Regulation of neural responses in the canine pyloric sphincter by opioids

Transcription

1 Br. J. Pharmacol. (1993), 108, " Macmillan Press Ltd, 1993 Regulation of neural responses in the canine pyloric sphincter by opioids Orline Bayguinov & 'Kenton M. Sanders Department of Physiology, University of Nevada School of Medicine, Reno, Nevada 89557, U.S.A. Keywords: 1 Regulation of excitatory and inhibitory junction potentials (ej.ps and ij.ps) by opioid peptides was studied in isolated muscle strips from the pyloric sphincter of the dog. 2 Methionine enkephalin (MetEnk; 1010 to 10-6 M) and [D-Ala2, D-Leu5] enkephalin (DADLE; 10-" to 10-' M), a 6-specific opioid agonist, inhibited ij.ps and ej.ps recorded from cells in the myenteric and submucosal regions of the circular muscle layer. These compounds had no effect on resting potential or slow wave activity suggesting that the effects on junction potentials were not due to direct effects on smooth muscle cells. 3 MetEnk and DADLE caused similar effects on junction potentials in preparations in which the myenteric plexus was removed, suggesting that opioids inhibit pre-junctional effects on nerve fibres within the muscularis externa. 4 Inhibition of junction potentials by MetEnk and DADLE was blocked by approximately the same extent by naloxone (106 M) and ICI 174,864 (106M), a 6-specific antagonist. 5 MetEnk and DADLE blocked a portion of the ij.p. that was sensitive to arginine analogues; after treatment with N0-nitro-L-arginine methyl ester (L-NAME, 10-4 M), MetEnk and DADLE had no further effect on i.j.ps. These data suggest that opioids regulate nitric oxide-dependent neurotransmission. 6 Naloxone (10-6 M) alone had no effect on ij.ps elicited by short trains of electrical field stimuli. 7 Ij.p. amplitude was reduced after a period of conditioning stimulation (2 min, 30 Hz, 30 V). Naloxone blocked the post-stimulation inhibition. Repetitive stimulation at high frequencies (30 Hz) resulted in sustained hyperpolarization. Naloxone increased the amplitude of the hyperpolarization responses elicited by high frequency stimulation. 8 These results show that ej.ps and ij.ps in the canine pylorus are inhibited by opioids. A portion of the inhibitory effects appears to be mediated via 6 receptors. 9 Although pyloric muscles are richly innervated by nerves containing opioid peptides, brief trains of stimuli do not appear to release concentrations of opioids that are effective in regulating junction potentials. Higher frequency stimulation (or longer durations of stimulation) appear to be necessary to release concentrations of opioids that are effective in modulating the amplitude of junction potentials. Enkephalins; enteric nervous system; pylorus; gastric emptying; gastric smooth muscle; nitric oxide; gastrointestinal motility Introduction The major, naturally-occurring opioids are derived from three classes of precursor polypeptides that have all been found in gastrointestinal tissues (for review see Miller & Hirning, 1989). Much of the expression of these peptides has been localized to intrinsic neurones (e.g. Schultzberg et al., 1980; Furness et al., 1983), and several studies have shown that opioids can be released by stimuli such as electrical field stimulation (Glass et al., 1986) and during peristalsis (Donnerer et al., 1984). Opioid peptides affect gut function through a variety of specific receptors, and functional 1i, 6 and K receptors have been found in neurones of the gastrointestinal tract (see Duggan & North, 1984). Opioid peptides may regulate the contractile behaviour of gastrointestinal muscles by reducing the excitability of enteric neurones by hyperpolarization (i.e. enhancement of K conductance) or inhibition of voltage-dependent Ca2" currents in cell bodies (Morita & North, 1981; Cherubini & North, 1985; Mihara & North, 1986; North et al., 1987; McFadzean, 1988) and by reducing neurotransmitter release by pre-junctional effects at axonal varicosities (Cowie et al., 1978; Vizi et al., 1984). Support for the latter mechanisms is provided by recent studies in which several naturally-occurring and syn- ' Author for correspondence. thetic opioid agonists were shown to reduce the amplitude of inhibitory junction potentials in strips of intestinal muscle from which the myenteric and submucosaal ganglia were removed (Bauer & Szurszewski, 1991; Bauer et al., 1991). The inhibitory effects of opioids appeared to be mediated by both 1L and 6 receptors. Opioids had no effect on the resting membrane potentials or slow wave activity of smooth muscle cells suggesting that these compounds do not directly affect smooth muscle cells in canine, human and baboon small intestines (Bauer & Szurszewski, 1991; Bauer et al., 1991). Similar results were obtained in experiments on human colonic muscles (Hoyle et al., 1990), but in these muscles the inhibitory effects of opioids appeared to be mediated predominantly by 6 receptors. Although exogenous opioids can clearly reduce junction potentials, regulation of neural transmission by endogenous opioids has been difficult to demonstrate. For example, the opioid receptor antagonist, naloxone, did not affect junction potentials elicited by single pulses or brief trains of electrical field stimulation in the dog intestine (Bauer & Szurszewski, 1991). It may be that the concentrations of opioids released during brief periods of stimulation are ineffective in regulating transmitter release. Mechanical studies suggest that relatively high frequencies of stimulation may be needed to elicit naloxone-sensitive responses (e.g. Puig et al., 1977). Both excitatory and inhibitory junction potentials can be

2 ENDOGENOUS OPIOIDS REGULATE JUNCTION POTENTIALS 1025 elicited in the circular muscle of the canine pylorus (Vogalis & Sanders, 1990), and a high density of enkephalinergic nerve fibres exists in the muscularis externa of the dog (Allescher et al., 1988), as well as in man (Ferri et al., 1987) and cat (Edin et al., 1980). Opioids may have a physiological role in pyloric function since opioid agonists have been shown to delay gastric emptying (Sullivan et al., 1981). In the present study we have examined the regulation of junction potentials in the canine pylorus by opioids. Recent studies have suggested that nitric oxide, or a related compound, might serve as an inhibitory transmitter in gastrointestinal muscles (for review, see Sanders & Ward, 1992), but regulation of nitric oxide release by opioids has not yet been addressed. Therefore, we have also attempted to determine whether inhibitory junction potentials, which appear to be mediated by nitric oxide in the canine pylorus (Bayguinov & Sanders, 1992), are regulated by endogenous opioids. Methods Mongrel dogs of either sex were anaesthetized with pentobarbitone sodium (100 mg kg-'). The abdomen of each animal was opened by a midline incision and the entire stomach and a short segment of the proximal duodenum was removed. The excised organs were placed in a dish filled with oxygenated, Krebs-Ringer buffer. The pyloric region and adjoining portions of the terminal antrum and duodenum were dissected from the rest of the stomach. The pyloric sphincter was clearly visible, as previously described (Vogalis et al., 1991). The pyloric canal was opened along the lesser curvature. The resulting sheet of tissue was pinned-out flat, and strips of muscle (1-1.5 mm in thickness) were cut parallel to the longitudinal muscle layer. These strips were laid 'on-side' in an electrophysiological chamber in such a manner as to provide a cross-sectional view of the entire muscularis externa. These preparations allowed impalement of smooth muscle cells at any point through the thickness of the circular layer (see Sanders & Vogalis, 1989). For some experiments, muscle strips were dissected to remove the myenteric plexus. These strips were prepared from the crosssectional muscle strips by cutting away the entire longitudinal