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Effect of (±)-DOI on neuromuscular transmission: a microelectrode study
European Journal o/Pharmacology, 195 (1991) 171-174 0 1991 Elsevier Science Publishers B.V. OOW2999/91/$03.50 ADONIS 001429999100278C
171
UP 20795
Short communication
ectof(f)-
on neuromuscular transmission: a Bula J. Bhattacharyya,
Martin
D. Sokoll and John P. Long
Departments of Anesthesia and Pharmacology, College of Medicine.The University of Iowa, Iowa City, IA 52242, U.S.A. Received 18 December 1990, accepted 29 January 1991
DO1 (I-(2,5-dimethoxy-4iodophenyl)-2-aminopropane) significantly depressed end plate current (EPC) amplitude. It decreased quantum content, increased the extent of neurally evoked EPC rundown during the train, produced a nonlinear current-voltage relationship, shortened time constant of decay, and depressed iontophoretically evoked EPC. The depressant response of DO1 on EPC amplitude was antagonized by S-HT,-like receptor antagonists, but was resistant to 5-HT, and 5-HT, receptor antagonists. This suggests that inhibitory 5-HT receptors roughly correspond to 5-HT,-like receptors.
Neuromuscular transmission; 5-HT receptor agonists: DO1 (I-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane); 5-HT,-like receptor antagonists
1. Introduction In central as well as peripheral tissues, serotonin (S-HT) produces profound synaptic actions. The interaction between 5-HT and another major neurotransmitter, acetylcholine (ACh), has been reported for both central and peripheral tissues (Maura and Raiter, 1986; Akasu et al., 1983). Modulation of ACh induced responses by 5-HT at the neuromuscular junction has been well documented (Akasu ei al., 1983; Colomo et al., 1968). In peripheral tissues, at least three types of S-HT receptors have been identified; 5-HT,-like, 5-HT, and 5-HT, (Bradley et al., 1986). We recently observed that three different 5-HT receptor agonists (&OH-DPAT, RU-24969 and DOI (l(2,5-dimethoxy-4-iodophenyl)-2-aminopropane)) mimicked the depressant effect of 5-HT in the neuromuscular junction of frogs: among them, DO1 had the most significant effect (Bhattacharyya et al., 1988b). Radioligand binding studies suggested that DO1 had high affinity for 5-HT, receptors (Ashby et al., 1989). Hence in order to understand the pharmacologic profile of serotonergic involvement and the pre- and postsynaptic effect of 5-HT, we used DO1 as well as 5-HT,-like, 5-HT, and 5-HT, receptor antagonists in our study.
Correspondence to: B.J. Bhattacharyya. Department of Anesthesia, 301 Medical Laboratories, The University of Iowa. Iowa City, IA 52242. U.S.A.
2. Materials and methods All experiments were carried out in vitro with sciatic nerve-sartorius muscle preparation from frogs (Runa @ens). The preparation was mounted in a 1 ml lucite chamber, the bottom of which was coated with Sylgard. The muscle was constantly perfused (3 ml/min) with Ringer solution (mM), NaCl 116, KC1 2.0, CaCl, 1.19, NaH,PO, 1.3 and Na,HPO, 0.7, pH 6.9-7. Glass capillary microelectrodes were filled with 3 M KCI; the tip resistance varied from 7-15 MS2. Miniature end plate currents (MEPCs) and end plate currents (EPCs) were recorded as described by Bhattacharyya et al. (1988a). EPCs were elicited either by stimulating sciatic nerves with rectangular pulses of 0.05 ms duration, or by using an ionophoretic electrode of resistance 30-60 MSZ when filled with 2 M ACh chloride. Iontophoretically induced EPC was monitored on an oscilloscope and recorded with a model 81 ElemaSchonander mingograph. Parameters for stimulations were either a single shock at 0.4 Hz, or tetanic stimulation at 40 Hz for 1 s. We recorded MEPC and EPC by voltage clamping end plates at different membrane potentials ( - 40, - 60. -80, - 100 and - 120 mv). Tetanic trains for nerve evoked and iontophoretically induced EPCs were recorded at -80 mV clamped membrane potential. The extent o!’ neurally evoked EPC rundown during the train was estimated ‘using the ratio of the mean amphtude of 21st to 30th EPC in each train divided by
EPC ;ImpIitu&). The voltage dependence of 7 e) was calculated according to the following
where V is the vaIue of membrane potential, ~(0) is the time constant of EPC decay at 0 mV, and H is the ge in membrane potential for e fold change in the decay time constant. The foBtowing impounds and their receptor type ~Iati~~nsb~p were evaIuated using the concentrations shown: 5-HT: receptor antagonists ketanserin, ICI 169396 and Lv^-53857 (10 nM-100 PM): 5-HT, receptor antagonists MDL-72222 and KS 205-930 (10 nM-100 FM)_ 5-HT,-like receptor antagonists mahysergide. mcs~Ierg~~e and mianserin (50 nM-20 p&l) were tested against the effect of DOI on EPC. Data snaIysis of MEPC and EPC was done using a computer program which determined peak amplitude, rise rime and haIf decay time. The time constant of decay ( 7) was determined by fitting a single exponentiai curve to the decay I(t) = I, e-‘,.JT, where I(t) is the current at time t. I, is the amplitude and 7 is the time constant (that is, the time it takes for I(t) to fall to 36.8% of lthc amplitude 1,).
