Opioids at low concentration decrease openings of K+ channels in sensory ganglion neurons

Brain Research, 558 (1991) 166-170 (~) 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939124825L

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Opioids at low concentration decrease openings of K ÷ channels in sensory ganglion neurons S.-F. Fan 1'3, K.-F. S h e n 1 and S.M. Crain 1'2 Departments of lNeuroscience and 2physiology/Biophysics, and Rose E Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Yeshiva University, Bronx, N Y 10461 (U.S.A.) and 3Department of Physiology and Biophysics, Health Sciences Center, State University of New York at Stony Brook, N Y 11794 (U.S.A.)

(Accepted 4 June 1991) Key words: Dorsal root ganglion neuron;/~-Opioid; 6-Opioid; r-Opioid; Opioid excitatory effect; Patch-clamp recording;

Voltage-sensitive K + channel

Previous studies showed that low concentrations of opioids prolong the calcium-dependent component of the action potential duration (APD) of dorsal root ganglion (DRG) neurons, whereas higher concentrations shorten the APD. In the present study whole-cell voltage-clamp, as well as cell-attached membrane-patch voltage-clamp, recordings demonstrate that application of picomolar to nanomolar concentrations of/~, 6 or r opioid agonists (DAGO, DPDPE or dynorphin) to DRG neurons in dissociated cell cultures reversibly decreased the activities of voltage-sensitive K + channels. Pretreatment of DRG neurons with the opioid receptor antagonists, naloxone (30 nM) or diprenorphine (1 nM) prevented 1~/6 or r opioid-induced decreases in K + channel activities, respectively. Since opioids added to the bath solution decreased the activities of K + channels in the membrane patch sealed off by the pipette tip, our results provide strong evidence that some modes of excitatory modulation of the action potential of DRG neurons are mediated by diffusible second messengers. The data are consonant with our previous studies indicating that opioids can elicit excitatory effects on sensory neurons via cholera toxin-sensitive Gs-linked excitatory opioid receptors coupled to cyclic AMP-dependent ionic channels. Opioid receptor agonists at/~M or higher concentrations shorten the duration of the calcium-dependent component of the action potential ( A P D ) in cultures of dorsal root ganglion ( D R G ) neurons of chick 9 and mouse 2'15-18. In recent intracellular current-clamp studies, Shen and Crain 12'13 found that lower concentrations (nM) of opioids (kt, 6 and r) prolong, instead of shorten, the A P D of about 75% of the D R G neurons in mouse D R G - c o r d explants 4. After treating the neurons with K + channel blockers, nanomolar concentrations o f / t and 6 opioids as well as r opioid, dynorphin (1-13) could no longer prolong the A P D , suggesting that this excitatory effect is the result of decreasing K ÷ conductance of the cell m e m b r a n e 12'13. This interpretation of action potential data has been confirmed in the present study by whole-cell voltage-clamp and by ceU-attached patchclamp single channel recordings. A preliminary report of part of the results has been published 5. Monolayer cultures of dissociated fetal rodent D R G neurons devoid of non-neuronal cells were used after 2 - 4 weeks in vitro (see details of culture techniques in Chen et al.3). The conventional tight-seal patch-clamp method 6 was used. Pipettes were made from microhematocrit

capillary tubes (1.3-1.4 m m o.d., Fisher) with a Narishige electrode puller (Model PP-83). The tip diameter of the pipettes was 1-3/~m. For single channel recordings, the pipette solution contained (in m M ) NaC1 136, KC1 5.4, CaCI 2 5, MgCI 2 0.5, H E P E S 10 ( p H 7.2). For whole-cell current recordings, pipette solution contained (in m M ) KC1 140, CaCI 2 0.1, E G T A 0.6, Mg~+-ATP 2, H E P E S 10, p H 7.2. T h e bath solution used had the same composition as the pipette solution used for single channel recording with 1 ~ M tetrodotoxin added to block the Na ÷ current. For both whole-cell current and singlechannel activity recordings, a patch-clamp amplifier (Axon Instruments, Model Axopatch l-D) was used. For whole-cell voltage-clamp studies, c o m p u t e r software (pCLAMP, Axon Instruments) and associated analogdigital converter and interface were used to provide the c o m m a n d electrical pulses as well as to digitize the cell response and store the data in an I B M AT-compatible computer. Correction of leakage current was made by digital subtraction of currents produced by scaled pulses (scale of 10 or 15). The results were analyzed with p C L A M P c o m p u t e r software. For single-channel recordings, the signals from the cell, after passing through the

Correspondence: S.M. Crain, Department of Neuroscience, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY 10461,

U.S.A.

