OOZE-3908/79/0401-0415602.00/0
COOPERATIVE INTERACTIONS OF HISTAMINE AND COMPETITIVE ANTAGONISM BY CIMETIDINE AT NEURONAL HISTAMINE RECEPTORS IN THE MARINE MOLLUSC, APLYSIA CALIFORNICA DONNA L. GRUOL* and D. WEINREICH University of Maryland School of Medicine, Department of Pharmacology and Experimental Therapeutics, 660 West Redwood Street, Baltimore, MD 21201, U.S.A. (Accepted
12 October
1978)
Summary-Dose-response curves and Hill plots were used to analyze the interaction between histamine (HA), two HA agonists and an HA antagonist, and the HA receptors mediating membrane hyperpolarizations in Apl~Ga neurons. Dose-response curves for both HA and the HA agonists (Zmethylhistamine and 4-methylhistamine) were sigmoidal and the corresponding Hill plots had slopes greater than one (mean = 2). This result is interpreted as evidence for a cooperative type of interaction between HA and HA agonists and the neuronal HA receptors. Cimetidine (10m6 to 4 x 10m6M), an HA antagonist, produced reversible. dose-dependent, parallel shifts in the HA dose-response curves. Modified Schild plots constructed from this data were linear. These results demonstrate that cimetidine is a competitive antagonist at the neuronal HA receptors. The slope of the modified Schild plots, which gives an estimate of the minimum number of antagonist molecules that interact with each receptor, was greater than one (mean = 1.7). This result is interpreted as evidence for a cooperative type of interaction between cimetidine and the neuronal HA receptors.
Much neurochemical evidence has accumulated to suggest that histamine (HA) may function as a neurotransmitter in vertebrate (Green, 1970; Snyder and Taylor, 1972; Schwartz, 1975) and invertebrate (Weinreich, 1978) nervous systems. Physiological evidence has recently been reported for histaminergic synaptic transmission at identified synapses within the CNS of the marine mollusc Aplysin caltjbrnica (Weinreich, 1977; see also McCaman and McKenna, 1978). The transmitter function for HA at these identified synapses might further be established if the kinetic properties of the postsynaptic receptors activated by the natural transmitter were shown to be similar to the properties of HA-receptor interactions. When a transmitter substance interacts with its postsynaptic receptors, a primary event following receptor activation is a change in the ionic conductance of the postsynaptic membrane. Werman (1969) and other investigators (see Triggle and Triggle, 1976) have shown that Hill plots or log-log plots of transmitter doseresponse data provide useful information on the behavior of transmitter-receptor interactions in situ. Further insights about the transmitter-receptor interactions are obtained from studies describing the actions of antagonist drugs with transmitter receptors. This quantitative information may be particularly useful for subsequent comparisons between a suspected transmitter and the natural transmitter.
Although little quantitative information is currently available concerning the nature of HA-receptor interactions on neuronal membranes, it has been observed that the neurons within the A-cluster of the cerebral ganglia of Aplysia respond to exogenously applied HA with mono- or biphasic hyperpolarizing responses (Gruol and Weinreich, 1979). These responses are generated by conductance increases to specific ions and they are selectively blocked by certain pharmacological antagonists. Furthermore, these HA responses have ionic and pharmacological properties similar to the two-component hyperpolarizing synaptic potentials evoked by stimulating presynaptic HAcontaining neurons in the cerebral ganglion (Weinreich, 1977; and unpublished). Since the A-cluster neurons are relatively large (> 15Opm) cells and readily accessible for electrophysiological investigations, they provide a favorable model to begin characterizing the stoichiometric relations of HA-receptor interactions on neuronal membranes. In the present work, it is shown that: (1) HA and 2 HA agonists interact with neuronal HA receptors in a cooperative manner; (2) the HA antagonist, cimetidine, is a competitive antagonist at these same receptors; and (3) cimetidine interacts with neuronal HA receptors in a cooperative manner. METHODS
* Present address: NINCDS, NIH, Bldg. 36, Rm. 2CO7. Bethesda, MD 20014, U.S.A. Key words: histamine neuronal receptors, histamine agonist and antagonists, molluscan central neurons,
Aplysia californica (150-500 g) were obtained from Pacific Biomarine Supply Co., Venice, CA, and maintained in a marine holding tank at 16°C. Cerebral ganglia were removed from the animals and pinned,
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DONNA L. GRUOL and D. WEINREICH
dorsal side up, to the silastic (Sylgard 184, Dow Corning Corp., Midland, MI) base of a 2ml Plexiglas recording chamber containing artificial seawater (ASW) of the following composition (mM): NaCl, 494; KCI, 11; MgC12, 19; MgSO.+, 30; CaCl,, 11, Tris, 10. During experiments, the ASW level was lowered to a minimum bath volume (-0.5 ml) and the chamber continuously perfused with ASW. In some experiments, ASW containing 4 times normal Mg*+ and one-third normal Ca’ + was used to ensure that drug responses were a result of activation of receptors on the impaled neuron and not due to activation of an interneuron, and also to abolish spontaneous synaptic activity that occasionally interfered with recording of drug responses. The pH of all perfusion solutions was 7.8. Perfusion rates ranged from 1 to 5 ml/min depending on the type of experiment. All experiments were performed on an identifiable group of neurons designated the A-cluster neurons of the cerebral ganglia (Jahan-Parwar and Fredman, 1976). These neuronal clusters are symmetrically located on the dorsal surface at the entrance of the cerebral-pleural connectives (see Figs 1 in Weinreich, McCaman, McCaman and Vaughn, 1973, and in Jahan-Parwar and Fredman, 1976). The connective tissue capsule surrounding the neurons was removed to facilitate placement of the recording and iontophoretic microelectrodes. Neurons were impaled with two separate K+-citrate (2M, acidified to pH 6.5 with citric acid)-filled glass microelectrodes (15-30 Ma), one electrode for voltage recording and one for current injection. The recording electrode was connected through a unity gain electrometer to a Tektronix Type 5103 oscilloscope and to a Brush 2400 pen recorder from which all responses were analyzed. Current injection was used to vary membrane potential and to monitor changes in membrane input resistance. Currents (iontophoretic and transmembrane) were measured with an operational amplifier which held the bath potential at a virtual ground. The inverting input of the amplifier (Analog Devices, 515) was connected to the bath by a Ag/AgCl wire via an ASW-agar bridge and the noninverting input was grounded. Experiments were performed year round on 37 neurons from 16 animals. Resting potentials ranged from -40 to - 82 mV with a mean value of 61.8 f 7.3 mV. All quantitative data is expressed as mean + S.D.; N = number of neurons tested. Application of agonists and antagonists For the majority of studies, histamine (HA 0.5 M, pH 5.0) was applied by iontophoresis from fine-tipped (< 1 pm tip diameter) glass microelectrodes. Iontophoretic currents were generated by WPI microiontophoresis programmer units (Model 160:WPI Instruments, Inc., New Haven, Conn.) and typically ranged from 10 to lOOnA for control experiments and 20 to 300 nA when cimetidine was present. When current pulses were not rectangular, the tip of the pipette was broken off by gentle bumping on connective tissue.
