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Journal of Physiology (1992), 447, pp. 119-131 With 6 figures Printed in Great Britain

BOTH ATP AND THE PEPTIDE VIP ARE INHIBITORY NEUROTRANSMITTERS IN GUINEA-PIG ILEUM CIRCULAR MUSCLE

BY JEFFREY R. CRIST, XUE D. HE AND RAJ K. GOYAL From the Center for Swallowing and Motility Disorders, Harvard-Thorndike Laboratory, Charles A. Dana Research Institute, Department of Medicine, Division of Gastroenterology, Beth Israel Hospital and Harvard Medical School, Boston, MA 02215, USA

(Received 15 February 1991) SUMMARY

1. Intracellular membrane potential recordings were made from circular smooth muscle cells of the guinea-pig ileum in the presence of atropine (1 #M) and nifedipine (01I,tM) at 30 'C. 2. Perfusion with adenosine triphospate (ATP, 100 /LM) and vasoactive intestinal peptide (VIP, 2 ,SM) resulted in membrane hyperpolarizations of 6-4 + 0 3 and 6'8 + 03 mV, respectively. Picospritzes of ATP (10 mm in pipette) and VIP (100 tim in pipette) resulted in membrane hyperpolarizations of 6-9 + 04 and 6-3 + 04 mV, respectively. 3. The ATP-induced hyperpolarizations were antagonized by ac, ,3-methylene ATP desensitization (100 ,tM for 30 min) and the ATP antagonist Reactive Blue 2 (200 /tM), but were unaffected by the VIP antagonist VIP 10-28 (1 JiM). 4. The VIP-induced hyperpolarizations were antagonized by VIP 10-28, but unaffected by a, ,-methylene ATP desensitization and Reactive Blue 2. 5. A single pulse of transmural nerve stimulation (2 ms, 15 mA) resulted in an inhibitory junction potential (IJP) that reached a maximal amplitude of 12-9 + 0-5 mV at 378 + 20 ms from the stimulus. This fast IJP was abolished by apamin (2 /zM) or tetrodotoxin (1 pM), antagonized by a, fl-methylene ATP desensitization or Reactive Blue 2, but unaffected by VIP 10-28. 6. In the presence of apamin (1 fM), four pulses of transmural stimulation (2 ms, 20 Hz, 15 mA) resulted in an IJP that reached a maximal amplitude of 4-8 + 0-2 mV at 14 + 0-1 s from the stimulus. This slow IJP was antagonized by tetrodotoxin (1 /LM) or VIP 10-28 (1 /bM), augmented by Reactive Blue 2 (200 jm), and unaffected by ax, ,J-methylene ATP desensitization. 7. These findings provide evidence that both ATP and VIP are inhibitory neurotransmitters in the circular muscle layer of the ileum and that ATP may be the neurotransmitter responsible for the fast IJP and VIP the neurotransmitter responsible for the slow IJP.

MS 9154

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J. R. CRIST, X. D. HE AND R. K. GOYAL INTRODUCTION