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Fig. 1. Bath application of low concentrations of opioids decreases the outward K + current of DRG neurons during pulsed depolarization. Top records: graded outward currents during application of depolarization pulses of increasing strength as shown in the lower right corner of each bottom record. Middle records: attenuation of outward currents after addition of opioid for 10 rain. Bottom records: recovery of responses 20-30 rain after washout of opioid. A: application of I nM dynorphin (1-13). B: application of I nM DPDPE. C: application of 10 pM DAGO.

patch-clamp amplifier, were recorded on videotape with a video-cassette recorder containing a pulse-code modulation processor (Toshiba Model DX-900). After each experiment, the tape was played back and analyzed for current distribution histograms. The programs used were kindly provided by Drs. P. Brink and R a m a n a n TM. The following drugs were used: Tyr-o-Ala-Gly-NMethyl-Phe-Gly-ol ( D A G O ) , Tyr-D-Pen-Gly-Phe-D-Penicillamine ( D P D P E ; Peninsula Labs); dynorphin (1-13) (Dyn; Sigma or Peninsula Labs); naloxone (NLX; Endo); diprenorphine (DPN; a gift from Dr. E.J. Simon). All other chemicals were from Sigma. The effects of low concentrations of opioids on wholecell currents during pulsed depolarization were first analyzed. The membrane current of a D R G neuron during pulsed depolarization contained both inward and outward components. The inward component could be blocked by Cd 2÷ and often ran down gradually. Drug effects were tested only on cells whose outward currents were stable for at least 5 to 10 rain after making a tight-seal connection with the patch pipette. In the majority of D R G neurons tested, bath application of 1 nM dynorphin (Fig. 1A) or 1 nM D P D P E (Fig. 1B) decreased the outward current reversibly (Table I), whereas 1 nM D A G O usually increased the outward current. However, D A G O also decreased the outward current when the concentration was lowered to 10 pM (Fig. 1C; Table I). The effect of opioids on the activity of single K + channels was then tested in cell-attached membrane

patches. During membrane-patch voltage-clamp recordings, voltage-sensitive K + channels with different conductances were detected. Usually two subtypes coexisted

TABLE I Effects of low concentrations of opioids on whole-cell outward K ÷ currents and the activities of K + channels in cell-attached membrane patches

All tests were carried out on DRG neurons after 2-4 weeks in dissociated cell culture. Number of cells tested with opioids Dynorphin (1-13) 1 nM

DPDPE 1 nM

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* All decreases or increases in K + current or single channel activity were >10%.

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Fig. 2. Bath application of low concentrations of opioids decreases the open probability of K + channels in cell-attached membrane patches of DRG neurons. First row: current record with cells in control medium. C denotes the closed state. O denotes the open states. Second row: current distribution histogram obtained from cells bathed in control medium. Sampling time for histograms shown both in this row and the last row was 100/~s. Third row: current record with cells in opioid solution for about 10 min. Fourth row: current distribution histogram obtained from cells bathed in opioid solution. A: 1 nM dynorphin (1-13) decreased the open probability of small-conductance K + channels and increased that of large-conductance K ÷ channels. ( $ ) and ( $ $ ) denote the currents of small- and large-conductance channels, respectively. Due to variances of currents, the dosed- and the first open-state current distributions merged into one unsymmetrical peak in the histogram. Insets are histograms with the ordinate expanded. Holding potential was 60 mV. B: 1 nM DPDPE decreased the open probability of both small- and large-conductance K + channels. Holding potential was 30 mV. C: 10 pM DAGO decreased the open probability of both small- and large-conductance K ÷ channels. Holding potential was 40 mV.