Drug leakage was prevented by applying 5 to 1OnA holding currents to the iontophoretic pipettes. During experiments the polarity of the iontophoretic current was occasionally reversed to test for current artifacts. The duration of the iontophoretic current (typically 100 to 1OOOmsec) was set so that the response for all HA doses reached a steady value before declining. The interval between each HA application was set so that consistent responses were evoked for a fixed iontophoretic pulse of HA. For superfusion studies, HA or HA agonists were dissolved in ASW (10-8-10-3M) and applied as a 1 or 2 ml pulse (< 30 set duration) through the perfusion system. Several minutes of ASW wash followed each application. A six-way switching manifold enabled rapid changes of solutions without significant alterations in the perfusion rate. Under these conditions consistent responses could be obtained for a fixed concentration of agonist. Prolonged applications (> 1 min) of agonists were avoided to prevent possible desensitization effects. The HA antagonist, cimetidine, was applied by continuous perfusion for a minimum of 2min before its effect on agonist responses was assessed. When agonists were also applied by superfusion, they were dissolved in ASW plus antagonist and applied as described above. The chamber was thoroughly washed with drug-free ASW after each series of tests with a fixed concentration of cimetidine. Cimetidine had little (< 5%) or no discernible effect on membrane input resistance in the range of concentrations tested (1O-7-1O-5 M). Dose-response data In electrophysiological studies, drug-receptor interactions are commonly measured as a change in membrane conductance or a change in membrane potential; conductance is the more direct measurement since membrane potential changes are a function of the conductance change, the driving force for the ionic currents, and the time constant of the membrane. Thus, in an initial series of experiments it was determined whether voltage changes sufficiently reflected the underlying conductance changes to be useful for analysis of drug-receptor interactions. For these studies, the change in membrane conductance was calculated from membrane input resistance measurements made before and at the peak of the voltage response by passing constant hyperpolarizing current pulses. In all neurons tested, the peak amplitude of the voltage response varied as a linear function of the underlying conductance change over a wide range of drug concentrations. A deviation from linearity occurred most commonly at high agonist concentrations when large responses were evoked (Fig. lB,). This nonlinearity did not appear, however, to alter the interpretation of the data because Hill plots constructed from voltage data were similar to plots constructed from conductance data (Fig. 1B3). From these initial experiments it was concluded that
HA receptors on mohuscan neurons
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Fig. 1. Sample records showing responses of A-cluster neurons to increasing doses of HA applied by superfusion (A) or iontophoresis (B,). Downward deflections occurring at regular intervals are electrotonic potentials evoked by constant, hyperpolarizing current pulses which were used to measure changes in membrane conductance, Magnitudes of iontophoretic current shown on line I. The last trace in Br shows a response to HA when the membrane was polarized to -82 mV, the approximate membrane potential reached by the largest HA response in this series. The observed HA-induced hyperpolarization at a preset membrane potential of -82 mV indicates that the plateau of the HAinduced hy~~olari~tions is not a consequence of voltage saturation. B,, Graph showing the relationship between the change in membrane potential (v) vs the change in membrane conductance (g) measured at the peak of the HA response for increasing doses of HA. Both measurements (v and g) are expressed as a fraction of the value obtained for the maximum HA response (Vmax and Gmax). Same cell as Bt. Ba, Hill plot of data shown in B2. The slope of the line drawn through the experimental points was the same (slope = 1.7) whether the response was expressed as a change in membrane voltage (0) or a change in membrane conductance (0). Current-voltage curves were linear in the region where responses were measured.