muscle layer and a small portion of the circular muscle layer to remove the entire myenteric plexus region. The muscles strips were continuously perfused with warmed, oxygenated Krebs solution at a rate of 5 ml min-'. The bath temperature was maintained at 37.5 ± 0.5 C, and the muscle strips were allowed to equilibrate for at least 2 h before intracellular recording was started. Smooth muscle cells of the circular layer were impaled with glass microelectrodes filled with 3 M KC1 and having resistances of MO. Impalements were accepted based on previously described criteria (Sanders & Vogalis, 1989). Transmembrane potential was measured with a standard high-input impedance electrometer (WPI M-707), and the output was displayed on an oscilloscope (Tektronix 5111 A). Data were simultaneously recorded by a chart recorder (Gould 2200) and an FM tape recorder (Hewlett Packard). Data were analyzed from chart recordings or from taped records. Intrinsic nerves were stimulated by electrical field stimulation (EFS) applied transmurally with platinum wires. Stimulating electrodes were connected via a stimulus isolation unit to a stimulator (Grass S48), and stimulus parameters were chosen for selective nerve stimulation as judged by blockade of responses by tetrodotoxin (pulses 0.5ms, 30Hz, 30-1OOV). Solutions and drugs The standard Krebs-bicarbonate solution (KRB) used in this study contained (in mm): Na' 137.4; K+ 5.9, Ca2+ 2.5, Mg2+ 1.2, CY 134, HC , H2PO4-1.2 and dextrose This solution achieved a final ph of 7.3 to 7.4 after equilibration with 97% 02:3% CO2. Methionine enkephalin (MetEnk; Sigma) and [D-Ala2, D-Leu5] enkephalin (DADLE; Sigma), were used as acetate salts. ICI 174,864 (N,N-diallyl-Tyr-Aib- Aib-Phe-Leu; Cambridge Research Biochemicals) and naloxone hydrochloride salt (Sigma) were used to block opioid receptors. Nw-nitro L-arginine methylester (L-NAME), and propranolol (all Sigma) were used as hydrochloride salts, and NG-monomethyl-L-arginine (L-NMMA) (Calbiochem) was used as the acetate salt. The sulphate salt of atropine (Sigma) and the mesylate salt of phentolamine (Ciba Geigi) were used. Drugs were dissolved in distilled water as stock solutions of 10-2 or 10-3 M and further serial dilutions were made in KRB as required. Oxyhaemoglobin was prepared as a haemolysate of canine blood as previously described (Ward et al., 1992). Data were expressed as mean ± s.e.mean, and paired or unpaired Student's t tests were used for determination of statistical significance where appropriate; n values refer to the number of muscle strips used in each experiment. Results Effects of opioids on circular muscle cells near myenteric border Circular muscle cells near the myenteric border had average resting potentials of - 64 ± 3 mv (n = 7). As previously described (Sanders & Vogalis, 1989), spontaneous electrical activity (slow waves) were recorded in this region. MetEnk and DADLE (10 `0 to 106 M) did not affect resting potential (- 64± 4 mv in the presence of MetEnk and - 65 ± 1 mv in DADLE) or spontaneous slow wave activity (n = 7) suggesting that these agonists did not have direct effects on the ionic conductances of smooth muscle cells (Figures 1). Electrical field stimulation elicited primarily inhibitory junction potentials (i j.ps) in the myenteric region that a Control b MetEnk10-M NW -28 mv -69 mv -90 mv -26 mv _1-69 mv I -69 mv -79 mv 10 s Figure 1 Effects of methionine enkephalin (MetEnk) on spontaneous electrical activity and inhibitory junction potentials (ij.ps). Top trace in (a) shows spontaneous slow waves recorded from a cell within the myenteric portion of the circular muscle layer. Bottom trace shows ij.ps elicited by field stimulation (30 Hz, 3 pulses, 60 V). (b) Shows activities after MetEnk (10-6 M). MetEnk did not affect slow wave activity or resting potentials, but inhibited ij.ps. Initial transient upstrokes in records from cells in myenteric region are stimulus artifacts (also in Figures 4, 7 and 8).