in a single m e m b r a n e patch, one with unitary conductance below 40 pS and the o t h e r close to 100 pS. Bath application of 1 n M dynorphin (Fig. 2 A ) , 1 nM D P D P E (Fig. 2B) or 10 p M D A G O (Fig. 2C) decreased the open probability of small-conductance K + channels in most of the cells tested, while the effect on the open probability of large-conductance channels varied (Table I). In the case of dynorphin, the activities of large-conductance K ÷ channels were generally increased. The s u m m a t e d effect of the decreases in small-channel activities was greater than that of the increase in large-channel activities, resulting in a net decrease in the whole-cell K ÷ conductance. In tests with D P D P E and D A G O , on the o t h e r

hand, the o p e n p r o b a b i l i t y of large-conductance as well as small-conductance K ÷ channels b o t h generally decreased. T h e opioid r e c e p t o r antagonists, N L X and D P N , blocked the effects of b o t h la/6 and ~: o p i o i d agonists, respectively. A f t e r bathing D R G n e u r o n s in 30 n M N L X for > 1 0 min, coperfusion of either 1 n M D P D P E or D A G O with N L X showed no changes in o u t w a r d current or open probabilities of K ÷ channels (4 out of 6 cells had shown characteristic effects when tested initially with D P D P E and 4 out of 5 cells with D A G O ) . Similarly, after bathing D R G neurons in 1 n M D P N for > 1 0 min, coperfusion of 1 nM d y n o r p h i n with D P N did not show

169 any change in the probability of opening of both types of K + channels. Two of these 5 cells had been tested initially with 1 nM dynorphin and had shown characteristic dynorphin-induced decrease in the openings of the smaller conductance K + channels and increase in the openings of the large channels. Although higher concentrations of dynorphin also appeared to activate 6 and/~ receptors, the K + channel-blocking effects of 1 nM dynorphin could only be prevented by the more universal opioid antagonist 1°'12 DPN, but not by NLX, indicating that the excitatory effects of dynorphin were most probably mediated by r receptors. During cell-attached patch-clamp recordings, the m e m b r a n e patch underneath the pipette tip is segregated from the bath solution. A n y effect caused by bathapplied membrane-impermeable agents could only be accounted for by mediation via diffusible intracellular second messenger system(s). Bath application of D A G O and D P D P E decreased the activities of both small- and large-conductance K + channels inside the patch sealed off by the pipette tip. Furthermore, bath application of dynorphin increased the activity of large-conductance K ÷ channels, in addition to decreasing the activity of smallconductance K + channels. The latency of the opioid effects was of the order of 1-2 min. The results indicate strongly that these opioid excitatory effects are mediated via diffusible second messengers, as suggested by previous evidence that injection of an inhibitor of cyclic A M P - d e p e n d e n t protein kinase blocked D A D L E - i n duced prolongation of the A P D of D R G neurons 3 following activation of Gs-linked excitatory opioid

1 Brink, P.R. and Fan, S.-F., Patch clamp recordings from membranes which contain gap junction channels, Biophys. J., 56 (1989) 579-593. 2 Chalazonitis, A. and Crain, S.M., Maturation of opioid sensitivity of fetal mouse dorsal-root ganglion neuron perikarya in organotypic cultures: regulation by spinal cord, Neuroscience, 17 (1986) 1181-1198. 3 Chen, G.G., Chalazonitis, A., Shen, K.-E and Crain, S.M., Inhibitor of cyclic AMP-dependent protein kinase blocks opioidinduced prolongation of the action potential of mouse sensory ganglion neurons in dissociated cell cultures, Brain Research, 462 (1988) 372-377. 4 Crain, S.M. and Shen, K.-E, Opioids can evoke direct receptormediated excitatory effects on sensory neurons, Trends Pharmacol. Sci., 11 (1990) 77-81. 5 Fan, S.-F., Shen, K.-F. and Crain, S.M., Dynorphin-induced suppression of the activity of a specific subtype of voltagesensitive K ÷ channels in mouse dorsal-root ganglion neurons may underlie prolongation of the action potential duration, Soc. Neurosci. Abstr., 15 (1989) 147. 6 HamiU, O.P., Marty, A., Nehr, E., Sakmann, B. and Sigworth, F.J., Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches, Pfliigers Arch., 391 (1981) 85-100. 7 Miyake, M.J., Christie, M.J. and North, R.A., Single potassium channels opened by opioids in rat locus ceruleus neurons, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 3419-3422.