DONNA L. GRUOL and D. WEINREICH
418
voltage changes could be used for analyzing the interaction between HA and HA receptors on A-cluster neurons. For most A-cluster neurons, the maximum HA response (typically ~25 mV) polarized the membrane to a level which was 5 to 10mV more positive than the reversal potential for the response (h - 89 mV; see Gruol and Weinreich, 1978). When the maximum HA response was closer to the reversal potential, constant depolarizing current was applied before doseresponse data was obtained to bring the membrane potential further from the reversal potential. This insured that the maximum response represented a saturation of the HA receptors and not a saturation of the response (Fig. 1Br). Current-voltage curves for A-cluster neurons were linear in the hyperpolarizing direction where measurements of agonists responses were made (i.e. - 55 to -90 mV). Drug source Cimetidine, 2-methylhistamine and 4-methylhistamine were gifts from Smith, Kline & French and their generosity is gratefully acknowledged. Histamine-HCl was purchased from Sigma Chemical Corp. (St Louis, MO). RESULTS
Dose-response curves: HA and HA agonists The most common response of A-cluster neurons to exogenously applied HA (iontophoresis or superfusion) was a slowly developing hyperpolarization mediated by an increased K+ conductance (Fig. lA, B,; Gruol and Weinreich, 1978). The amplitude of the response varied as a function of applied HA (Fig. lA, B,) and dose-response curves (iontophoretic current or molar concentration vs change in membrane Table
1. Comparisons
Drugs
and method
A. Histamine of auulication
B. Control of application
Histamine-Iontophoresis Control Cimetidine (10-6-10-5 M) Histamine-Superfusion Control Cimetidine (10e6-2 x 10e6 M) C. Cimetidine Analysis Modified schild plot? Los-log plot
for histamine, cimetidine
and histamine agonists No. of cells
2-Methylhistamine-superfusion 4-Methylhistamine-superfusion and method
tervals are electrotonic potentials produced by constant, hyperpolarizing current pulses. (B) Hill plot of data shown in (A). The slope of the line drawn through the experimental points is 2.0.
of n or m values obtained or the histamine antagonist,
Histamine-iontophoresis Histamine-superfusion
Drugs
Fig. 2(A) Sample record of responses of an A-cluster neuron to the HA agonist, 4-MeHA, applied by superfusion. Similar responses were obtained with 2-MeHA (data not shown). Downward deflections occurring at regular in-
histamine
n
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No. of trials
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* Values represent means + SD. t Modified Schild plot: log [(Dose ratio)” - l] vs log cimetidine concentration where n equals slope of Hill plot for histamine interaction with histamine receptors.
419
HA receptors on molluscan neurons
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Fig. 3. Effect of cimetidine on HA log dose-response curves (A, and B,) and corresponding Hill plots (A, and BZ). A,. HA applied by iontophoresis. Data shown are mean values from 2 cells. A,, Slope of Hill plot = 1.6. B,, HA applied by superfusion. Data shown are mean values from 2 cells. Bz, Slope of Hill plot = 1.3. potential) were sigtnoidal (data not shown). When Hill plots were constructed from dose-response data, the slope of the line (n) drawn through the experimental points was greater than 1 (Figs IB,, 3A, and 3B,). Values for n ranged from 1.2 to 2.7 with a mean of 1.8 f 0.4 (N = 30) when HA was applied by iontophoresis and 1.7 f 5 (N = 11) when HA was applied by superfusion (Table 1). Similar values for n were obtained when the response was measured as the change in membrane conductance (Fig. 1B3; see Methods). The HA agonists, 2-methylhistamine Qmethylhistamine (4MeHA), (2-MeHA) and although less potent than HA also produced dosedependent, K+-mediated membrane hyperpolarizations in the A-cluster neurons (Fig. 2A; Gruol and Weinreich, 1978). Dose-response curves for these agonists were also sigmoidal and the slope of Hill
plots were greater than 1 (Fig. 2B). Mean values for n were 1.9 + 0.3 (N = 6) for 2-MeHA and 1.9 k 0.3 (N = 8) for 4-MeHA (Table 1A). These data indicate thatr (1) 2 or more molecules of HA interact with each neuronal HA receptor to produce the observed voltage change; and (2) the HA agonists, 2-MeHA and 4-MeHA, interact with the receptor in a manner similar to HA. HA dose-response curves: effect of cimetidine It has previously been reported that cimetidine, a HA antagonist, specifically and reversibly blocks the K+-mediated hyperpolarizations evoked by HA or HA agonists in A-cluster neurons (Gruol and Weinreich, 1978). In the present series of experiments, doseresponse data has been utilized to study the type of interaction between cimetidine and neuronal HA
DONNA L. GRUOL
420
receptors. When applied at 10m6M concentrations, cimetidine produced parallel shifts in the HA log dose-response curves and in the Hill plots (Fig. 3). Mean values for n, the slope of the Hill plot, were similar for control data and data obtained in the presence of cimetidine (Table 1B). At higher cimetidine concentrations, parallel shifts in the HA log dose-response curves were also observed, but frequently the maximum response was somewhat less than the control maximum (data not shown). These experiments suggest that, at low concentrations, cimetidine is a competitive antagonist at neuronal HA receptors on the A-cluster neurons. The competitive action of cimetidine was more rigorously tested by construction of Schild plots from dose-response data @child, 1957). For this analysis, the dose ratio (taken at 50% of the maximum response) was raised to a power equal to n (the slope of the Hill plot) in order to take into account the cooperative interaction between the HA molecules and the HA receptors*. As shown in Figure 4A, the modified Schild plots are linear, a characteristic which is interpreted as indicative of a competitive type of interaction @child, 1957). The slope of the Schild plot (m) is analogous to the slope of the Hill plot (n) and gives an estimate of the minimum number of antagonist molecules which interact with each receptor to produce the observed inhibition. In 4 neurons where this analysis was successfully carried out, m was greater than 1. The mean value of m for these 4 neurons was 1.7 f 0.3 which is similar to that obtained for n, the estimate for the minimum number of HA or HA agonist molecules which- interact with each HA receptor (Table 1). An m value greater than 1 was further confirmed in two additional experiments where dose-response curves were constructed by varying the cimetidine concentration while holding the HA dose constant. The limiting slope of log-log plots (change in amplitude of HA response vs cimetidine concentration) gives an estimate of m (Werman, 1969). For both neurons tested m was greater than 1 (mean = 1.7; Fig. 4B; Table IC). DISClJS.SION
If it is assumed that the relationship between drugreceptor activation and the measured response is linear, dose-response data and Hill plots can be used to analyze the interaction between drug molecules and their receptors (see reviews by Waud, 1968; Werman, 1969; Triggle and Triggle, 1976). When dose-response curves are sigmoidal and Hill plots have a slope greater than 1, the interaction is thought to be cooperative and the value of n, the slope of the Hill plot, is indicative of the minimum number of drug molecules which interact with the receptor to produce *The authors thank Dr Neville Brookes for pointing out the necessity for this modification.
and D.