3 BAYGUINOV & K.M. SANDERS averaged 13 ± 6 mv in amplitude (n = 7 representative preparations) as previously described (Vogalis & Sanders, 1990). These events persisted after the addition of atropine, phentolamine and propranolol (all at 10-6 M), and therefore resulted from the activation of non-adrenergic, noncholinergic (NANG) enteric inhibitory nerves (see Figure la). Addition of MetEnk (10-0 to 10-6 M) caused a concentration-dependent reduction of the amplitude of ij.ps (IC50 = 5 x 10-8 M). At the maximum effect, MetEnk reduced i.j.ps to an average of 28 ± 2% of the control amplitude. Figure 2b shows the effects of MetEnk on ij.ps and a concentration-response curve summarizing the effects in 7 muscles is shown in Figure 3. The 6-selective agonist, DADLE (10-" to 10-7 M) had effects similar to MetEnk on ij.ps (Figure 2e). At the maximum effective concentration, ij.ps were reduced to an average of 26 ± 3% of the control amplitude. Concentrationresponse curves showed that DADLE was about 2 orders of magnitude more potent (ICm, = 5 x M) than MetEnk in reducing ij.ps (Figure 3). We have recently shown that L-NAME and L-NMMA, arginine analogues that block nitric oxide synthesis, decrease the amplitude of ij.ps in the circular muscle of the canine pylorus (Bayguinov & Sanders, 1992). Similar effects were caused by oxyhaemoglobin (HbO). Four muscles were treated with L-NAME (10- M) and then exposed to MetEnk (10-6 M; n = 3) and DADLE (10-6 M; n = 3). After washing out L-NAME, three of the muscles were pretreated with HbO (1%) and then exposed to either MetEnk (n = 2) or DADLE (n = 1). When ij.ps were suppressed with drugs to block nitric oxide neurotransmission, MetEnk and DADLE caused no further reduction in ij.p. amplitude (Figure 4). These data suggest that opioid antagonists act on a population of enteric inhibitory nerves that release nitric oxide. Naloxone (10-6 M) had no effect on resting membrane potential, slow wave activity, or the ij.ps elicited by EFS. This compound reduced the maximim inhibitory effects of MetEnk (10-6 M) and DADLE (10-6 M) on ij.ps restoring the amplitudes of ij.ps to 90± 4% (n = 4) and 94± 2% (n = 4) of control levels, respectively (Figure 2c and f). The specific 6 opioid receptor antagonist, ICI 174,864 (10-6 M) had effects similar to naloxone. ICI 174, 864 did not affect resting potential, slow wave activity, or ij.ps elicited by EFS, but it antagonized the effects of MetEnk and DADLE, restoring the amplitudes of ij.ps to 91 ± 3% (n = 3) and 97 ± 2% (n = 3) of control levels, respectively _ log (Agonist) Figure 3 Concentration-response relationships for the effects of methionine enkephalin (MetEnk, *) and DADLE (0) on inhibitory junction potentials. DADLE was about 2 orders of magnitude more potent than MetEnk in reducing the amplitude of junction potentials. Data are means ± s.e.mean; numbers of experiments given in text. Effects of opioids on circular muscle cells near the submucosal border As observed previously, cells in the submucosal region of the circular muscle layer were not spontaneously active. Resting membrane potentials averaged - 70 ± 2 mv, and the application of MetEnk or DADLE did not significantly affect resting potential (i.e ± 2 mv and - 69 ± 3 mv in the presence of MetEnk (n =4) and DADLE (n = 3), respectively). Field stimulation of this region produced predominantly cholinergically-mediated ej.ps that averaged 36 ± 2 mv (n = 7) as previously described (Vogalis & Sanders, 1990). Both opioids suppressed ej.ps in a concentration-dependent manner. At the maximum effect, MetEnk (10-6 M) decreased the amplitude of ej.ps by an a Control b MetEnk 10M c MetEnk 106m + Nal 1O 6M -_68mV I1 mv d Control e DADLE 10-M f DADLE lom + Nal 10 6M -.6 mv 10S Figure 2 Effects of methionine enkephalin (MetEnk) and DADLE on i.j.ps; (a) shows ij.ps elicited by EFS (30 Hz, 3 pulses, 60 V); (b) shows reduction in ij.ps by MetEnk (10-6 M). The effect of MetEnk was blocked by naloxone (Nal 10-6 M; C), (d) and (e) show that DADLE also reduced ij.p. amplitude, and these effects were reversed by naloxone (10-6 M).