Intraluminal distension of the intestine results in relaxation of circular smooth muscle immediately anal to the distending stimulus. This relaxation is due to reflex activation of intrinsic inhibitory nerves in the intestine (Costa & Furness, 1976; Grider, 1989; Smith, Bornstein & Furness, 1990). The nature of the inhibitory nerves responsible for this relaxation reflex is a matter of considerable controversy and has been examined using isometric tension and electrophysiological membrane potential recording techniques (Costa & Furness, 1976; Bauer & Kuriyama, 1982; Bornstein, Costa, Furness & Lang, 1986; Grider & Makhlouf, 1986; Costa, Furness & Humphreys, 1986). Electrophysiological studies of the inhibitory responses of guinea-pig ileum circular muscle to transmural nerve stimulation have shown two distinct components of hyperpolarization (Niel, Bywater & Taylor, 1983; Taylor & Bywater, 1986; Bywater & Taylor, 1986). One of thee components, known as the fast inhibitory junction potential (IJP), is observed in response to a single pulse of transmural nerve stimulation. This fast IJP reaches a peak amplitude approximately 0 4 s from the stimulus, and is abolished by the potassium channel blocker apamin (Bulbring & Tomita, 1967; Bauer & Kuriyama, 1982; Bywater & Taylor, 1986; Hoyle & Burnstock, 1989). Another more recently described component of hyperpolarization, known as the slow IJP, requires a short train of stimulation and is observed in the presence of apamin. This slow IJP reaches a peak amplitude more than 1 s following the stimulus and is smaller in amplitude than the fast IJP (Niel, Bywater & Taylor, 1983; Taylor & Bywater, 1986; Bywater & Taylor, 1986). The distinct nature of these two inhibitory junction potentials raises the possibility that they might be due to the release of different neurotransmitters. There is presently considerable debate concerning the possible roles of adenosine triphosphate (ATP) and vasoactive intestinal peptide (VIP) as inhibitory neurotransmitters in the guinea-pig intestine (MacKenzie & Burnstock, 1980; Bauer & Kuriyama, 1982; Hills, Collis & Burnstock, 1983; Costa et al. 1986; Grider & Makhlouf, 1986; Grider, 1989; Hoyle & Burnstock, 1989). The purpose of our study was to determine the role the putative neurotransmitters ATP and VIP might be playing in producing the fast and slow IJPs observed in response to transmural nerve stimulation in the guinea-pig ileum. METHODS

Twenty (male or female) guinea-pigs weighing between 250 and 400 g were anaesthetized by means of CO2 narcosis and subsequently stunned and bled via the carotid arteries. A segment of ileum 15-20 cm in length was taken from between 5 and 40 cm oral to the ileo-caecal junction and the anal aspect was marked with a black silk suture. The segment was then cleansed with an intraluminal flush of Krebs solution and cut open along the mesenteric border of its longitudinal axis. The removed ileal segment was trimmed to a length of 20 mm and transferred to a bath and pinned with its mucosal surface facing down. The bath consisted of a chamber 30 mm in length and 11 mm in width with a floor of Sylgard (Dow-Corning, Midland, MI, USA). The bath had a volume of 1-4 ml and was continuously perfused with Krebs solution which was oxygenated and heated prior to entry in the bath. The rate of flow was maintained constant at 3 ml/min. The bath fluid was further oxygenated by bubbling of 95% 02-5% C02 directly into the bath. The bath temperature was maintained at 30 0 + 0 5 °C rather than 37 °C so as to decrease muscle contractions and displacement of the recording microelectrode.