WEINREICH
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B
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cimetidine
X,O-‘M
Fig. 4(A) Modified Schild plot. DR = dose ratio taken at 50% of the maximum response. n = Hill slope. HA applied by iontophoresis. Slope of the modified Schild plot = 2. (B) Log-log plot showing the relationship between the de-’ crease in amplitude of the control HA response in the presence of cimetidine vs the cimetidine concentration. HA applied by superfusion at 5 x 10m6M. Control values were measuted before and after each cimetidine dose. Limiting slope = 1.6.
the response (Brookes and Werman, 1973). Cooperative interactions between putative neurotransmitters and their receptors have been demonstrated using electrophysiological techniques in a variety of vertebrate and invertebrate preparations including molluscan neurons (Katz and Thesleff, 1957; Takeuchi and Takeuchi, 1969; Brookes and Werman, 1973; Diamond and Roper, 1973; Oomura, Ooyama and Sawada, 1974; Dudel, 1975, 1977; Hartzell, Kuffler and Yoshikami, 1975; Ziskind and Werman, 1975; McLennan and Wheal, 1976; Constanti, 1977). The present authors’ data indicates that a cooperative type
HA receptors on molluscan neurons
of interaction also occurs between the putative neurotransmitter HA and neuronal HA receptors. When agonists are applied by iontophoresis, interpretation of dose-response data may be complicated by nonlinearity of the current ejected from the iontophoretic pipette and/or uncertainties as to the equivalence of the receptor population which is exposed to low vs high doses of the drug. In order to rule out these possible sources of error, an additional series of experiments were initiated where HA was applied by superfusion. The value of n, the Hill slope, for data obtained by superfusion of HA agreed closely with that obtained by iontophoresis of HA. Thus, it is unlikely that technical errors can account for observed cooperativity. It has previously been shown that cimetidine specifically blocks the K+-mediated HA response in A-cluster neurons but the type of antagonism was not characterized (Gruol and Weinreich, 1979). In the present study, it has been shown that cimetidine is a competitive antagonist at these neuronal HA receptors by two commonly used criteria: (1) a parallel shift in the HA dose-response curve, and (2) a linear Schild plot. The data also indicates that 2 or more molecules of cimetidine interact with each HA receptor to produce the observed inhibition. Considering the close similarity in structure between HA and cimetidine (Brimblecombe, Ducan, Durant, Emmett, Ganellin and Parsons, 1975) it is not surprising that a cooperative type of interaction occurs for both agonist and antagonist at the HA receptors. Acknowledgrments-The authors wish to thank Drs N. Brookes and T. Smith for critical reading of this manuscript. This work was supported by NSF grant (BNS 77-13034) to D. W. and by Smith, Kline & French Co. REFERENCES
Brimblecombe, R. W., Ducan, W. A. M., Durant, G. J., Emmett, J. C., Ganellin, C. R. and Parsons, M. E. (1975). A non-thiourea H,-receptor antagonist. J. inc. Med. Res. 312: 86-92. Brookes, N. and Werman, R. (1973). The cooperativity of y-aminobutyric acid action on the membrane of locust muscle fibers. Molec. Pharmac. 9: 571-579. Constanti, A. (1977). A quantitative study of the y-aminobutyric acid (GABA) dose conductance relationship at the lobster inhibitory neuromuscular junction. Neuropharmacology 16: 351-366. Diamond, J. and Roper, S. (1973). Analysis of Mauthner cell response to iontophoretically delivered pulses of GABA, glycine and L-glutamate. J. Physiol., Lond. 232: 113-128. Dudel, J. (1975). Kinetics of postsynaptic action of glutamate pulses applied iontophoretically through high resistance micropipettes. Pfliigers Arch. ges. Physiol. 3% 329-346.