4 ENDOGENOUS OPIOIDS REGULATE JUNCTION POTENTIALS 1027 a Control d L-Arg 10-3M b L-NAME 10-4M C L-NAME 10-4M + MetEnk 106M -69 mv mv -93 mv -90OmV e MetEnk 10M -69 mv '-75mV '-9m f MetEnk 10O, Nal 106M -92 mv 10 s Figure 4 Methionine enkephalin (MetEnk) inhibited nitric oxidedependent ij.ps. (a) shows ij.ps evoked by EFS (30Hz, 3 pulses, 60 V) in a cell from myenteric region of circular muscle layer; (b) shows reduction in ij.p. amplitude by treatment (20min) with N0- nitro-l-arginine methyl ester (L-NAME). (c) Shows that after L- NAME, MetEnk had little or no effect on remaining i j.p. The inhibitory effects of MetEnk and L-NAME were reversible. To increase the rate of ij.p. recovery from the effects of L-NAME, L- arginine (10-3M) was added to the bath solution for 15min. (d) I.j.ps were inhibited by MetEnk (10-6 M) after the wash-out of L-NAME and L-arginine (e) and Panel F (f) shows that naloxone (10-6 M; given in continued presence of MetEnk) blocked the inhibitory effects of MetEnk. All recordings were from the same impalement. a Control MetEnk JLX b Atr 106, Phent 10-6, Propr 106M DADLE k ICI m I-31 mv 108 +ICI mv 500 ms -69 mv -81 mv los Figure 5 Effects of methionine enkephalin (MetEnk) on ej.p. and i.j.p. amplitudes recorded from a cell in the submucosal region: (a) shows responses elicited by EFS (30 Hz, 3 pulses, 60 V). The predominant response to EFS in the submucosal region was an ej.p. MetEnk (l0-8_ 10-6 M) decreased ej.p. amplitude. Representative e.j.ps shown during MetEnk exposure. ICI 174,864 (10-6 M) blocked the effects of MetEnk. Representative i.j.ps elicited during the same impalement after treatment with atropine, phentolamine and propranolol (all at 10-6 M) are shown in (b). Atropine blocked the ej.p. response unmasking large non-adrenergic, non-cholinergic ij.ps. DADLE reduced the amplitude of ij.ps (similar results were obtained with MetEnk; see text), and these effects were blocked by ICI 174, average of 83 ± 2% (Figure 5a; n =4) and DADLE decreased ej.ps by % (n = 3; data not shown). Concentration-response curves showed that DADLE was more than 2 orders of magnitude more potent (IC50 = 5 X 10-0 M) than MetEnk in reducing ej.ps (Figure 6). Application of naloxone (10-6 M) had no effect on resting membrane potential or ej.p. amplitude, but this compound antagonized the inhibitory effects of MetEnk and DADLE, restoring the amplitude of ej.ps to 96± 3% (n =4) and 92 ± 1% (n = 3) of control responses, respectively, in the continued presence of MetEnk and DADLE. ICI 174,864 (10-6 M) also had no effect on the resting potential, but it blocked the effects of MetEnk and DADLE (i.e. restoration to 94 ± 1% (n = 4) and % (n = 3) of control responses, respectively). Figure 5a shows effects of ICI 174,864 on e.j.ps. Besides an effect on ej.ps, we noted that MetEnk and DADLE also reduced the amplitude of the small ij.p. responses that often follow the initial ej.p. in submucosal muscles. In order to study the effects of opioids on ij.ps in this region, muscles were exposed to atropine, phentolamine and propranolol (all at 10-6 M). Atropine eliminated ej.ps, leaving the non-adrenergic, non-cholinergic ij.ps. These events averaged 10 ± 0.5 mv in peak amplitude (n = 7). MetEnk and DADLE suppressed ij.ps (i.e. MetEnk reduced i.j.ps by an average of %, n = 4 and DADLE reduced i.j.ps by %, n = 3) with the same effectiveness as they suppressed the ij.ps recorded near the myenteric border (Figure Sb). These data suggest that opioid receptors are present on cholinergic and enteric inhibitory nerves in the -o ol/j 50 0 Xa 0W log (Agonist) Figure 6 Concentration-response relationships for the effect of methionine enkephalin (MetEnk, 0) and DADLE (0) on excitatory junction potentials elicited in circular muscle cells near the submucosal border. DADLE was about 2 orders of magnitude more potent than MetEnk in reducing the amplitude of excitatory junction potentials. Data are means ± s.e.mean; numbers of experiments given in text. pylorus. Naloxone and the specific 5 opioid antagonist, ICI 174,864, eliminated the effects of MetEnk and DADLE on submucosal i.j.ps. Naloxone restored ij.p. amplitude to

