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Noradrenergic-mediated potentiation of acetylcholine release from the phrenic nerve: Evidence for presynaptic α1-adrenoceptor involvement
Life Sciences, Vol. 31, pp. 853-857 Printed in the U.S.A.
Pergamon Pres~
NORADRENERGIC-MEDIATED POTENTIATION OF ACETYLCHOLINE RELEASE FROM THE PHRENIC ~ R V E : EVIDENCE FOR PRESYNAPTIC ~I-ADRENOCEPTOR INVOLVEMENT R. Michael Snider I and Michael C. Gerald 2 Division of Pharmacology, College of Pharmacy The Ohio State University Columbus, Ohio 43210 (Received in final form June 16, 1982) Summary This study, conducted in the rat phrenic nerve-diaphragm preparation, was designed to establish more direct evidence that norepinephrine enhances acetylcholine (ACh) release from motor neurons and characterize the ~-adrenoceptor type mediating this action. Norepinephrine (50 ~M, ~I + ~2 agonist) increased nervestimulated release by 183%, as determined by radioenzymatic assay. This effect was completely abolished by pretreatment with the ~-adrenoceptor antagonists phentolamine (~I + ~2) and by WB 4101 (~l) but only modestly reduced by yohimbine (~2). Clonidine (~2 agonist) did not enhance ACh release or nervestimulated muscle contractions, while phenylephrine (~l agonist) and norepinephrine increased muscle contractions up to 19.5-22.4%. These results support the hypothesis that norepinephrine increases ACh release from somatic motor nerves via a presynaptic ~I interaction. Cateeholamines exert a biphasic effect on mammalian neuromuscular transmission, namely, an initial facilitatory effect of relatively rapid onset followed by a slowly developing depressant effect (1,2). The results of muscle contraction and electrophysiological studies support the hypothesis that the faeilitatory effects of norepinephrine and epinephrine result from enhanced release of acetylcholine (ACh) from motor nerve terminals (2-4). The ability of phentolamine and phenoxybenzamine, but not pronethalol, to block the facilitatory effects of cateeholamines suggests an interaction involving the ~-adrenoceptor on motor nerve endings (1,4,5). The present study was designed to directly test the hypothesis that norepinephrine increases ACh release from motor nerves. Moreover, using selective ~-adrenoceptor agonists and antagonists, an attempt was made to characterize the ~-adrenoceptor type (~i or ~2) which mediates the stlmulatory effects of norepinephrine on ACh release and on muscle contractions.
IPresent address: Department of Pharmacology, Mayo Medical School, Rochester, Minnesota. ZTo whom correspondence should be addressed. 0024-3205/82/090853-05503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
854
~l-adrenergic Potentiation of ACh Release
Vol. 31, No. 9, 1982
Materials and Methods General Considerations Male albino Sprague-Dawley rats (Lab Supply, Indianapolis, Ind.) weighing 175-300g were used in these studies. Rats were killed with dlethyl ether and, unless otherwise indicated, the left hemidiaphragm and phrenic nerve were removed as described by Bulbrlng (6). Krebs' solution was of the following composition (mM): NaCI, 125; KCI, 5.0; CaCI2, 2.0; MgCI2, 1.0; NaH2PO~, 1.0; NaHCO3, 15; and glucose, ii; the resulting solution was aerated with 95% O2 + CO2 and had a pH = 7.3 ± 0.i. Data are expressed as a percentage of control or predrug baseline. Statistical comparisons were performed on raw data using the Students' t-test (two-tailed) for paired comparisons or by Dunnett's procedure when several treatments were compared to a single control. Values were considered significant if p < 0.05. Isolated phrenic nerve-diaphragms were suspended with 500 mg tension in a 50 ml organ bath containing Krebs' solution which was gassed with 95% O2 and maintained at 32 ± 0.5°C. The phrenic nerve was stimulated with supramaximal 5 V monophaslc square wave pulses of 0.4 msec duration at a frequency of 0.2 Hz for the 45-60 min equilibration period. Muscle contractions were recorded via a FT.03C isometric transducer coupled to a Grass polygraph. Acetylcholine Release Experiments The cannulated phrenic nerve-diaphragm preparation employed to study ACh release was previously described (7,8). The preparation was perfused at room temperature (24 ~ I°C) at a rate of 40 ~i/min with aerated Krebs' solution containing physostigmine (30 ~M) to prevent ACh hydrolysis. The phrenlc nerve was stimulated with 5 V pulses of 0.4 msec duration at a frequency of i0 Hz. A total of six 10-min samples were collected for ACh assay: 2 pre-drug control periods, 2 periods in the presence of amphetamine, and 2 post-drug washout periods, and the results presented represent the average of these 2 intervals for each condition. ACh was quantified employing a radioenzymatic assay (9). In all experiments, blanks and standard amounts of ACh were processed in the same manner as the tissue perfusates, thus generating a standard curve from which the quantity of ACh in the samples was calculated. The practical limit sensitivity of the assay (3-10 pmoles ACh) was twice the CPM measured in blank solutions. Results Norepinephrine (50 ~M) increased nerve-stimulated release of ACh by 183% (Table i). This enhancement was completely antagonized by tissue treatment with phentolamine and the selective ~1-adrenoceptor antagonist WB 4101 but was only modestly reduced by yohimbine (by 27%), ~ a relatively selective ~2antagonist (Table I). When tested alone at the concentrations employed in these studies (i0 BM), these antagonists produced less than a ± 2% change in release. Similarly, clonidine, a selective ~2-agonlst, failed to enhance neurotransmitter release.