Fig. I. Efkt af DO1 on end plate sxwren&. (A) Effect on ~~at~on~p between membrane potentiat and peak EPC ampiitude (symbols represent: control, 0; 5 pM. r; 10CM. 0; 20 pM, Cl; and 40 PM, a). (B) Effect on relationship between membrane potential and TEPC (symbols represent: control PM, a). (Cf Effect of DOI on trains neurally evoked (40 Hz) EPC at - 60 mV clamped membrane potentiaIs: fa) controf, vertical bar 100 ti horizontal bar 100 ms: (b) DOI, 10 FM, vertical bar 50 nA, horizontal bar 100 ms. (D) Effect of DOI on trains iontophoretically evoked EPC. 40 Hz at - 80 mV clemped membrane potential (vertical bar, 100 nA; horizontal bar. 100 ms; (a) control; (b) 10 PM, (c) 40
Under voltage clamped conditions. exposure of the end plate to the 5-HT receptor agonist (DOI) produced a s~gn~~cant decrease of MEPC and EPC amplitude and time coastant of decay (7). These effects were concentration dependent. At -SO mV, the KS, values for the inhibition of peak EPC and MEPC amplitudes were 12.7 (10.5-16.3 PM, confidence limits) and 39.9 (31.5-59.7 PM, confidence limits). The IC, values for the decrease of time constant of decays for EPC and MEPCs were 30.7 (25.2-39.5 PM, confidence limits) and 29.7 (26.4-33 PM, confidence limits) respectively. In presence of DOI. EPC amplitude was affected more than the MEPC ampiirude. Quantum content, measured directly, showed a dose dependent decrease after DO1 application. At 10 FM DO1 concentration, quantum content was decreased by 39 f 5% of control. Under control conditions, the relationship between peak MEPC and EPC amplitude vs membrane potential was appro~ma~ely linear. This current voftage linearity was maintained at Iow concent~tions of DOI, but at higher concentrations departure from linearity was observed (fig. 1A). A concentration-dependent shortening of the time constant of decay of EPC and MEPC was observed.
This decay was in single exponentiaIs in presence of DO1 (fig, 1B). A shift in voltage dependence was observed as the cancentration of DO1 was increased (controI,H= -102_7*6mV; DOI4OpM,H=-12125 mV). A rundown in the ampIitude of EPC in a train evoked by 40 Ha stimulation of the sciatic nerve usuaIly produced 15 + 3% of rundown of trains in control. At 10 PM of DO1 concentration, 37.4 f 10% rundown of tetanic trains was observed (fig. 1C). In contrast to its effect on neuraIIy evoked EPC DO1 produced no rundown of trains of iontopboretitally induced EPCs, but simply reduced the amplitude of each EPC in the train compared to the corresponding EPC in the control train (fig. 1D). This postjunctional response of DOI was prominent at higher concentrations (40 FM). When 5-HT receptor antagonists were tested to antagonize DO1 (10 FM) induced depression of EPC (0.4 Hz), both the 5-HT, receptor antagonists (ketanserin, ICI 169,369 and LY-53857; 100 nM-100 FM) and the S-HT, receptor antagonists (MDL-72222 and ICS 205-930; 100 nM-100 PM) were inactive. The 5-HT,-like receptor antagonists (methysergide, mesulergine and mianserin; 50 nM-15 PM) produced significant antagonism against DO1 (fig. 2A,B). This antagonism was more prominent in EPC amplitude
~
Membrane Potcalial ( mV I
173 60
c *8
b
-f
Pig. 2. Antagonism of inhibitory action of DO1 on end plate currents. (A) Percentage antagonism of inhibitory action of DOI (10 pM; caused by different concentrations of methysergide (@). mesukrgine (0) and mianserin (A). 0% is the effect of DOI in the absence of antagonist. and 100% represents the amplitude of EPC. Vertical bar represents+S.E.M. (Bf I~ustration of the depressant effect of DO1 (10 pM) on EPC. and antagonism with different antagonist; (a. control; b, DO1 10 FM: c, methyser~de 12 pM: d. mesulergine 7.5 PM; e. mesulergine 9 FM: c. d and e are recorded in the presence of both DO1 and the antagonist). (C) Illustration of the enhanced depressive effect of 5-HT and DO1 on end plate current (a. control; b. serotonin 50 CM; f, DO1 10 pM). Bach EPC shown represents the average from 10 EPCs; verttcal bar. 200 nA: horizontal bar. 1 ms.