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The Krebs solution consisted of (in mM): glucose (1 15), bicarbonate (21-9), phosphate (1 2), sodium (138-5), calcium (2 5), magnesium (1-2), potassium (4 6) and chloride (125). The pH of the Krebs solution after 30 min of bubbling with 95% 02-5% CO2 ranged between 7 34 and 7 39. Atropine (1 /M) and nifedipine (0-1 /M) were added to the perfusate to block muscarinic transmission and reduce spontaneous and evoked contraction of the muscle. Organic calcium entry blockers such as nifedipine (0-1 /eM) have been shown to lack any significant effects on neuroneuronal synaptic transmission (Smith & Furness, 1988; Smith, Furness, Costa & Bornstein, 1988; Wood, 1989) and neuro-muscular synaptic transmission in enteric nerves of the guinea-pig small intestine (Bywater & Taylor, 1986). The following method was used to antagonize ATP responses by means of desensitization using the ATP-analogue a, fi-methylene ATP which is resistant to enzymatic degradation. Perfusion with a, /J-methylene ATP (100,UM) resulted in a membrane hyperpolarization that gradually returned to resting membrane potential over the subsequent 15-20 min of continued perfusion. Desensitization of ATP receptors was verified after 30 min of perfusion by the absence of any hyperpolarizing effect on resting membrane potential upon increasing the perfusion concentration of a, f8-methylene ATP tenfold to 1 mm. Intracellular recordings Intracellular recordings of membrane potential were obtained from smooth muscle cells using microelectrodes made from glass of 1-2 mm external diameter (Frederick Haer & Co., Brunswick, ME, USA) and filled with 3 M-potassium chloride. The resistance of the microelectrodes was between 30 and 80 MQ. The microelectrode was connected to the probe of a high-input-impedance electrometer (Neuroprobe 1600, A-M Systems Inc., Everett, WA, USA), the output of which was displayed on a digitizing storage oscilloscope (Tektronix 5223, Tektronix Inc., Beaverton, OR, USA). Permanent records were made by passing the oscilloscope signals onto a strip chart recorder (Gould 220, Gould inc., Cleveland, OH, USA). Impalement of a circular smooth muscle cell was made by advancing the microelectrode attached to a micromanipulator (E. Leitz Inc., Rockleigh, NJ, USA) through the surface longitudinal muscle and then into the deeper cell layer of circular muscle. Previous studies have demonstrated that transmural stimulation results in an inhibitory junction potential (IJP) in circular muscle impalements and not longitudinal muscle impalements (Bywater, Holman & Taylor, 1981; Bywater & Taylor, 1986). Hence, a successful impalement of a circular smooth muscle cell was defined as a negative deflection in the oscilloscope trace with subsequent maintenance of a stable negative potential for longer than 10 min and an inhibitory junction potential (IJP) in response to a transmural stimulus. All membrane potential values were determined by the difference between the stable potential recorded within the cell compared to the balanced zero potential upon withdrawal. Transmural stimulation of intramural nerves within the intestinal strip was performed by means of two Ag-AgCl electrodes (0-26 mm diameter) placed above and below the intestinal preparation and perpendicular to its longitudinal axis. These electrodes were insulated up to 2 mm from their tips and connected to a stimulator (Grass S-88, Grass Instr., Quincy, MA, USA) in series with a stimulus isolation unit (Grass SIU5) and a constant-current unit (Grass CCU1). Two types of transmural nerve stimulation were used in this study. One type consisted of a single pulse (2-0 ms pulse duration, 30 mA) and the other type consisted of a short train of four pulses (2-0 ms pulse duration, 30 mA, 20 Hz). These stimulus parameters were chosen as they produced maximal responses when recordings were made within 3 mm of the stimulating electrode and were abolished by tetrodotoxin (1 ,SM).

Local application of drugs Brief pulses of pressure (air) from a pressure application device (Picospritzer II, General Valve Corp., Fairfield, NJ, USA) attached to a micropipette were used to locally apply small amounts of drug onto the surface of the circular muscle layer of the ileum. The picospritzer pressure was maintained at 20 lbf/in2. The pressure-ejecting pipettes were made form borosilicate capillary tubing having an external diameter of 1-2 mm, internal diameter of 0 9 mm (Frederick Haer Corp, Brunswick, ME, USA) and a truncated tip diameter of 25-35 #M. The amount of drug discharged from a given micropipette was varied by changing the duration of the pulse of pressure (range 8-15 ms) or the number of pulses delivered (one to five). The preparation was viewed under a binocular microscope (Bausch and Lomb, Rochester, NY, USA) with the recording electrode positioned within 0-15-1-0 mm of the drug ejection pipette. Once a membrane potential change

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was observed in response to a drug ejection, the duration and number of pulses and the relative position of the drug ejection pipette to the recording electrode were maintained without alteration. This resulted in highly reproducible membrane potential responses to the local application of drugs.

Statistics Statistical comparisons were made using Student's standard paired and unpaired t statistics. All data are expressed as mean+s.E.M.