3Value derived by the following formula: [ i-- % change by noreplnephrlne + yohimblne ) X 100% % change by norepinephrine
Vol. 31, No. 9, 1982
el-adrenergic Potentiation of ACh Release
855
To further investigate the nature of the presynaptic ~-adrenoceptor hypothesized to mediate the enhancement of ACh release from motor neurons, phenylephrine and clonidine were compared with norepinephrine for their ability to augment nerve-stimulated muscle contractions. Cumulative addition of norepinephrine and the selective ~l-adrenoceptor agonist phenylephrine produced concentration-dependent facilitation of twitch height by up to 22.4 and 19.5%, respectively (Fig. I). By contrast, clonidine failed to augment twitch; inhibition of contractions by 30% was observed at the highest concentration tested.
TABLE I Effects of e-Adrenoceptor Agonists and Antagonists on Acetylcholine Release a Treatment
Norepinephrine (50 ~M) + Phentolamine (I0 ~M) +WB
4101 (i0 ~M)
+ Yohimbine (I0 ~M) Clonidine (30 ~M)
Acetylcholine release
283 ± 42
b
(N )
(ii)
102 ± 7.8
(4)
93.9 ± 7.7
(4)
233 ± 70
b
87.4 ± 16
(4) (3)
l.a,values represent the mean ± S.E.M. compared to pre-drug ACh release, I employing each tissue as its own control, and expressed as a percentage of control. Mean pre-drug ACh release was 4.6 ± 0.4 pmol/min (N=27 preparations). 'IblValues significantly different from control (p < 0.05). '
Discussion In the rat phrenic nerve-diaphragm preparation, norepinephrine (25 ~M) increases the frequency of miniature end-plate potentials 2-fold but not their amplitude; these electrophysiological findings are consistent with enhanced release of neurotransmitter (4). In the present study, norepinephrine (50 ~M) increased ACh release, as determined by radioenzymatic assay, to approximately the same extent. Post- and presynaptic adrenergic receptors have generally been classified as el- and ~-adrenoceptors, respectively (i0,ii). Stimulation of presynaptic ez-receptors at the autonomic neuroeffector junction decreases transmitter release (i0). By contrast, the results of the present study suggest the involvement of a presynaptic el-adrenoceptor which mediates increased neurotransmitter release at the somatic neuromuscular junction. Malta et al. reached a similar conclusion employing experiments which compared the potency of selective e-adrenoceptor agonists and antagonists in modifying neuromuscular transmission with an autonomic vascular response. On the basis of the results of these in vivo experiments in cats, the character-
856
~l-adrenergic Potentiation of ACh Release
,~o
70
Vol. 31, No. 9, 1982
/x-----x
I
I
i
~
I
I
I
_~
,
[~,,ist] (u) FIG. i Effects of norepinephrine ( X ) , phenylephrine ( • ) , and clonidine ( m ) on nerve-stimulated muscle contractions. Drugs were added in a cumulative manner at 5 min intervals. Values represent the mean contraction height of 3 tissues for each treatment. S.E.M. ranged from 0.3-1.9% with exception of clonidine at 10-3M (3.2%).