than in T. When serotonin (l-100 CM) was applied along with DOI, an enhanced effect for depression of EPC was observed (fig. 2C).
4. Discussion 5-HT has been found to produce significant depressant effect on frog neuromuscular transmission (Akasu et al., 1983). Our previous electrophysiological study (Bhattacharyya et al., 1988b) also demonstrated that 5-I-IT receptor agonists mimicked the effect of serotonin on MEPCs from the motor end plates of frogs. The rank order of potency for these agonists was DO1 > RU-24969 > 8-OH-DPAT. In our present study, depression of peak EPC amplitude and time constant of decay was observed after DO1 application. The IC, values indicated a greater depression of nerve evoked EPC amplitudes than MEPC amplitude. DO1 produced an increase in rundown of amplitude of EPC when elicited by nerve stimulation and not when evoked by application of ACh iontophoreti~ally to the end plate. Qu~tum content was also decreased. These data indicate that release of ACh induced by presynapnc stimulation is attenuated by DOI. However, at higher concentrations DOI also reduced the time constant of EPC decay. The shift in voltage dependence, the decrease in peak height in EPC amplitude and decay, and the nonlinearity of the current-voltage relationship, along with decreased iontophoretically induced ACh response, suggest a postsynaptic action at higher concentrations of this 5-HT agonist. These depressant effects of DO1 on cholinergic ~eurotransnlission are consistent with the
previously reported depressant effect of S-HT on neuromuscular transmission, in which both pre- and postsynaptic involvement was observed (Akasu et al., 1986). Enhanced responses were obtained when 5-NT was added to DOI, suggesting a possible common site of action. A wide variety of 5-HT receptor antago~sts have been tested to characterize the depressant effect of DO1 on cholinergic neurotransmission. In peripheral tissues, Bradley et al. (1986) subdivided 5-HT receptors into three distinct groups; 5-HT,-like, 5-HT, and 5-HT,. They suggested the following antagonists for different 5-HT subtypes: methysergide and methioth~in (5 HT,-like); ketanserin and cyproheptadine (5-HT& and MDL-72222 and ICS 205-930 (5-HT,). However. SHT,-like receptors have further been subclassified as: 5-HT,, (antagonists spiroperidol and methiotbepin): 5-HT,, ~antag~nists 21-909 and methiothepin~; and 5HT,, (antagonists mesulergine and met~othepin~. Although not all of these drugs could be used in the present investigation, the ineffectiveness of MDL-72222 and ICS 205-930 allows us to exclude the possibility of the involvement of 5-HT, receptors. Since DO1 is known to have high affinity for N-IT, sites, the failure by selective 5-HT, receptor antagonists (ketanserin, ICI 169,369 and LY-53857, antagonists for ketanserin-insensitive sites: Blackburn et al.. 1988; Cohen et al.. 1985) to attenuate the depressant effect of DOI, and effective antagonism by methysergide. mesulergine and mianserin (5I-IT, and 5-HT,, antagonists; Hoyer, 1988) raises questions about the selectivity of DOI as a 5-HT, rather than !I-HT,, receptor agonist (Lyon et al., 1987). Since the DO1 induced depressant effect was mimicked by specific 5-HT, receptor agonists @-OH