Drugs Drugs used in this study included apamin, atropine, tetrodotoxin, nifedipine, Reactive Blue 2 (Cibacron Blue 3GA; has A ring o-sulphonic acid structure), a, ,-methylene adenosine 5'triphosphate (a, fl-methylene ATP), and vasoactive intestinal peptide (VIP), which were obtained from Sigma Chemical Co. (St Louis, MO, USA) and VIP 10-28, which was obtained from Bachem Inc. (Torrance, CA, USA). Nifedipine was dissolved in 95 % ethanol at 10 mm and stored in a lightfree container. All other drugs were made up fresh on the day of the experiment. RESULTS

Effects of VIP on resting membrane potential Perfusion of VIP (2 /M) resulted in a membrane hyperpolarization of 6-8 + 0 3 mV (n = 8 in four animals). This hyperpolarization reached maximal amplitude within 5 min of initiating perfusion and terminated within 5 min following wash-out with Krebs solution (Fig. I A). Local application of VIP using pressure ejection techniques (100 /SM in pipette) resulted in a maximum membrane hyperpolarization of 6-3 + 0-4 mV (n = 20 in four animals) (Fig. 1B). The time to maximal hyperpolarization following the pressure ejection was 6-2 + 0 9 s and the duration of this hyperpolarization was 23-1 + 3-2 s (n = 20 in four animals). The onset, maximum amplitude and duration of hyperpolarization were highly reproducible for successive pressure ejection pulses from a particular electrode, but varied greatly with changes in the positioning of the pressure ejection pipette in relation to the recording microelectrode. The variability in onset, maximum amplitude and duration of pressure ejection-induced hyperpolarization therefore appeared to be related primarily to the positioning of the pressure-ejecting pipette next to the recording electrode.

Effects of ATP on resting membrane potential Perfusion of ATP (100 /tM) resulted in a membrane hyperpolarization of 6-4 + 0-3 mV (n = 8 in four animals). This hyperpolarization reached maximal amplitude within 2 min of initiating perfusion and terminated within 2 min following wash-out with Krebs solution (Fig. 1C). Local application of ATP using pressure ejection techniques (10 mm in pipette) resulted in a membrane hyperpolarization of 69 + 04 mV (n = 20 in four animals) (Fig. ID). The time to maximal hyperpolarization following the pressure ejection was 8-56 + 1-5 s and the duration of this hyperpolarization was 21P9 + 3-3 s (n = 20 in four animals).

Effect of VIP and ATP antagonism on membrane responses to local application of VIP and ATP Perfusion with the VIP antagonist VIP 10-28 (Grider & Rivier, 1990) (1 ,UM) had no effect on resting membrane potential (control RMP = -51.0+15 mV, 15 min;

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-60 mV

110 mV VIP

B

2 min

-57 mV

mV

VIP

5s

VIP

C

-57 mV

10 mV ATP

D

30s

-62 mV 4

ATP

~|10 mV 1

2s

Fig. 1. Effect of VIP and ATP on resting membrane potential. A, perfusion with VIP (2 /,M) resulted in membrane hyperpolarization. B, local application of VIP by pressure ejection technique (100 ,UM in pipette) resulted in a brief membrane hyperpolarization. C, perfusion with ATP (100 ,UM) resulted in membrane hyperpolarization. D, local application of ATP by pressure ejection technique (10 mm in pipette) resulted in a brief membrane hyperpolarization. TABLE 1. Influence of a, fl-methylene ATP desensitization, Reactive Blue 2 and VIP 10-28 on hyperpolarization amplitudes due to VIP or ATP picospritzes a, /J-Methylene ATP desens. Reactive Blue 2 VIP 10-28 (100,UM for Control 30 min) Control (200 ,UM) Control (1 /LM) Amplitude of 53+05 6-3+0-6 6-2+04 4-7+03 6-2+0 4 1-7+0-2 VIP picospritz n=6 n=4 n=4 n=4 n=3 n = 20 hyperpolarization (P > 005) (P 005) (mV) Amplitude of 8-2+0-4 3-1+0 4 8-0+0-4 1P5+0-3 8-3+05 8-7+07 ATP picospritz n=5 n=4 n=5 n=4 n=4 n=5 hyperpolarization (P < 001) (P < 001) (P > 0 05) (mV) Values are mean+ S.E.M. n = number of cells. P values = significance as compared to control values.

