced neuronal defect appears to be mediated through a noncompetitive type of antagonism similar to that reported with nerve stimulation to the cat nictitating membrane (Trendelenburg et al., 1962; Haefely et al., 1964). It should be noted that the preparations obtained from dogs Rl and R2 (table 1) suffered severe impairment of adrenergic nerve function, whereas the preparations obtained from dogs R3 and R4 suffered a lesser depression. Thus at fherapeutic dose levels the effects of reserpine appear to be approximately dose related. These findings on sympathetic neuronal function are consistent with in vivo studies made on dogs in another aspect of this study (Jandhyala et al., unpublished). The response to tilt, bilateral carotid occlusion and hexamethonium were significantly
L
I
I
Y
I
3 LOG
6 FREOUENCY
9
I2
24
46
(SHOCKI/SLC.)
Fig. 1. The effect of chronic reserpine treatment for 1 year on the frequency-response curve to sympathetic nerve stimulation to the isolated perfused mesenteric and intestinal blood vessels of dogs. o-o control; l -m reserpine treated. *p
380
D. E. Clarke et al., Chronic reserpine treatment
Table 1 The effect of chronic reserpine treatment for 1 year on the responses to injected norepinephrine (NE) and nerve stimulation (NS) in the isolated perfused mesenteric and Intestinal blood vessels of dogs. _ Reserpine Mean response (mm Hg) to NE Mean response (mm Hg) to NS Control Reserpine dose n dogs dogs 1.0 2.0 4.0 8.0 6 9 12 24 bdkdday) (shocks/set)
01g) Rl R2 R3 R4
34.0 * 25.0 26.7 21.5
4 3 2 4
Mean+ SE.
3
Mean f S.E.
10
39.8 31.2 85.0 60.0
58.1 48.7 125.0 88.3
54.0 f 12.0 ____~_ 25.6 f 3.0
80.0 + 17.2 41.1 + 4.9
82.3 78.1 151.0 105.3
110.6 106.2 175.0 131.6
104.3 + 130.9 f 16.1 15.7 _ 71.9 + 103.1 f. 6.0 6.3
1.25 11.6 20.0 17.5
1.25 14.2 22.0 26.9
3.7 27.5 30.0 33.1
5.6 16.0 30.0 38.7
12.6 f 4.2
16.2 + 5.6
23.6 + 6.7
22.6 + 7.3
36.4 rt 11.0
61.2 + 9.2
90.1 + 6.3
107.2 + 3.9
* The dose of reserpine was reduced to 17 pg/kg/day after 8 months (see text). The mean responses to norepinephrine in reserp ine-treated preparations are not significantly greater @ = 0.05) than those obtained from control preparations.
bretylium, acetylcholine and phentolamine all blocked the responses to nerve stimulation in reserpine treated preparations it is evident that other adrenergic mechanisms had not been materially altered over the one-year period of reserpine treatment. The acetylcholine-induced block of adrenergic neurotransmission occurred without any concomitant reduction in the response to injected norepinephrine and could be fully prevented by atropine. Thus, these observations extend the recent reports of Malik and Ling (1969) and Lbffelholz and Muscholl (1969) that muscarinic receptors are present on postganglionic sympathetic neurones. The depressant effect of reserpine on adrenergic neurotransmission was significantly restored by injections of norepinephrine. Such a reversal of adrenergic function is not usually possible if extensive damage to intraneuronal vesicular storage and uptake mechanisms is present; as is the case after large doses of reserpine (Haggendal and Malmfors, 1969; Malmfors, 1969). This observation indicates that small daily doses of reserpine do not summate to produce pharmacological effects equivalent to large acute doses of this agent. A similar conclusion has been made from studies on the cardiovascular sympathetic tone of rats given small daily doses of reserpine (Clarke, 1967). Dog Rl was the smallest dog in the study, and
therefore received the largest mg/kg dose of reserpine (table 1). After 8 months of treatment, this dog exhibited a marked Parkinsonism-like syndrome with pronounced depression and loss of body weight. During the last four months of treatment the reserpine dose was reduced by 50% but the dog continued to exhibit extensive depression and tremors although body weight was regained. In spite of this dose reduction, the preparations from this dog consistently exhibited the most severe depression of adrenergic nerve function. It is possible therefore that the initial regimen of chronic reserpine dosage may modify the intensity of effect obtained from subsequent changes in dose level. Evidence for such an effect has been obtained in rats (Clarke, 1967), although in this species small daily doses of reserpine appeared to antagonize the effects of a subsequently administered large dose.
ACKNOWLEDGEMENTS This work was supported by U.S. Public Health Service grant number GM 15587 from the National Institutes of General Medical Sciences. Reserpine (Serpasil) was kindly donated by Dr. Plummer, Ciba Pharmaceutical Company, Summit, NJ., U.S.A.
D.E.Clarke et al., Chronic reserpine treatment
REFERENCES Clarke, D.E., 1967, The effect of differing reserpine pretreatments on the cardiovascular response to tyramine, J. Pharm. Pharmacol. 19,474. Haefely, W., A. Hurliian and H. Thoenen, 1964, A quantitative study of the effect of cocaine on the response of the cat nictitating membrane to nerve stimulation and to injected noradrenaline, Brit. J. Pharmacol. 22,s. Haggendal, J. and T. Mahnfors, 1969, The effect of nerve stimulation on catecholamines taken up in adrenergic nerves after reserpine pretreatment, Acta Physiol. &and. 75,33. Iversen, L.L., 1967, The Uptake and Storage of Noradrenaline in Sympathetic Nerves (Cambridge University Press, Cambridge).
381
Mffelholz, K. and E. Muscholl, 1969, A muscarinic inhibition of the noradrenaline release evoked by postganglionic sympathetic nerve stimulation, Arch. Exptl. Pathol. Pharmakol. 265, 1. Malik, K.U. and G.M. Ling, 1969, Modification by acetycholine of the response of rat mesenteric arteries to sympathetic stimulation, Circulation Res. 25, 1. Malmfors, T., 1969, Histochemical studies on the effect of nerve impulses on exogenous catecholamines taken up into the adrenergic nerves of reserpine-pretreated animals, Pharmacology 2, 193. Trendelenburg, U., A. Muskus, W.W. Fleming and Gomez Alonso De La Sierra, 1962, Modification by reserpine of the action of sympathomimetic amines in spinal cats; a classification of sympathomimetic amines, J. Pharmacol. Exptl. Therap. 138, 70.