5 BAYGUINOV & K.M. SANDERS 94 3% of control amplitude in the presence of MetEnk (n = 4), and it restored ij.p. amplitude to 96 ± 4% of control amplitude in the presence of DADLE (n = 3). ICI 174,864 restored ij.p. amplitude to 91 ± 4% of control amplitude in the presence of MetEnk (n = 4), and it restored ij.p. amplitude to 95 ± 1% of control amplitude in the presence of DADLE (n = 3; see Figure 5b). Effects of opioids on responses to EFS in muscles free of the myenteric plexus Studies on intact muscles suggest that opioids may affect excitatory and inhibitory transmitter release. The site of action of opioids could either be within ganglia or at nerve terminals. We studied this question in preparations from which the myenteric plexus had been removed. Cells in the remaining myenteric region of the circular muscle layer were impaled and ij.ps were elicited by EFS. Application of MetEnk (10-6 M) and DADLE (10-6 M) inhibited ij.p. by an average of 73 ± 3% and 74 ± 5% (both n = 5). These data suggest that opioids exert actions directly upon nerve processes within the muscle layer. Effects of endogenous opioids on responses to field stimulation The experiments described above do not clarify the role of endogenous opioids that may be released in response to EFS. The observations that naloxone did not affect spontaneous electrical activity or responses to EFS suggest that endogenous opioids do not affect pyloric smooth muscle cells directly nor are they released in sufficient quantity during brief periods of EFS to affect the release of transmitter. Therefore we tested the effects of more sustained EFS. I.j.p. amplitude was compared before and after a conditioning stimulation period. Ij.ps were elicited with 3 pulses (30 Hz; 60 V). Then a conditioning train of stimuli was applied for 2 min (30 Hz; 30 V). After cessation of the conditioning train and repolarization of the resting potential, ij.ps were again elicited with 3 pulses (30 Hz; 60 V). Ij.ps elicited after the conditioning train were reduced in amplitude by an average of 17 ± 3% (n = 5; P<0.01; Figure 7a). The muscles were then exposed to DADLE (10-6 M). This reduced ij.p amplitude as discussed above, and it also inhibited the reduction in ij.p. amplitude caused by the conditioning train (Figure 7b). Further addition of naloxone (10-6 M in the continued presence of DADLE) antagonized the effects of DADLE on ij.ps. In the presence of naloxone the conditioning train did not reduce ij.p. amplitude (i.e. ij.ps were 97 ± 3% of control amplitude; P> 0.05; Figure 7c). In another series of experiments muscles were stimulated for 20 s (0.5 ms, 60 V, 30 Hz). This caused a sustained hyperpolarization averaging 10 ± 4 mv in amplitude (n = 4). Then muscles were treated with naloxone (10-6 M) for 5 min and the same stimulus protocol was repeated. Naloxone significantly increased the amplitude of the sustained hyperpolarization by an average of 28 ± 11% (n =4; P< 0.01; Figure 8). Taken together these results suggest that effective concentrations of opioids may be released when higher frequency stimuli are applied for a sustained period. Discussion A number of studies have shown that enteric neurones contain opioid peptides (Polak et al., 1977; Schultzberg et al., 1980; Furness et al., 1980; 1983), and these compounds can be released by EFS (Glass et al., 1986) and during peristalsis (Donnerer et al., 1984). In the guinea-pig intestine, enkephalins and dynorphin are expressed along with other transmitter substances in excitatory and inhibitory neurones (Brookes et al., 1991), suggesting the possibility that opioids may be co-released with excitatory and inhibitory transmit- a b Control DADLE 1O-6 M AA- V~ c DADLE 10 M, Nal 10 M W0s -58 mv -m7ov -58 mv -64 mv -59 mv Figure 7 Effects of conditioning stimuli on ij.p. amplitude: (a) shows 2 superimposed traces of ij.ps elicited by field stimulation (30 Hz, 3 pulses, 60 V) before (trace denoted by arrows) and after a conditioning period of stimulation (30 Hz, 2 min, 30 V). The amplitude i.j.ps decreased following the conditioning stimuli. (b) Shows superimposed records before and after the conditioning stimulation in the presence of DADLE (10-6 M). DADLE reduced the amplitude of ij.ps and blocked the effect of the conditioning stimulation. The effects of naloxone are shown in (c). Superimposed records were again taken before and after the conditioning stimulation. Naloxone blocked the reduction in ij.p. amplitude caused by DADLE, and it also blocked the post-stimulation inhibition seen in the control record. All records were taken during a single impalement. Naloxone 106M 10s -60 mv -75 mv Figure 8 Effects of naloxone on sustained hyperpolarization