VIP 10-28 perfusion RMP = -5241+0-9 mV, P > 0.05, n = 10 in five animals). Following 15 min of perfusion with VIP 10-28, the membrane hyperpolarization produced by local application of VIP (100 ftM in micropipette) was markedly

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decreased (P < 0.05, n = 10 in five animals), but that produced by local application of ATP (10 mm in pipette) was not significantly altered (P > 0 05, n = 10 in five animals; see Table 1 and Fig. 2). Perfusion with the known antagonist of P2Y-purinoceptors, Reactive Blue 2 (Burnstock & Warland, 1987; 100 JtM), produced a small decrease in RMP (control VIP 10-28

Control A -48 mV

___

-49 mV

VIP

VIP

-65 mV -

B -62 mV

ATP

ATP

J 10 mV 5s Fig. 2. Effect of VIP 10-28 (1 /uM) on the responses to local application of VIP and ATP. VIP 10-28 abolished the hyperpolarization induced by VIP but had no effect on the hyperpolarization induced by ATP.

RMP = -53-1 + 1-8 mV, 15 min; Reactive Blue 2 perfusion RMP =-50 9 +1 0 mV, P < 0 05, n = 8 in four animals). Desensitization of ATP (P2) receptors by means of prolonged perfusion with x, /8-methylene ATP (100 daM) had no effect on RMP after 30 min (control RMP = -54*3 + 1-2 mV, 30 min; a, /8-methylene ATP perfusion RMP = -53-9 + 1-3 mV, P > 0 05, n = 10 in five animals). ATP antagonism, by either Reactive Blue 2 or a, ,-methylene ATP desensitization, significantly antagonized the membrane hyperpolarization produced by local application of ATP (10 mm in pipette), but had no effect on the hyperpolarization produced by local application of VIP (100/,M in pipette; see Table 1 and Fig. 3). Membrane potential responses to transmural nerve stimulation In the presence of atropine and nifedipine, single pulses of transmural nerve stimulation resulted in an inhibitory junction potential (IJP) that reached a maximal amplitude of 12-9 + 0 5 mV at 378 + 20 ms from the stimulus and had a total duration (time from stimulus to return of hyperpolarization to RMP) of 1-5 + 01 s (n = 20 in five animals, Fig. 4). This IJP was abolished by apamin (1 gM) and has been previously designated the fast IJP (Niel et al. 1983; Bywater & Taylor, 1986; Crist & He, 1991; Crist, He & Goyal, 1991 a). As shown in Fig. 4, in the presence of apamin, increasing the stimulus train to four pulses at 20 Hz resulted in a different-appearing inhibitory junction potential which has previously been referred to as the slow IJP (Niel et al. 1983; Bywater & Taylor, 1986; Crist & He, 1991; Crist et al. 1991 a). This slow IJP was smaller in amplitude (maximal amplitude of 4-8 +0-2 mV), exhibited a greater time to maximum amplitude (time from stimulus to maximal amplitude of 14 + 041 s) and had a longer

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a, ,B-Methylene ATP

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-62 mV

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ATP

Reactive Blue 2 -59 mV

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-55 mV

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VIP

Reactive Bluai 2 LO I

-48mV

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A I VIP

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5s

Fig. 3. Effect of a, 8-methylene ATP desensitization (100 /zM for 30 min) and Reactive Blue 2 (200 IeM) on the responses to local application of VIP and ATP. A, the hyperpolarizations induced by ATP were antagonized by a, fl-methylene ATP desensitization and Reactive Blue 2. B, the hyperpolarizations induced by VIP were not affected by a, fl-methylene ATP desensitization or Reactive Blue 2. Control

Apamin

Apamin

-52 mV

-40 mV

-40 mV

J 10 mV 2s Fig. 4. A single pulse of transmural nerve stimulation (2 ms, 15 mA) resulted in an inhibitory junction potential (IJP) having a total duration of less than 2 s. This IJP was essentially abolished following perfusion with apamin (1 uM). In the presence of continued apamin perfusion, a short train of four pulses (2 ms, 15 mA, 20 Hz) resulted in a much smaller amplitude and longer duration IJP followed by an excitatory junction potential

(EJP).

duration of hyperpolarization (time from stimulus to return of hyperpolarization to RMP of 26 + 02 s) than the apamin-sensitive fast IJP (P < 005, n = 20 in five

animals).