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Dudel, J. (1977). Dose-response curves of glutamate applied by superfusion to crayfish muscle synapses. Pjiigers Arch. ges. Physiol. 36% 49-54. Green, J. P. (1970). Histamine. In: Handbook of Neurochemistry (Lajtha, A., Ed.), pp. 221-250. Plenum Press, New York. Gruel, D. L. and Weinreich, D. (1979). Two pharmacologically distinct histamine receptors mediating membrane hyperpolarization on identified neurons of Aplysia californica. Brain Res. In press. Hartzelf, H. C., Kuffler, S. W. and Yoshikami, D. (1975). Post-synaptic potentiation: interaction between quanta of acetylcholine at .the skeletal neuromuscular synapse. J. Physiol., Load. 251: 427463. Jahan-Parwar, B. and Fredman, S. M. (1976). Cerebral ganglion of Aplysia: cellular organization and origin of nerves. Comp. Biochem. Physiol. 54: 347-357. Katz, B. and Thesleff, S. (1957). A study of the “desensitization” produced by acetylcholine at the motor end-plate. J. Physiol, Load. 13& 63-80. McCaman, R. E. and McKenna, D. G. (1978). Monosynap tic connections between histamine-containing neurons and their various follower cells. Brain Res. 141: 165171. McLennan, H. and Wheal, H. V. (1976). The interaction of glutamic and aspartic acids with excitatory amino acid receptors in the mammalian central nervous system. Can. J. Physiol. Pharmac. 54: 7&72. Oomura, Y., Ooyama, H. and Sawada, M. (1974). Analysis of hyperpolarizations induced by glutamate and acetylcholine on Onchidium neurons. J. Physiol., Land. 243: 321-341. Schild, H. 0. (1957). Drug antagonism and PAX. Pharmac. Rev. 9: 242-246. Schwartz, J. C. (1975). Histamine as a transmitter in brain. Life Sci. 17: 503-518. Snyder, S. H. and Taylor, K. M. (1972). Histamine in the brain: a neurotransmitter. In: Perspectioes in NeuroPharmacology (Snyder, S. H., Ed.), pp. 43-73. Oxford University Press. Takeuchi, A. and Takeuchi, N. (1969). A study of the action of picrotoxin on the inhibitory neuromuscular junction of the crayfish. J. PhysioL, Lond. 205: 377-391. Triggle, D. J. and Triggle, C. R. (1976). Chemical Pharmacology of Synapses, pp. 124231. Academic Press, New York. Waud, D. R. (1968). Pharmacological receptors. Pharmac. Rev. 20: 49-88. Weinreich D. (1977). Synaptic responses mediated by identified histamine-containing neurons. Nature, Land. M7: 854-856. Weinreich, D. (1978). Histamine-containing neurons in Aplysia. In: Biochemistry of Characterized Neurons (Osborn, N. N., Ed.), pp. 153-175. Pergamon Press. England. Weinreich, D., McCaman, M. W., McCaman, R. E. and Vaughn, J. (1973). Chemical enzymatic and ultrastructural characterization of 5-hydroxytryptamine-containing neurons from the ganglia of Aplysia californica and Tritonia diomedia. J. Neurochem. 20: 969-976. Werman, R. (1969). An electrophysiological approach to drug-receptor mechanisms. Comp. Biochem. Physiol. 30: 997-1017. Ziskind, L. and Werman, R. (1975). At least 3 molecules of carbamylchohne are needed to activate a cholinergic receptor. Brain Res. 88: 177-180.
COOPERATIVE INTERACTIONS OF HISTAMINE AND COMPETITIVE ANTAGONISM BY CIMETIDINE AT NEURONAL HISTAMINE RECEPTORS IN THE MARINE MOLLUSC, APLYSIA CALIFORNICA DONNA L. GRUOL* and D. WEINREICH University of Maryland School of Medicine, Department of Pharmacology and Experimental Therapeutics, 660 West Redwood Street, Baltimore, MD 21201, U.S.A. (Accepted
12 October
1978)
Summary-Dose-response curves and Hill plots were used to analyze the interaction between histamine (HA), two HA agonists and an HA antagonist, and the HA receptors mediating membrane hyperpolarizations in Apl~Ga neurons. Dose-response curves for both HA and the HA agonists (Zmethylhistamine and 4-methylhistamine) were sigmoidal and the corresponding Hill plots had slopes greater than one (mean = 2). This result is interpreted as evidence for a cooperative type of interaction between HA and HA agonists and the neuronal HA receptors. Cimetidine (10m6 to 4 x 10m6M), an HA antagonist, produced reversible. dose-dependent, parallel shifts in the HA dose-response curves. Modified Schild plots constructed from this data were linear. These results demonstrate that cimetidine is a competitive antagonist at the neuronal HA receptors. The slope of the modified Schild plots, which gives an estimate of the minimum number of antagonist molecules that interact with each receptor, was greater than one (mean = 1.7). This result is interpreted as evidence for a cooperative type of interaction between cimetidine and the neuronal HA receptors.
Much neurochemical evidence has accumulated to suggest that histamine (HA) may function as a neurotransmitter in vertebrate (Green, 1970; Snyder and Taylor, 1972; Schwartz, 1975) and invertebrate (Weinreich, 1978) nervous systems. Physiological evidence has recently been reported for histaminergic synaptic transmission at identified synapses within the CNS of the marine mollusc Aplysin caltjbrnica (Weinreich, 1977; see also McCaman and McKenna, 1978). The transmitter function for HA at these identified synapses might further be established if the kinetic properties of the postsynaptic receptors activated by the natural transmitter were shown to be similar to the properties of HA-receptor interactions. When a transmitter substance interacts with its postsynaptic receptors, a primary event following receptor activation is a change in the ionic conductance of the postsynaptic membrane. Werman (1969) and other investigators (see Triggle and Triggle, 1976) have shown that Hill plots or log-log plots of transmitter doseresponse data provide useful information on the behavior of transmitter-receptor interactions in situ. Further insights about the transmitter-receptor interactions are obtained from studies describing the actions of antagonist drugs with transmitter receptors. This quantitative information may be particularly useful for subsequent comparisons between a suspected transmitter and the natural transmitter.