istics of prejunctional ~-adrenoceptors on somatic motor nerve terminals in potentiating neuromuscular transmission were found to be similar to the postsynaptic ~-adrenoceptors in the blood vessel vasculature, ~.~., ~l-type adrenoceptors. In the present study, the nonselective s-antagonist phentolamine and the selective ~1-antagonist WB 4101 completely blocked noreplnephrlne-enhanced ACh release, whereas release was only slightly decreased by yohimbine. Since yohlmbine antagonizes both ~I- and ~2-adrenoceptors, with only about a 25-fold greater selectivity for ~2-adrenoceptors (12), it is possible that the partial inhibition observed results solely from its ~I antagonistic activity. Our neurochemical findings are further supported by the inability of the selective ~2-agonist clonldine to potentiate acetylcholine release or augment nervestimulated muscle contractions. By contrast, the ~l-agonlst phenylephrine significantly increased contraction height, as did norepinephrlne, a drug with ~I + ~2 agonist properties. Thus, based upon the selective potency differences between clonldlne and phenylephrlne (12,13), it is concluded that those presynaptic receptors enhancing release are of the ~ type.
Vol. 31, No. 9, 1982
~l-adrenergic Potentiation of ACh Release
857
Acknowledgment This work was supported by National Institute of Drug Abuse Grant DA-01477.
References I. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13.
W.C. BOWMAN and C. RAPER, Br. J. Pharmac. 27 313-331 (1966). W.C. BOWMAN, Handbook of Experimental Pharmacology, L. SZEKERES, Ed., 54/11 pp. 47-128, Springer-Verlag, New York (1981). K. KRNJEVlC and R. MILEDI, J. Physiol. 141 291-304 (1958). K. KUBA, J. Physiol. 211 551-570 (1970). E..MAL.TA, G.A. McPHERSON and C. RAPER, Br. J. Pharmac. 6 5 2 4 9 - 2 5 6 (1979). E. BULBRING, Br. J. Pharmac. 1 38-61 (1946). G.G. BIERKAMPER and A.M. GOLDBERG, J. Electrophysiol. Techniques 6 40-46 (1978). R.M. SNIDER and M.C. GERALD, J. Pharmac. Exp. Ther. 221 14-21 (1982). A.M. GOLDBERG and R.E. McCAMAN, J. Neurochem. 20 1-8 (1973). S.Z. LANGER, Br. J. Pharmac. 60 481-497 (1977). S. BERTHELSEN and W.A. PETTINGER, Life Sci. 21 595-606 (1977). R.R. RUFFOLO, JR., E.L. YADEN and J.E. WADDELL, J. Pharmac. Exp. Ther. 213 557-561 (1980). J. WIKBERG, Acta Physiol. Scand 103 225-239 (1978).
Pergamon Pres~
NORADRENERGIC-MEDIATED POTENTIATION OF ACETYLCHOLINE RELEASE FROM THE PHRENIC ~ R V E : EVIDENCE FOR PRESYNAPTIC ~I-ADRENOCEPTOR INVOLVEMENT R. Michael Snider I and Michael C. Gerald 2 Division of Pharmacology, College of Pharmacy The Ohio State University Columbus, Ohio 43210 (Received in final form June 16, 1982) Summary This study, conducted in the rat phrenic nerve-diaphragm preparation, was designed to establish more direct evidence that norepinephrine enhances acetylcholine (ACh) release from motor neurons and characterize the ~-adrenoceptor type mediating this action. Norepinephrine (50 ~M, ~I + ~2 agonist) increased nervestimulated release by 183%, as determined by radioenzymatic assay. This effect was completely abolished by pretreatment with the ~-adrenoceptor antagonists phentolamine (~I + ~2) and by WB 4101 (~l) but only modestly reduced by yohimbine (~2). Clonidine (~2 agonist) did not enhance ACh release or nervestimulated muscle contractions, while phenylephrine (~l agonist) and norepinephrine increased muscle contractions up to 19.5-22.4%. These results support the hypothesis that norepinephrine increases ACh release from somatic motor nerves via a presynaptic ~I interaction. Cateeholamines exert a biphasic effect on mammalian neuromuscular transmission, namely, an initial facilitatory effect of relatively rapid onset followed by a slowly developing depressant effect (1,2). The results of muscle contraction and electrophysiological studies support the hypothesis that the faeilitatory effects of norepinephrine and epinephrine result from enhanced release of acetylcholine (ACh) from motor nerve terminals (2-4). The ability of phentolamine and phenoxybenzamine, but not pronethalol, to block the facilitatory effects of cateeholamines suggests an interaction involving the ~-adrenoceptor on motor nerve endings (1,4,5). The present study was designed to directly test the hypothesis that norepinephrine increases ACh release from motor nerves. Moreover, using selective ~-adrenoceptor agonists and antagonists, an attempt was made to characterize the ~-adrenoceptor type (~i or ~2) which mediates the stlmulatory effects of norepinephrine on ACh release and on muscle contractions.