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Effect of A TP and VIP antagonists on membrane potential responses to transmural nerve stimulation Fast IJP As shown in Fig. 5A and B, the fast IJP decreased in amplitude following desensitization to a, ,I-methylene ATP or perfusion with Reactive Blue 2. The effect Control A

-56 mV

a, ,B-Methylene ATP desensitization -54 mV

Wash

-54 mV

Reactive Blue 2

B

-57 mV

-53 mV

-56 mV

VIP 10-28

C

-52 mV

-47 mV

-48 mV

-0 mV 2s

Fig. 5. Effect of a, /3-methylene ATP desensitization, Reactive Blue 2 and VIP 10-28 on the IJP observed in response to a single pulse of transmural stimulation. A, a,,b/methylene ATP desensitization (100 #M for 30 min) caused a decrease in IJP amplitude that was reversed within 15 min of wash-out with Krebs solution. B, Reactive Blue 2 (200 /tM) caused a decrease in IJP amplitude that was only partially reversed following 30 min of wash-out with Krebs solution. C, VIP 10-28 (1 /tM) had no effect on the IJP

amplitude.

of desensitization of a, ,3-methylene ATP was rapidly reversed following wash-out with Krebs solution, whereas a more prolonged wash-out period (> 30 min) was required to entirely reverse the effect of Reactive Blue 2. As shown in Fig. 5C, VIP antagonism by means of perfusion with VIP 10-28 (1 sam) had no effect on the amplitude of the fast IJP Quantitative data concerning the effect of ATP and VIP antagonism on the fast IJP are provided in Table 2. Slow IJP As shown in Fig. 6A, the slow IJP decreased in amplitude following perfusion with VIP 10-28. ATP antagonism by means of desensitization to ac, ,-methylene ATP, however, had no effect on the amplitude of this slow IJP (Fig. 6B). Perfusion with

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< -45 mV

-45 mV a, ,B-Methylene ATP desensitization

B

-36 mV

-38 mV

-40 mV

Reactive Blue 2

C

-47 mV

--I

-45 mV

-45 mV 10 mV 2s

Fig. 6. Effect of a, /3-methylene ATP desensitization, Reactive Blue 2 and VIP 10-28 on the IJP observed in response to a short train of four pulses (2 ms, 15 mA, 20 Hz) in the presence of apamin (1 ,M). A, VIP 10-28 (1 ,UM) caused a decrease in IJP amplitude that was reversed following 15 min of wash-out with Krebs solution. B, a, ,l-methylene ATP desensitization (100 ,UM for 30 min) had no effect on IJP amplitude. C, Reactive Blue 2 (200 ,UM) caused an increase in IJP amplitude that persisted following 30 min of wash-out with Krebs solution. TABLE 2. Influence of a,,/-methylene ATP desensitization, Reactive Blue 2 and VIP 10-28 on amplitudes of fast and slow IJPs a, ,I-Methylene Reactive ATP desens. VIP 10-28 Blue 2 (100 uM for (1 ,tM) (200 /LM) Control 30 min) Control Control 6-7+0-2 12-4+06 12-1+1-9 5-3+07 13-8+0 7 Amplitude of 11 7+09 n = 18 n=9 n=7 fast IJP n=9 n=9 n=8 (P > 0 05) (mV) (P < 0 01) (P < 0 01) 6-6+0-8 1-0+0-3 7-2 +0-2 4-7+03 5-7+0-2 6-9+0-2 Amplitude of n=4 n=5 n=6 n=9 slow IJP n=3 n=3 (P > 0 05) (mV) (P < 001) (P > 0 05) Values are mean+ S.E.M. n = number of cells. P values = significance as compared to control values.