Although little quantitative information is currently available concerning the nature of HA-receptor interactions on neuronal membranes, it has been observed that the neurons within the A-cluster of the cerebral ganglia of Aplysia respond to exogenously applied HA with mono- or biphasic hyperpolarizing responses (Gruol and Weinreich, 1979). These responses are generated by conductance increases to specific ions and they are selectively blocked by certain pharmacological antagonists. Furthermore, these HA responses have ionic and pharmacological properties similar to the two-component hyperpolarizing synaptic potentials evoked by stimulating presynaptic HAcontaining neurons in the cerebral ganglion (Weinreich, 1977; and unpublished). Since the A-cluster neurons are relatively large (> 15Opm) cells and readily accessible for electrophysiological investigations, they provide a favorable model to begin characterizing the stoichiometric relations of HA-receptor interactions on neuronal membranes. In the present work, it is shown that: (1) HA and 2 HA agonists interact with neuronal HA receptors in a cooperative manner; (2) the HA antagonist, cimetidine, is a competitive antagonist at these same receptors; and (3) cimetidine interacts with neuronal HA receptors in a cooperative manner. METHODS
* Present address: NINCDS, NIH, Bldg. 36, Rm. 2CO7. Bethesda, MD 20014, U.S.A. Key words: histamine neuronal receptors, histamine agonist and antagonists, molluscan central neurons,
Aplysia californica (150-500 g) were obtained from Pacific Biomarine Supply Co., Venice, CA, and maintained in a marine holding tank at 16°C. Cerebral ganglia were removed from the animals and pinned,
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416
DONNA L. GRUOL and D. WEINREICH
dorsal side up, to the silastic (Sylgard 184, Dow Corning Corp., Midland, MI) base of a 2ml Plexiglas recording chamber containing artificial seawater (ASW) of the following composition (mM): NaCl, 494; KCI, 11; MgC12, 19; MgSO.+, 30; CaCl,, 11, Tris, 10. During experiments, the ASW level was lowered to a minimum bath volume (-0.5 ml) and the chamber continuously perfused with ASW. In some experiments, ASW containing 4 times normal Mg*+ and one-third normal Ca’ + was used to ensure that drug responses were a result of activation of receptors on the impaled neuron and not due to activation of an interneuron, and also to abolish spontaneous synaptic activity that occasionally interfered with recording of drug responses. The pH of all perfusion solutions was 7.8. Perfusion rates ranged from 1 to 5 ml/min depending on the type of experiment. All experiments were performed on an identifiable group of neurons designated the A-cluster neurons of the cerebral ganglia (Jahan-Parwar and Fredman, 1976). These neuronal clusters are symmetrically located on the dorsal surface at the entrance of the cerebral-pleural connectives (see Figs 1 in Weinreich, McCaman, McCaman and Vaughn, 1973, and in Jahan-Parwar and Fredman, 1976). The connective tissue capsule surrounding the neurons was removed to facilitate placement of the recording and iontophoretic microelectrodes. Neurons were impaled with two separate K+-citrate (2M, acidified to pH 6.5 with citric acid)-filled glass microelectrodes (15-30 Ma), one electrode for voltage recording and one for current injection. The recording electrode was connected through a unity gain electrometer to a Tektronix Type 5103 oscilloscope and to a Brush 2400 pen recorder from which all responses were analyzed. Current injection was used to vary membrane potential and to monitor changes in membrane input resistance. Currents (iontophoretic and transmembrane) were measured with an operational amplifier which held the bath potential at a virtual ground. The inverting input of the amplifier (Analog Devices, 515) was connected to the bath by a Ag/AgCl wire via an ASW-agar bridge and the noninverting input was grounded. Experiments were performed year round on 37 neurons from 16 animals. Resting potentials ranged from -40 to - 82 mV with a mean value of 61.8 f 7.3 mV. All quantitative data is expressed as mean + S.D.; N = number of neurons tested. Application of agonists and antagonists For the majority of studies, histamine (HA 0.5 M, pH 5.0) was applied by iontophoresis from fine-tipped (< 1 pm tip diameter) glass microelectrodes. Iontophoretic currents were generated by WPI microiontophoresis programmer units (Model 160:WPI Instruments, Inc., New Haven, Conn.) and typically ranged from 10 to lOOnA for control experiments and 20 to 300 nA when cimetidine was present. When current pulses were not rectangular, the tip of the pipette was broken off by gentle bumping on connective tissue.
Drug leakage was prevented by applying 5 to 1OnA holding currents to the iontophoretic pipettes. During experiments the polarity of the iontophoretic current was occasionally reversed to test for current artifacts. The duration of the iontophoretic current (typically 100 to 1OOOmsec) was set so that the response for all HA doses reached a steady value before declining. The interval between each HA application was set so that consistent responses were evoked for a fixed iontophoretic pulse of HA. For superfusion studies, HA or HA agonists were dissolved in ASW (10-8-10-3M) and applied as a 1 or 2 ml pulse (< 30 set duration) through the perfusion system. Several minutes of ASW wash followed each application. A six-way switching manifold enabled rapid changes of solutions without significant alterations in the perfusion rate. Under these conditions consistent responses could be obtained for a fixed concentration of agonist. Prolonged applications (> 1 min) of agonists were avoided to prevent possible desensitization effects. The HA antagonist, cimetidine, was applied by continuous perfusion for a minimum of 2min before its effect on agonist responses was assessed. When agonists were also applied by superfusion, they were dissolved in ASW plus antagonist and applied as described above. The chamber was thoroughly washed with drug-free ASW after each series of tests with a fixed concentration of cimetidine. Cimetidine had little (< 5%) or no discernible effect on membrane input resistance in the range of concentrations tested (1O-7-1O-5 M). Dose-response data In electrophysiological studies, drug-receptor interactions are commonly measured as a change in membrane conductance or a change in membrane potential; conductance is the more direct measurement since membrane potential changes are a function of the conductance change, the driving force for the ionic currents, and the time constant of the membrane. Thus, in an initial series of experiments it was determined whether voltage changes sufficiently reflected the underlying conductance changes to be useful for analysis of drug-receptor interactions. For these studies, the change in membrane conductance was calculated from membrane input resistance measurements made before and at the peak of the voltage response by passing constant hyperpolarizing current pulses. In all neurons tested, the peak amplitude of the voltage response varied as a linear function of the underlying conductance change over a wide range of drug concentrations. A deviation from linearity occurred most commonly at high agonist concentrations when large responses were evoked (Fig. lB,). This nonlinearity did not appear, however, to alter the interpretation of the data because Hill plots constructed from voltage data were similar to plots constructed from conductance data (Fig. 1B3). From these initial experiments it was concluded that
HA receptors on mohuscan neurons
417
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Fig. 1. Sample records showing responses of A-cluster neurons to increasing doses of HA applied by superfusion (A) or iontophoresis (B,). Downward deflections occurring at regular intervals are electrotonic potentials evoked by constant, hyperpolarizing current pulses which were used to measure changes in membrane conductance, Magnitudes of iontophoretic current shown on line I. The last trace in Br shows a response to HA when the membrane was polarized to -82 mV, the approximate membrane potential reached by the largest HA response in this series. The observed HA-induced hyperpolarization at a preset membrane potential of -82 mV indicates that the plateau of the HAinduced hy~~olari~tions is not a consequence of voltage saturation. B,, Graph showing the relationship between the change in membrane potential (v) vs the change in membrane conductance (g) measured at the peak of the HA response for increasing doses of HA. Both measurements (v and g) are expressed as a fraction of the value obtained for the maximum HA response (Vmax and Gmax). Same cell as Bt. Ba, Hill plot of data shown in B2. The slope of the line drawn through the experimental points was the same (slope = 1.7) whether the response was expressed as a change in membrane voltage (0) or a change in membrane conductance (0). Current-voltage curves were linear in the region where responses were measured.