IPresent address: Department of Pharmacology, Mayo Medical School, Rochester, Minnesota. ZTo whom correspondence should be addressed. 0024-3205/82/090853-05503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
854
~l-adrenergic Potentiation of ACh Release
Vol. 31, No. 9, 1982
Materials and Methods General Considerations Male albino Sprague-Dawley rats (Lab Supply, Indianapolis, Ind.) weighing 175-300g were used in these studies. Rats were killed with dlethyl ether and, unless otherwise indicated, the left hemidiaphragm and phrenic nerve were removed as described by Bulbrlng (6). Krebs' solution was of the following composition (mM): NaCI, 125; KCI, 5.0; CaCI2, 2.0; MgCI2, 1.0; NaH2PO~, 1.0; NaHCO3, 15; and glucose, ii; the resulting solution was aerated with 95% O2 + CO2 and had a pH = 7.3 ± 0.i. Data are expressed as a percentage of control or predrug baseline. Statistical comparisons were performed on raw data using the Students' t-test (two-tailed) for paired comparisons or by Dunnett's procedure when several treatments were compared to a single control. Values were considered significant if p < 0.05. Isolated phrenic nerve-diaphragms were suspended with 500 mg tension in a 50 ml organ bath containing Krebs' solution which was gassed with 95% O2 and maintained at 32 ± 0.5°C. The phrenic nerve was stimulated with supramaximal 5 V monophaslc square wave pulses of 0.4 msec duration at a frequency of 0.2 Hz for the 45-60 min equilibration period. Muscle contractions were recorded via a FT.03C isometric transducer coupled to a Grass polygraph. Acetylcholine Release Experiments The cannulated phrenic nerve-diaphragm preparation employed to study ACh release was previously described (7,8). The preparation was perfused at room temperature (24 ~ I°C) at a rate of 40 ~i/min with aerated Krebs' solution containing physostigmine (30 ~M) to prevent ACh hydrolysis. The phrenlc nerve was stimulated with 5 V pulses of 0.4 msec duration at a frequency of i0 Hz. A total of six 10-min samples were collected for ACh assay: 2 pre-drug control periods, 2 periods in the presence of amphetamine, and 2 post-drug washout periods, and the results presented represent the average of these 2 intervals for each condition. ACh was quantified employing a radioenzymatic assay (9). In all experiments, blanks and standard amounts of ACh were processed in the same manner as the tissue perfusates, thus generating a standard curve from which the quantity of ACh in the samples was calculated. The practical limit sensitivity of the assay (3-10 pmoles ACh) was twice the CPM measured in blank solutions. Results Norepinephrine (50 ~M) increased nerve-stimulated release of ACh by 183% (Table i). This enhancement was completely antagonized by tissue treatment with phentolamine and the selective ~1-adrenoceptor antagonist WB 4101 but was only modestly reduced by yohimbine (by 27%), ~ a relatively selective ~2antagonist (Table I). When tested alone at the concentrations employed in these studies (i0 BM), these antagonists produced less than a ± 2% change in release. Similarly, clonidine, a selective ~2-agonlst, failed to enhance neurotransmitter release.
3Value derived by the following formula: [ i-- % change by noreplnephrlne + yohimblne ) X 100% % change by norepinephrine
Vol. 31, No. 9, 1982
el-adrenergic Potentiation of ACh Release
855
To further investigate the nature of the presynaptic ~-adrenoceptor hypothesized to mediate the enhancement of ACh release from motor neurons, phenylephrine and clonidine were compared with norepinephrine for their ability to augment nerve-stimulated muscle contractions. Cumulative addition of norepinephrine and the selective ~l-adrenoceptor agonist phenylephrine produced concentration-dependent facilitation of twitch height by up to 22.4 and 19.5%, respectively (Fig. I). By contrast, clonidine failed to augment twitch; inhibition of contractions by 30% was observed at the highest concentration tested.