receptors 14. It has been reported that dynorphin can pass through the cell m e m b r a n e 8 and directly activate G proteins. However, the effects of dynorphin applied in the bath were blocked in the presence of D P N , indicating that dynorphin acted on opioid receptors located in the extra-patch membrane of the D R G neuron. Thus, these inhibitory effects of dynorphin, mediated by increasing the openings of large-conductance K ÷ channels, also appear to involve diffusible second messengers. In contrast, in locus ceruleus neurons, K + channel activity was modulated only when the opioid was added directly to the pipette solution contacting the m e m b r a n e patch 7. The present demonstration that low (picomolar to nanomolar) concentrations of opioids can block K ÷ channel activities in a DPN/NLX-sensitive manner provides further strength to the hypothesis that opioids can elicit direct concentration-dependent receptor-mediated excitatory effects on sensory neurons 4'12. It will be of great interest to apply these patch-clamp techniques to membranes of central nervous system neurons to determine if opioids can evoke direct excitatory effects on specific types of brain cells, in addition to the disinhibitory effects mediated by opioid inhibition of non-opioid inhibitory interneurons 19. This work was supported by Research Grants DA-05203 and DA-02031 to S.M.C. The DRG cell cultures were prepared by Peter Vanamee in tissue culture facilities kindly provided by Dr. Murray B. Bornstein. Analyses of the single-channel activities of the DRG neurons were carried out by S.EE, using computer facilities kindly provided by Dr. Peter R. Brink, Department of Physiology and Biophysics, State University of New York at Stony Brook, NY, U.S.A.

8 Mousli, M., Bueb, J.-L., Bronner, C., Rouot, B. and Landry, Y., G protein activation: a receptor-independent mode of action for cationic amphiphilic neuropeptides and venom peptides, Trends Physiol. Sci., 11 (1990) 358-362. 9 Mudge, A.W., Leeman, S.E. and Fischbach, G.D., Enkephalin inhibits release of substance P from sensory neurons in culture and decreases action potential duration, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 526-530. 10 Paterson, S.J., Robson, L.E. and Kosterlitz, H.W., Classification of opioid receptors, Br. Med. Bull., 39 (1983) 31-36. 11 Ramanan, S.V. and Brink, P.R., Alternate methods of representing single-channel data, Biophys. J., 57 (1990) 893-901. 12 Shen, K.-E and Crain, S.M., Dual opioid modulation of the action potential duration of mouse dorsal root ganglion neurons in culture, Brain Research, 491 (1989) 227-242. 13 Shen, K.-E and Crain, S.M., Dynorphin prolongs the action potential of mouse sensory ganglion neurons by decreasing a potassium conductance whereas another specific r opioid does so by increasing a calcium conductance, Neuropharmacology, 29 (1990) 343-349. 14 Shen, K.-F. and Crain, S.M., Cholera toxin-A subunit blocks opioid excitatory effects of sensory neuron action potentials indicating mediation by Gs-linked opioid receptors, Brain Research, 525 (1990) 225-231. 15 Werz, M.A. and Macdonald, R.L., Opioid peptides decrease calcium dependent action potential duration of mouse dorsal root ganglion neurons in cell culture, Brain Research, 239 (1982)

170 315-321. 16 Werz, M.A. and MacDonald, R.L., Opioid peptides with differential affinity for /z- and 6-receptors decrease sensory neuron calcium-dependent action potentials, J. Pharmacol. Exp. Ther., 227 (1983) 394-402. 17 Werz, M.A. and MacDonald, R.L., Opioid peptides selective for ,M- and 6-opiate receptors reduce calcium-dependent action potential duration by increasing potassium conductance, Neurosci. Lett., 42 (1983) 173-178.

18 Werz, M.A. and MacDonald, R.L., Dynorphin and neoendorphin peptides decrease dorsal root ganglion neuron calciumdependent action potential duration, J. PharmacoL Exp. Ther., 234 (1985) 49-56. 19 Zieglgansberger, W., French, E.D., Siggins, G.R. and Bloom, EE., Opioid peptides may excite hippoeampal pyramidal neurons by inhibiting adjacent inhibitory interneurons, Science, 205 (1979) 415-417.