DONNA L. GRUOL and D. WEINREICH
418
voltage changes could be used for analyzing the interaction between HA and HA receptors on A-cluster neurons. For most A-cluster neurons, the maximum HA response (typically ~25 mV) polarized the membrane to a level which was 5 to 10mV more positive than the reversal potential for the response (h - 89 mV; see Gruol and Weinreich, 1978). When the maximum HA response was closer to the reversal potential, constant depolarizing current was applied before doseresponse data was obtained to bring the membrane potential further from the reversal potential. This insured that the maximum response represented a saturation of the HA receptors and not a saturation of the response (Fig. 1Br). Current-voltage curves for A-cluster neurons were linear in the hyperpolarizing direction where measurements of agonists responses were made (i.e. - 55 to -90 mV). Drug source Cimetidine, 2-methylhistamine and 4-methylhistamine were gifts from Smith, Kline & French and their generosity is gratefully acknowledged. Histamine-HCl was purchased from Sigma Chemical Corp. (St Louis, MO). RESULTS
Dose-response curves: HA and HA agonists The most common response of A-cluster neurons to exogenously applied HA (iontophoresis or superfusion) was a slowly developing hyperpolarization mediated by an increased K+ conductance (Fig. lA, B,; Gruol and Weinreich, 1978). The amplitude of the response varied as a function of applied HA (Fig. lA, B,) and dose-response curves (iontophoretic current or molar concentration vs change in membrane Table
1. Comparisons
Drugs
and method
A. Histamine of auulication
B. Control of application
Histamine-Iontophoresis Control Cimetidine (10-6-10-5 M) Histamine-Superfusion Control Cimetidine (10e6-2 x 10e6 M) C. Cimetidine Analysis Modified schild plot? Los-log plot
for histamine, cimetidine
and histamine agonists No. of cells
2-Methylhistamine-superfusion 4-Methylhistamine-superfusion and method
tervals are electrotonic potentials produced by constant, hyperpolarizing current pulses. (B) Hill plot of data shown in (A). The slope of the line drawn through the experimental points is 2.0.
of n or m values obtained or the histamine antagonist,
Histamine-iontophoresis Histamine-superfusion
Drugs
Fig. 2(A) Sample record of responses of an A-cluster neuron to the HA agonist, 4-MeHA, applied by superfusion. Similar responses were obtained with 2-MeHA (data not shown). Downward deflections occurring at regular in-
histamine
n
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antagonism No. of cells
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1.7 k 0.3* 1.7
* Values represent means + SD. t Modified Schild plot: log [(Dose ratio)” - l] vs log cimetidine concentration where n equals slope of Hill plot for histamine interaction with histamine receptors.