~&by. C.R.. E. Edwards. K. Harkins and R.Y. Wang, 1989, Effects .J f f ) DO1 on mudial prefrontal cortical cells: a microiontophoretic study. Brain Res. 498. 393. Bhattacharyya. B.. M.D. Sokoll. J.G. Cannon and J.P. Long, 1988a, Neur~~n~us~uiar blocking action of two hemicholinium-3 analogs. European J. Pharmacol. 146. 155. Bhstrachar>ya. 8.. D. Zwagerman, L.R. Davies. J.P. Long, J.G. Cannon and M.D. Sokoll. 1988h. Effect of serotonergic receptor agonists on miniature end plate currents (MEFCs) of frog neuromuscular junction. FASEB J. 2(4). A 792. Blackburn. T.P., C.W. Thornber. R.J. Pearce and B. Con, 1988. In vitro studies with ICI 169.369, a chemically novel 5-HT antagonist. European J. Pharmacol. 150. 247. Bradley. P.B.. G. Engel. J.R. Feniuk. P.P. Fozard, A. Humphrey. D.N. Middlemi~s. E.J. Mylecharane. B.P. Rochardson and P.R. Saxenr. 1986. Proposal for rhe classification and nomenclature of functional reeptcwsfew S.hydro~yt~ptamine. Neuropharmacol~y 25. 563. Cohen. M.L., W. Cdhert and L.A. Wittenauer. 1985, Receptor specificily of the S-NT, receptor antagonists. LY 53857. Drug Deve!. Res. 5. 313. Hover. D.. 198X. Molecular pha~a~#logv and biology of S-HT,, receptors, Trends Fharmacol. Sci. 9. 89. L:,on. R.A.. K.H. Davies and M. Titeler, 1987, “H-DOB (4.broma_ 2.5-dime~hoxyphenylisopropylamine) labels a guanyl neocleolidesensirive state of cortical 5-HT, receptors. Mol. Pharmacol. 31. 194. Maura. G. and M. Raiter, 1986, Choline@ terminal in rat hippocampus possess 5-HT,, receptors mediating inhibition of acetylcholine release. European J. Pharmacol. 129, 333. ALaw. T.. .X6. Karczmar and K. Kokctasu. 1913. Effects of serotonin ~~-b;d~~~~~~pFarnine) on amphibian neur~~mus~ular junction. Eur&ean‘J. ~Fharmacol. 88. 63.
171
UP 20795
Short communication
ectof(f)-
on neuromuscular transmission: a Bula J. Bhattacharyya,
Martin
D. Sokoll and John P. Long
Departments of Anesthesia and Pharmacology, College of Medicine.The University of Iowa, Iowa City, IA 52242, U.S.A. Received 18 December 1990, accepted 29 January 1991
DO1 (I-(2,5-dimethoxy-4iodophenyl)-2-aminopropane) significantly depressed end plate current (EPC) amplitude. It decreased quantum content, increased the extent of neurally evoked EPC rundown during the train, produced a nonlinear current-voltage relationship, shortened time constant of decay, and depressed iontophoretically evoked EPC. The depressant response of DO1 on EPC amplitude was antagonized by S-HT,-like receptor antagonists, but was resistant to 5-HT, and 5-HT, receptor antagonists. This suggests that inhibitory 5-HT receptors roughly correspond to 5-HT,-like receptors.
Neuromuscular transmission; 5-HT receptor agonists: DO1 (I-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane); 5-HT,-like receptor antagonists
1. Introduction In central as well as peripheral tissues, serotonin (S-HT) produces profound synaptic actions. The interaction between 5-HT and another major neurotransmitter, acetylcholine (ACh), has been reported for both central and peripheral tissues (Maura and Raiter, 1986; Akasu et al., 1983). Modulation of ACh induced responses by 5-HT at the neuromuscular junction has been well documented (Akasu ei al., 1983; Colomo et al., 1968). In peripheral tissues, at least three types of S-HT receptors have been identified; 5-HT,-like, 5-HT, and 5-HT, (Bradley et al., 1986). We recently observed that three different 5-HT receptor agonists (&OH-DPAT, RU-24969 and DOI (l(2,5-dimethoxy-4-iodophenyl)-2-aminopropane)) mimicked the depressant effect of 5-HT in the neuromuscular junction of frogs: among them, DO1 had the most significant effect (Bhattacharyya et al., 1988b). Radioligand binding studies suggested that DO1 had high affinity for 5-HT, receptors (Ashby et al., 1989). Hence in order to understand the pharmacologic profile of serotonergic involvement and the pre- and postsynaptic effect of 5-HT, we used DO1 as well as 5-HT,-like, 5-HT, and 5-HT, receptor antagonists in our study.