responses to EFS. Muscle was stimulated by repetitive pulses of EFS (30 Hz, 20 s, 60 V). This stimulation caused a sustained hyperpolarization (control record). Exposure to naloxone increased the amplitude of the hyperpolarization recorded from the same cell, suggesting that release of endogenous opioids during the control record limited the response. ters. Opioids could have effects either pre- or postjunctionally. The presence of opioid receptors in subcellular fractions containing neural markers, but not in fractions containing smooth muscle markers (Allescher et al., 1989), suggests that the primary site of action of these peptides may be pre-junctional. In support of the concept of pre-junctional modulation, opioids have been shown to decrease the release of enteric transmitter substances (Vizi et al., 1984; Grider & Maklouf, 1987a). In the present study we characterized the role of opioids in

6 ENDOGENOUS OPIOIDS REGULATE JUNCTION POTENTIALS 1029 regulating junction potentials in the circular muscle layer of the canine pyloric sphincter. This region has been shown to contain a very high density of opioid-containing neurones in several species (Edin et al., 1980; Ferri et al., 1987; Allescher et al., 1988). We found that exogenous opioids reduced the amplitude of excitatory and inhibitory junction potentials. Opioids specifically reduced the portion of the ij.ps that were inhibited by arginine analogues, suggesting that opioids may reduce the synthesis and release of nitric oxide just as they appear to reduce the release of traditional neurotransmitters (Vizi et al., 1984; Grider & Maklouf, 1987a). The fact that opioids affected both excitatory and inhibitory junction potentials suggests that opioid receptors are coupled to a basic signal for transmitter release. A common mechanism may be regulation of Ca2" concentration in varicosities since a rise in [Ca2+]i appears to be the signal for quantal release of most transmitters and for synthesis and release of nitric oxide (Stark et al., 1991). Naloxone, a non-specific opioid receptor antagonist, had little or no effect on junction potentials elicited by single or brief trains of stimuli. But naloxone: (i) increased the amplitude of hyperpolarization responses elicited by longer trains of stimulation, and (ii) blocked depression of ij.ps following periods of high frequency stimulation. These data suggest that repetitive stimuli may be necessary to release enough opioids to affect transmitter release. Others have suggested a similar role for endogenous opioids in the small intestine. For example, Puig et al. (1977) found that higher stimulus frequencies may be necessary for release of opioids in the guinea-pig ileum. These authors found that a conditioning period of stimulation at 10 Hz caused inhibition of electrically evoked twitch responses in the period immediately following the conditioning stimuli. Naloxone protected against the post-stimulus inhibition, suggesting that opioids released during the conditioning stimuli were responsible for the post-stimulus inhibition. Similar results were reported by Fosbraey & Johnson (1980). Others have failed to show effects of naloxone on junction potentials elicited by brief field stimulation. For example, Bauer & Szurszewski (1991) found that naloxone antagonized the reduction in ij.ps caused by exogenous opioid agonists in the canine duodenum, but opioid receptor antagonists did not significantly affect ij.ps. Similar results have been reported from experiments on guinea-pig intestine; naloxone had no effect on resting membrane potential or on junction potentials (Ito & Tajima, 1980). These findings also suggest that the concentrations of opioids released by brief stimuli are too low to affect transmitter release. For effective concentrations of opioids to be reached, relatively high frequency stimulation appears to be required. An important question is whether the enteric nervous system operates at high enough frequencies to utilize endogenous opioids as a means to regulate transmitter release. Bornstein et al. (1991) have shown that firing within motor neurones of the enteric nervous system can reach instantaneous frequencies of 100 Hz during reflex activation. Although high instantaneous frequencies are reached, it is unclear whether high frequency firing persists for sufficient periods to activate naloxone-sensitive responses. Physiological evidence, however, tends to