419
HA receptors on molluscan neurons
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Fig. 3. Effect of cimetidine on HA log dose-response curves (A, and B,) and corresponding Hill plots (A, and BZ). A,. HA applied by iontophoresis. Data shown are mean values from 2 cells. A,, Slope of Hill plot = 1.6. B,, HA applied by superfusion. Data shown are mean values from 2 cells. Bz, Slope of Hill plot = 1.3. potential) were sigtnoidal (data not shown). When Hill plots were constructed from dose-response data, the slope of the line (n) drawn through the experimental points was greater than 1 (Figs IB,, 3A, and 3B,). Values for n ranged from 1.2 to 2.7 with a mean of 1.8 f 0.4 (N = 30) when HA was applied by iontophoresis and 1.7 f 5 (N = 11) when HA was applied by superfusion (Table 1). Similar values for n were obtained when the response was measured as the change in membrane conductance (Fig. 1B3; see Methods). The HA agonists, 2-methylhistamine Qmethylhistamine (4MeHA), (2-MeHA) and although less potent than HA also produced dosedependent, K+-mediated membrane hyperpolarizations in the A-cluster neurons (Fig. 2A; Gruol and Weinreich, 1978). Dose-response curves for these agonists were also sigmoidal and the slope of Hill
plots were greater than 1 (Fig. 2B). Mean values for n were 1.9 + 0.3 (N = 6) for 2-MeHA and 1.9 k 0.3 (N = 8) for 4-MeHA (Table 1A). These data indicate thatr (1) 2 or more molecules of HA interact with each neuronal HA receptor to produce the observed voltage change; and (2) the HA agonists, 2-MeHA and 4-MeHA, interact with the receptor in a manner similar to HA. HA dose-response curves: effect of cimetidine It has previously been reported that cimetidine, a HA antagonist, specifically and reversibly blocks the K+-mediated hyperpolarizations evoked by HA or HA agonists in A-cluster neurons (Gruol and Weinreich, 1978). In the present series of experiments, doseresponse data has been utilized to study the type of interaction between cimetidine and neuronal HA
DONNA L. GRUOL
420
receptors. When applied at 10m6M concentrations, cimetidine produced parallel shifts in the HA log dose-response curves and in the Hill plots (Fig. 3). Mean values for n, the slope of the Hill plot, were similar for control data and data obtained in the presence of cimetidine (Table 1B). At higher cimetidine concentrations, parallel shifts in the HA log dose-response curves were also observed, but frequently the maximum response was somewhat less than the control maximum (data not shown). These experiments suggest that, at low concentrations, cimetidine is a competitive antagonist at neuronal HA receptors on the A-cluster neurons. The competitive action of cimetidine was more rigorously tested by construction of Schild plots from dose-response data @child, 1957). For this analysis, the dose ratio (taken at 50% of the maximum response) was raised to a power equal to n (the slope of the Hill plot) in order to take into account the cooperative interaction between the HA molecules and the HA receptors*. As shown in Figure 4A, the modified Schild plots are linear, a characteristic which is interpreted as indicative of a competitive type of interaction @child, 1957). The slope of the Schild plot (m) is analogous to the slope of the Hill plot (n) and gives an estimate of the minimum number of antagonist molecules which interact with each receptor to produce the observed inhibition. In 4 neurons where this analysis was successfully carried out, m was greater than 1. The mean value of m for these 4 neurons was 1.7 f 0.3 which is similar to that obtained for n, the estimate for the minimum number of HA or HA agonist molecules which- interact with each HA receptor (Table 1). An m value greater than 1 was further confirmed in two additional experiments where dose-response curves were constructed by varying the cimetidine concentration while holding the HA dose constant. The limiting slope of log-log plots (change in amplitude of HA response vs cimetidine concentration) gives an estimate of m (Werman, 1969). For both neurons tested m was greater than 1 (mean = 1.7; Fig. 4B; Table IC). DISClJS.SION
If it is assumed that the relationship between drugreceptor activation and the measured response is linear, dose-response data and Hill plots can be used to analyze the interaction between drug molecules and their receptors (see reviews by Waud, 1968; Werman, 1969; Triggle and Triggle, 1976). When dose-response curves are sigmoidal and Hill plots have a slope greater than 1, the interaction is thought to be cooperative and the value of n, the slope of the Hill plot, is indicative of the minimum number of drug molecules which interact with the receptor to produce *The authors thank Dr Neville Brookes for pointing out the necessity for this modification.
and D.
WEINREICH
cimetidine
-6
XIO M
B
‘V 4
cimetidine
X,O-‘M
Fig. 4(A) Modified Schild plot. DR = dose ratio taken at 50% of the maximum response. n = Hill slope. HA applied by iontophoresis. Slope of the modified Schild plot = 2. (B) Log-log plot showing the relationship between the de-’ crease in amplitude of the control HA response in the presence of cimetidine vs the cimetidine concentration. HA applied by superfusion at 5 x 10m6M. Control values were measuted before and after each cimetidine dose. Limiting slope = 1.6.
the response (Brookes and Werman, 1973). Cooperative interactions between putative neurotransmitters and their receptors have been demonstrated using electrophysiological techniques in a variety of vertebrate and invertebrate preparations including molluscan neurons (Katz and Thesleff, 1957; Takeuchi and Takeuchi, 1969; Brookes and Werman, 1973; Diamond and Roper, 1973; Oomura, Ooyama and Sawada, 1974; Dudel, 1975, 1977; Hartzell, Kuffler and Yoshikami, 1975; Ziskind and Werman, 1975; McLennan and Wheal, 1976; Constanti, 1977). The present authors’ data indicates that a cooperative type
HA receptors on molluscan neurons
of interaction also occurs between the putative neurotransmitter HA and neuronal HA receptors. When agonists are applied by iontophoresis, interpretation of dose-response data may be complicated by nonlinearity of the current ejected from the iontophoretic pipette and/or uncertainties as to the equivalence of the receptor population which is exposed to low vs high doses of the drug. In order to rule out these possible sources of error, an additional series of experiments were initiated where HA was applied by superfusion. The value of n, the Hill slope, for data obtained by superfusion of HA agreed closely with that obtained by iontophoresis of HA. Thus, it is unlikely that technical errors can account for observed cooperativity. It has previously been shown that cimetidine specifically blocks the K+-mediated HA response in A-cluster neurons but the type of antagonism was not characterized (Gruol and Weinreich, 1979). In the present study, it has been shown that cimetidine is a competitive antagonist at these neuronal HA receptors by two commonly used criteria: (1) a parallel shift in the HA dose-response curve, and (2) a linear Schild plot. The data also indicates that 2 or more molecules of cimetidine interact with each HA receptor to produce the observed inhibition. Considering the close similarity in structure between HA and cimetidine (Brimblecombe, Ducan, Durant, Emmett, Ganellin and Parsons, 1975) it is not surprising that a cooperative type of interaction occurs for both agonist and antagonist at the HA receptors. Acknowledgrments-The authors wish to thank Drs N. Brookes and T. Smith for critical reading of this manuscript. This work was supported by NSF grant (BNS 77-13034) to D. W. and by Smith, Kline & French Co. REFERENCES
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