Correspondence to: B.J. Bhattacharyya. Department of Anesthesia, 301 Medical Laboratories, The University of Iowa. Iowa City, IA 52242. U.S.A.
2. Materials and methods All experiments were carried out in vitro with sciatic nerve-sartorius muscle preparation from frogs (Runa @ens). The preparation was mounted in a 1 ml lucite chamber, the bottom of which was coated with Sylgard. The muscle was constantly perfused (3 ml/min) with Ringer solution (mM), NaCl 116, KC1 2.0, CaCl, 1.19, NaH,PO, 1.3 and Na,HPO, 0.7, pH 6.9-7. Glass capillary microelectrodes were filled with 3 M KCI; the tip resistance varied from 7-15 MS2. Miniature end plate currents (MEPCs) and end plate currents (EPCs) were recorded as described by Bhattacharyya et al. (1988a). EPCs were elicited either by stimulating sciatic nerves with rectangular pulses of 0.05 ms duration, or by using an ionophoretic electrode of resistance 30-60 MSZ when filled with 2 M ACh chloride. Iontophoretically induced EPC was monitored on an oscilloscope and recorded with a model 81 ElemaSchonander mingograph. Parameters for stimulations were either a single shock at 0.4 Hz, or tetanic stimulation at 40 Hz for 1 s. We recorded MEPC and EPC by voltage clamping end plates at different membrane potentials ( - 40, - 60. -80, - 100 and - 120 mv). Tetanic trains for nerve evoked and iontophoretically induced EPCs were recorded at -80 mV clamped membrane potential. The extent o!’ neurally evoked EPC rundown during the train was estimated ‘using the ratio of the mean amphtude of 21st to 30th EPC in each train divided by
EPC ;ImpIitu&). The voltage dependence of 7 e) was calculated according to the following
where V is the vaIue of membrane potential, ~(0) is the time constant of EPC decay at 0 mV, and H is the ge in membrane potential for e fold change in the decay time constant. The foBtowing impounds and their receptor type ~Iati~~nsb~p were evaIuated using the concentrations shown: 5-HT: receptor antagonists ketanserin, ICI 169396 and Lv^-53857 (10 nM-100 PM): 5-HT, receptor antagonists MDL-72222 and KS 205-930 (10 nM-100 FM)_ 5-HT,-like receptor antagonists mahysergide. mcs~Ierg~~e and mianserin (50 nM-20 p&l) were tested against the effect of DOI on EPC. Data snaIysis of MEPC and EPC was done using a computer program which determined peak amplitude, rise rime and haIf decay time. The time constant of decay ( 7) was determined by fitting a single exponentiai curve to the decay I(t) = I, e-‘,.JT, where I(t) is the current at time t. I, is the amplitude and 7 is the time constant (that is, the time it takes for I(t) to fall to 36.8% of lthc amplitude 1,).
Fig. I. Efkt af DO1 on end plate sxwren&. (A) Effect on ~~at~on~p between membrane potentiat and peak EPC ampiitude (symbols represent: control, 0; 5 pM. r; 10CM. 0; 20 pM, Cl; and 40 PM, a). (B) Effect on relationship between membrane potential and TEPC (symbols represent: control PM, a). (Cf Effect of DOI on trains neurally evoked (40 Hz) EPC at - 60 mV clamped membrane potentiaIs: fa) controf, vertical bar 100 ti horizontal bar 100 ms: (b) DOI, 10 FM, vertical bar 50 nA, horizontal bar 100 ms. (D) Effect of DOI on trains iontophoretically evoked EPC. 40 Hz at - 80 mV clemped membrane potential (vertical bar, 100 nA; horizontal bar. 100 ms; (a) control; (b) 10 PM, (c) 40
Under voltage clamped conditions. exposure of the end plate to the 5-HT receptor agonist (DOI) produced a s~gn~~cant decrease of MEPC and EPC amplitude and time coastant of decay (7). These effects were concentration dependent. At -SO mV, the KS, values for the inhibition of peak EPC and MEPC amplitudes were 12.7 (10.5-16.3 PM, confidence limits) and 39.9 (31.5-59.7 PM, confidence limits). The IC, values for the decrease of time constant of decays for EPC and MEPCs were 30.7 (25.2-39.5 PM, confidence limits) and 29.7 (26.4-33 PM, confidence limits) respectively. In presence of DOI. EPC amplitude was affected more than the MEPC ampiirude. Quantum content, measured directly, showed a dose dependent decrease after DO1 application. At 10 FM DO1 concentration, quantum content was decreased by 39 f 5% of control. Under control conditions, the relationship between peak MEPC and EPC amplitude vs membrane potential was appro~ma~ely linear. This current voftage linearity was maintained at Iow concent~tions of DOI, but at higher concentrations departure from linearity was observed (fig. 1A). A concentration-dependent shortening of the time constant of decay of EPC and MEPC was observed.