TABLE I Effects of e-Adrenoceptor Agonists and Antagonists on Acetylcholine Release a Treatment
Norepinephrine (50 ~M) + Phentolamine (I0 ~M) +WB
4101 (i0 ~M)
+ Yohimbine (I0 ~M) Clonidine (30 ~M)
Acetylcholine release
283 ± 42
b
(N )
(ii)
102 ± 7.8
(4)
93.9 ± 7.7
(4)
233 ± 70
b
87.4 ± 16
(4) (3)
l.a,values represent the mean ± S.E.M. compared to pre-drug ACh release, I employing each tissue as its own control, and expressed as a percentage of control. Mean pre-drug ACh release was 4.6 ± 0.4 pmol/min (N=27 preparations). 'IblValues significantly different from control (p < 0.05). '
Discussion In the rat phrenic nerve-diaphragm preparation, norepinephrine (25 ~M) increases the frequency of miniature end-plate potentials 2-fold but not their amplitude; these electrophysiological findings are consistent with enhanced release of neurotransmitter (4). In the present study, norepinephrine (50 ~M) increased ACh release, as determined by radioenzymatic assay, to approximately the same extent. Post- and presynaptic adrenergic receptors have generally been classified as el- and ~-adrenoceptors, respectively (i0,ii). Stimulation of presynaptic ez-receptors at the autonomic neuroeffector junction decreases transmitter release (i0). By contrast, the results of the present study suggest the involvement of a presynaptic el-adrenoceptor which mediates increased neurotransmitter release at the somatic neuromuscular junction. Malta et al. reached a similar conclusion employing experiments which compared the potency of selective e-adrenoceptor agonists and antagonists in modifying neuromuscular transmission with an autonomic vascular response. On the basis of the results of these in vivo experiments in cats, the character-
856
~l-adrenergic Potentiation of ACh Release
,~o
70
Vol. 31, No. 9, 1982
/x-----x
I
I
i
~
I
I
I
_~
,
[~,,ist] (u) FIG. i Effects of norepinephrine ( X ) , phenylephrine ( • ) , and clonidine ( m ) on nerve-stimulated muscle contractions. Drugs were added in a cumulative manner at 5 min intervals. Values represent the mean contraction height of 3 tissues for each treatment. S.E.M. ranged from 0.3-1.9% with exception of clonidine at 10-3M (3.2%).
istics of prejunctional ~-adrenoceptors on somatic motor nerve terminals in potentiating neuromuscular transmission were found to be similar to the postsynaptic ~-adrenoceptors in the blood vessel vasculature, ~.~., ~l-type adrenoceptors. In the present study, the nonselective s-antagonist phentolamine and the selective ~1-antagonist WB 4101 completely blocked noreplnephrlne-enhanced ACh release, whereas release was only slightly decreased by yohimbine. Since yohlmbine antagonizes both ~I- and ~2-adrenoceptors, with only about a 25-fold greater selectivity for ~2-adrenoceptors (12), it is possible that the partial inhibition observed results solely from its ~I antagonistic activity. Our neurochemical findings are further supported by the inability of the selective ~2-agonist clonldine to potentiate acetylcholine release or augment nervestimulated muscle contractions. By contrast, the ~l-agonlst phenylephrine significantly increased contraction height, as did norepinephrlne, a drug with ~I + ~2 agonist properties. Thus, based upon the selective potency differences between clonldlne and phenylephrlne (12,13), it is concluded that those presynaptic receptors enhancing release are of the ~ type.
Vol. 31, No. 9, 1982
~l-adrenergic Potentiation of ACh Release
857
Acknowledgment This work was supported by National Institute of Drug Abuse Grant DA-01477.
References I. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13.
W.C. BOWMAN and C. RAPER, Br. J. Pharmac. 27 313-331 (1966). W.C. BOWMAN, Handbook of Experimental Pharmacology, L. SZEKERES, Ed., 54/11 pp. 47-128, Springer-Verlag, New York (1981). K. KRNJEVlC and R. MILEDI, J. Physiol. 141 291-304 (1958). K. KUBA, J. Physiol. 211 551-570 (1970). E..MAL.TA, G.A. McPHERSON and C. RAPER, Br. J. Pharmac. 6 5 2 4 9 - 2 5 6 (1979). E. BULBRING, Br. J. Pharmac. 1 38-61 (1946). G.G. BIERKAMPER and A.M. GOLDBERG, J. Electrophysiol. Techniques 6 40-46 (1978). R.M. SNIDER and M.C. GERALD, J. Pharmac. Exp. Ther. 221 14-21 (1982). A.M. GOLDBERG and R.E. McCAMAN, J. Neurochem. 20 1-8 (1973). S.Z. LANGER, Br. J. Pharmac. 60 481-497 (1977). S. BERTHELSEN and W.A. PETTINGER, Life Sci. 21 595-606 (1977). R.R. RUFFOLO, JR., E.L. YADEN and J.E. WADDELL, J. Pharmac. Exp. Ther. 213 557-561 (1980). J. WIKBERG, Acta Physiol. Scand 103 225-239 (1978).