suggest that concentrations of opioids may reach effective levels during reflex activity. Opioid peptides and receptor antagonists have been shown to modulate the peristaltic reflex (Kromer et al., 1990), although a recent study suggests that opioids may only be important when the intestine is working against relatively high outflow resistances (Waterman et al., 1992). Grider & Makhlouf (1987b) have attempted to dissect the relative contribution of opioids to the descending relaxation and ascending contraction components of the peristaltic reflex. They found that naloxone caused a concentration-dependent increase in descending relaxation and decreased ascending contraction. The increase in descending relaxation was attributed to an increase in VIP release that coincided with naloxone treatment, and the decrease in ascending contraction may also have been related to an increase in VIP release. Since VIP appears to be expressed in the same inhibitory neurones that synthesize nitric oxide (Costa et al., 1991), it is possible that nitric oxide was also involved in the naloxonesensitive effects noted by Grider & Makhlouf (1987b). Donnerer et al. (1984) measured dynorphin, substance P and somatostatin released from the guinea-pig intestine. They showed that levels of these peptides increased during peristalsis. Release of substance P and dynorphin were blocked by TTX, suggesting these peptides originated from a neural source. Significant levels of substance P were observed only when naloxone was present, suggesting that opioids normally limit the release of substance P released during peristalsis. Limited evidence suggesting a physiological role for opioids in the pylorus exists. Emptying of solid meals was significantly slowed by the enkephalin analogue DAMME (D-Ala2, MePhe4, Met(O-Ol-enkephalin; FK33-824) (Sullivan et al., 1981), but others reported that naloxone did not affect gastric emptying (Feldman et al., 1980). Allescher et al. (1988) found that exogenous opioid agonists inhibited motor activity of the canine pylorus in vivo. Naloxone antagonized these effects, demonstrating the presence of functional opioid receptors in the canine pylorus. A variety of opioids, directed at ;t, 6 and ic receptors, however, did not affect basal motor activity in the pyloric region, and blockade of opioid receptors did not affect excitatory responses elicited by duodenal field stimulation or inhibitory responses elicited by antral field stimulation or vagal stimulation. These in vivo studies further illustrate the difficulties in demonstrating the role of endogenous opioids. Our results would predict that rather intense or prolonged stimuli would be necessary to elicit naloxone-sensitive responses. In some species opioids might have pre- and postjunctional effects. For example in the cat, atropine-resistant contractions of the pylorus elicited by vagal stimulation could be blocked by naloxone (Edin et al., 1980). Others have suggested that endogenous opioids in the pyloric region are involved in an excitatory reflex stimulated by duodenal acidification or amino acids (Reynolds et al., 1984; 1985). Taken together it appears that in the cat there may be (i) release of enkephalins from non-cholinergic excitatory nerves, and (ii) direct effects on the smooth muscle. These mechanisms are not apparent in the dog because we found no effect on either resting membrane potential or slow waves in canine pyloric muscles. In addition to regulation of physiological responses, the receptors and effectors for opioids provide an important means for therapeutic control of motor activity. For example, it has been known for many years that the peristaltic reflex elicited by distension of the bowel is inhibited by morphine (Gyang et al., 1964). In the pylorus it appears that a significant portion of the regulation of junction potentials by opioids occurs via 6 receptors since ICI 174,864, a specific 6 receptor antagonist, had nearly identical actions to those of the non-specific antagonist, naloxone. Similar results have been reported for the modulation of inhibitory junction potentials by exogenous opioids in the human colon (Hoyle et al., 1990). This study was supported by a grant from the National Institutes of Health (DK-40569). The authors are grateful to Drs C. William Shuttleworth and Kathleen Keef for many helpful comments on the manuscript.

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