This decay was in single exponentiaIs in presence of DO1 (fig, 1B). A shift in voltage dependence was observed as the cancentration of DO1 was increased (controI,H= -102_7*6mV; DOI4OpM,H=-12125 mV). A rundown in the ampIitude of EPC in a train evoked by 40 Ha stimulation of the sciatic nerve usuaIly produced 15 + 3% of rundown of trains in control. At 10 PM of DO1 concentration, 37.4 f 10% rundown of tetanic trains was observed (fig. 1C). In contrast to its effect on neuraIIy evoked EPC DO1 produced no rundown of trains of iontopboretitally induced EPCs, but simply reduced the amplitude of each EPC in the train compared to the corresponding EPC in the control train (fig. 1D). This postjunctional response of DOI was prominent at higher concentrations (40 FM). When 5-HT receptor antagonists were tested to antagonize DO1 (10 FM) induced depression of EPC (0.4 Hz), both the 5-HT, receptor antagonists (ketanserin, ICI 169,369 and LY-53857; 100 nM-100 FM) and the S-HT, receptor antagonists (MDL-72222 and ICS 205-930; 100 nM-100 PM) were inactive. The 5-HT,-like receptor antagonists (methysergide, mesulergine and mianserin; 50 nM-15 PM) produced significant antagonism against DO1 (fig. 2A,B). This antagonism was more prominent in EPC amplitude
~
Membrane Potcalial ( mV I
173 60
c *8
b
-f
Pig. 2. Antagonism of inhibitory action of DO1 on end plate currents. (A) Percentage antagonism of inhibitory action of DOI (10 pM; caused by different concentrations of methysergide (@). mesukrgine (0) and mianserin (A). 0% is the effect of DOI in the absence of antagonist. and 100% represents the amplitude of EPC. Vertical bar represents+S.E.M. (Bf I~ustration of the depressant effect of DO1 (10 pM) on EPC. and antagonism with different antagonist; (a. control; b, DO1 10 FM: c, methyser~de 12 pM: d. mesulergine 7.5 PM; e. mesulergine 9 FM: c. d and e are recorded in the presence of both DO1 and the antagonist). (C) Illustration of the enhanced depressive effect of 5-HT and DO1 on end plate current (a. control; b. serotonin 50 CM; f, DO1 10 pM). Bach EPC shown represents the average from 10 EPCs; verttcal bar. 200 nA: horizontal bar. 1 ms.
than in T. When serotonin (l-100 CM) was applied along with DOI, an enhanced effect for depression of EPC was observed (fig. 2C).