Reactive Blue 2 resulted in a small, but significant, increase in amplitude of the slow IJP (Fig. 6C). Quantitative data concerning the effect of ATP and VIP antagonism on the slow IJP are provided in Table 2.

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These studies provide evidence that both ATP and VIP are inhibitory neurotransmitters released from non-adrenergic, non-cholinergic nerves innervating the circular muscle layer of the guinea-pig ileum. Moreover, they suggest that ATP may be the neurotransmitter responsible for the fast IJP, and VIP the neurotransmitter responsible for the slow IJP observed in response to transmural nerve stimulation. There is considerable evidence in favour of ATP and VIP as inhibitory neurotransmitters in the circular muscle layer of the guinea-pig ileum. VIP-like immunoreactive myenteric neurons have been shown to run within the circular muscle layer (Costa & Furness, 1983). Similar immunohistochemical studies with ATP have not been performed due to the lack of a histochemical exploitable feature specific to a neuron that utilizes ATP as a transmitter (Furness & Costa, 1987). Isometric tension studies have shown that both ATP and VIP cause relaxation of circular muscle strips from the guinea-pig intestine (Cocks & Burnstock, 1979; Frew & Lundy, 1982; Costa et al. 1986; Grider & Makhlouf, 1986, 1987, 1988; Grider, 1989). Electrophysiological studies have shown that ATP, and in some studies VIP, cause membrane hyperpolarization of guinea-pig circular smooth muscle cells (Bauer & Kuriyama, 1982; Hills et al. 1983; Hoyle & Burnstock, 1989). Despite evidence that both ATP and VIP may be inhibitory neurotransmitters, there is presently no consensus as to the role of these two putative neurotransmitters in the inhibitory responses of the intestine. Some investigators (Cocks & Burnstock, 1979; Burnstock, Hokfelt, Gershon, Iversen, Kosterlitz & Szurszewski, 1979; MacKenzie & Burnstock, 1980; Hills et al. 1983) believe that ATP, and not VIP, is the non-adrenergic, noncholinergic inhibitory neurotransmitter. This is based on studies demonstrating that the relaxation (or IJP in electrophysiological studies) induced by transmural stimulation of intramural nerves is antagonized by the known potassium channel blocker apamin (Burnstock et al. 1979; MacKenzie & Burnstock, 1980; Hills et al. 1983). The ATP-induced relaxation (or hyperpolarization) could similarly be abolished by apamin, but the VIP-induced relaxation (or hyperpolarization) was not effected. Other investigators (Frew & Lundy, 1982; Grider, Cable, Said & Makhlouf, 1985; Grider & Makhlouf, 1986, 1988; Grider, 1989; Grider & Rivier, 1990) believe that VIP is the inhibitory neurotransmitter in the guinea-pig intestine. This is based on studies demonstrating that VIP antiserum antagonizes the descending inhibitory reflex in the guinea-pig colon (Grider & Makhlouf, 1986) and nerve-mediated relaxation of strips of circular smooth muscle from the guinea-pig stomach and colon (Grider et al. 1985; Grider & Makhlouf, 1988). In these studies, it was also observed that apamin significantly antagonized the nerve-mediated inhibitory responses and abolished ATP-induced muscle relaxation. Apamin, however, had no effect on VIPinduced relaxation (Grider & Makhlouf, 1987). This descrepancy in the ability of apamin to inhibit the nerve-mediated response and inability to inhibit the VIP response was explained by the finding that apamin acted pre-synaptically to inhibit the release of VIP (Grider & Makhlouf, 1987). This explanation, however, is contrary to other studies examining the effects of apamin in neuromuscular preparations. Other studies in the gastrointestinal tract, as well as a variety of other tissues,