4. Discussion 5-HT has been found to produce significant depressant effect on frog neuromuscular transmission (Akasu et al., 1983). Our previous electrophysiological study (Bhattacharyya et al., 1988b) also demonstrated that 5-I-IT receptor agonists mimicked the effect of serotonin on MEPCs from the motor end plates of frogs. The rank order of potency for these agonists was DO1 > RU-24969 > 8-OH-DPAT. In our present study, depression of peak EPC amplitude and time constant of decay was observed after DO1 application. The IC, values indicated a greater depression of nerve evoked EPC amplitudes than MEPC amplitude. DO1 produced an increase in rundown of amplitude of EPC when elicited by nerve stimulation and not when evoked by application of ACh iontophoreti~ally to the end plate. Qu~tum content was also decreased. These data indicate that release of ACh induced by presynapnc stimulation is attenuated by DOI. However, at higher concentrations DOI also reduced the time constant of EPC decay. The shift in voltage dependence, the decrease in peak height in EPC amplitude and decay, and the nonlinearity of the current-voltage relationship, along with decreased iontophoretically induced ACh response, suggest a postsynaptic action at higher concentrations of this 5-HT agonist. These depressant effects of DO1 on cholinergic ~eurotransnlission are consistent with the
previously reported depressant effect of S-HT on neuromuscular transmission, in which both pre- and postsynaptic involvement was observed (Akasu et al., 1986). Enhanced responses were obtained when 5-NT was added to DOI, suggesting a possible common site of action. A wide variety of 5-HT receptor antago~sts have been tested to characterize the depressant effect of DO1 on cholinergic neurotransmission. In peripheral tissues, Bradley et al. (1986) subdivided 5-HT receptors into three distinct groups; 5-HT,-like, 5-HT, and 5-HT,. They suggested the following antagonists for different 5-HT subtypes: methysergide and methioth~in (5 HT,-like); ketanserin and cyproheptadine (5-HT& and MDL-72222 and ICS 205-930 (5-HT,). However. SHT,-like receptors have further been subclassified as: 5-HT,, (antagonists spiroperidol and methiotbepin): 5-HT,, ~antag~nists 21-909 and methiothepin~; and 5HT,, (antagonists mesulergine and met~othepin~. Although not all of these drugs could be used in the present investigation, the ineffectiveness of MDL-72222 and ICS 205-930 allows us to exclude the possibility of the involvement of 5-HT, receptors. Since DO1 is known to have high affinity for N-IT, sites, the failure by selective 5-HT, receptor antagonists (ketanserin, ICI 169,369 and LY-53857, antagonists for ketanserin-insensitive sites: Blackburn et al.. 1988; Cohen et al.. 1985) to attenuate the depressant effect of DOI, and effective antagonism by methysergide. mesulergine and mianserin (5I-IT, and 5-HT,, antagonists; Hoyer, 1988) raises questions about the selectivity of DOI as a 5-HT, rather than !I-HT,, receptor agonist (Lyon et al., 1987). Since the DO1 induced depressant effect was mimicked by specific 5-HT, receptor agonists @-OH
~&by. C.R.. E. Edwards. K. Harkins and R.Y. Wang, 1989, Effects .J f f ) DO1 on mudial prefrontal cortical cells: a microiontophoretic study. Brain Res. 498. 393. Bhattacharyya. B.. M.D. Sokoll. J.G. Cannon and J.P. Long, 1988a, Neur~~n~us~uiar blocking action of two hemicholinium-3 analogs. European J. Pharmacol. 146. 155. Bhstrachar>ya. 8.. D. Zwagerman, L.R. Davies. J.P. Long, J.G. Cannon and M.D. Sokoll. 1988h. Effect of serotonergic receptor agonists on miniature end plate currents (MEFCs) of frog neuromuscular junction. FASEB J. 2(4). A 792. Blackburn. T.P., C.W. Thornber. R.J. Pearce and B. Con, 1988. In vitro studies with ICI 169.369, a chemically novel 5-HT antagonist. European J. Pharmacol. 150. 247. Bradley. P.B.. G. Engel. J.R. Feniuk. P.P. Fozard, A. Humphrey. D.N. Middlemi~s. E.J. Mylecharane. B.P. Rochardson and P.R. Saxenr. 1986. Proposal for rhe classification and nomenclature of functional reeptcwsfew S.hydro~yt~ptamine. Neuropharmacol~y 25. 563. Cohen. M.L., W. Cdhert and L.A. Wittenauer. 1985, Receptor specificily of the S-NT, receptor antagonists. LY 53857. Drug Deve!. Res. 5. 313. Hover. D.. 198X. Molecular pha~a~#logv and biology of S-HT,, receptors, Trends Fharmacol. Sci. 9. 89. L:,on. R.A.. K.H. Davies and M. Titeler, 1987, “H-DOB (4.broma_ 2.5-dime~hoxyphenylisopropylamine) labels a guanyl neocleolidesensirive state of cortical 5-HT, receptors. Mol. Pharmacol. 31. 194. Maura. G. and M. Raiter, 1986, Choline@ terminal in rat hippocampus possess 5-HT,, receptors mediating inhibition of acetylcholine release. European J. Pharmacol. 129, 333. ALaw. T.. .X6. Karczmar and K. Kokctasu. 1913. Effects of serotonin ~~-b;d~~~~~~pFarnine) on amphibian neur~~mus~ular junction. Eur&ean‘J. ~Fharmacol. 88. 63.