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provide strong evidence to suggest that apamin acts selectively as a potassium channel blocker and has no action presynaptically to inhibit neurotransmitter release (Maas & Den Hertog, 1979; Maas, Den Hertog, Ras & Van den Akker, 1980; Maas, 1981; Hills et al. 1983; Daniel, Helmy-Elkholy, Jager & Kannan, 1983; Castle, Haylett & Jenkinson, 1989). In fact, several studies have shown that potassium channel blockers such as apamin cause an increase in neurotransmitter release through depolarization of the nerve terminal (Molgo, Lemeignam & Lechat, 1977; Kumamoto & Kuba, 1985). Our finding that the fast IJP is abolished by apamin and the slow IJP persists following apamin provides further evidence that apamin acts solely as a potassium channel blocker, and by this means selectively inhibits the ATP-induced hyperpolarization and fast IJP with no antagonizing effect on the VIPinduced hyperpolarization and slow IJP. The ability of apamin to antagonize the fast IJP in our study confirms previous studies suggesting that ATP acts by opening potassium channels (Maas & Den Hertog, 1979; Maas et al. 1980; Maas, 1981; Hills et al. 1983; Daniel et al. 1983; Castle et al. 1989). The mechanism by which VIP causes hyperpolarization was not examined in the present study. However, recent findings by us raise the possibility that VIP may produce hyperpolarization through the closure of chloride channels (Crist, He & Goyal, 1991 b). Additional studies are needed to address this question. In our studies, ATP antagonism by means of a, fl-methylene ATP desensitization or the ATP antagonist Reactive Blue 2 (Burnstock & Warland, 1987) and VIP antagonism by means of VIP 10-28 resulted in an incomplete blockade of the fast IJP and slow IJP, respectively. Although this inability to completely block these IJPs may be simply due to an absence of complete ATP and VIP antagonism, it remains possible that inhibitory neurotransmitters in addition to ATP and VIP are involved in these junction potentials. Recent studies have suggested that nitric oxide might be an inhibitory transmitter in gastrointestinal smooth muscle (Bult, Boeckxstaens, Pelckmans, Jordaens, Van Maercke & Herman, 1990; Toda, Baba & Okamura, 1990). Studies examining the possible role of nitric oxide in the fast and slow IJP are needed. This work was supported by USPHS grant DKO1430 and DK31092. REFERENCES

BAUER, V. & KURIYAMA, H. (1982). The nature of non-cholinergic, non-adrenergic transmission in longitudinal and circular muscles of the guinea-pig ileum. Journal of Physiology 332, 375-391. BORNSTEIN, J. C., COSTA, M., FURNESS, J. B. & LANG, R,. J. (1986). Electrophysiological analysis of projections of enteric inhibitory motoneurones in the guinea-pig small intestine. Journal of Physiology 370, 61-74. BULBRING, E. & TOMITA, T. (1967). Properties of the inhibitory potential of smooth muscle as observed in the response to field stimulation of the guinea-pig taenia coli. Journal of Physiology 189, 299-315. BULT, H., BOECKXSTAENS, G. E., PELCKMANS, P. A., JORDAENS, F. H., VAN MAERCKE, Y. M. & HERMAN, A. G. (1990). Nitric oxide as an inhibitory non-adrenergic non-cholinergic neurotransmitter. Nature 345, 346-347. BURNSTOCK, G., HOKFELT, T., GERSHON, M. D., IVERSEN, L. L., KOSTERLITZ, H. W. & SZURSZEWSKI, J. H. (1979). Non-adrenergic, non-cholinergic autonomic neurotransmission mechanisms. Neuroscience Research Program Bulletin 17, 377-519. 7 PHY 447

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BURNSTOCK, G. & WARLAND, J. J. I. (1987). P2-purinoceptors of two subtypes in the rabbit mesenteric artery: reactive blue 2 selectively inhibits responses mediated via the P2y- but not the P2x-purinoceptor. British Journal of Pharmacology 90, 383-391. BYWATER, R. A., HOLMAN, M. E. & TAYLOR, G. S. (1981). Atropine-resistant depolarization in the guinea-pig small intestine. Journal of Physiology 316, 369-378. BYWATER, R. A. & TAYLOR, G. S. (1986). Non-cholinergic excitatory and inhibitory junction potentials in the circular smooth muscle of the guinea-pig ileum. Journal of Physiology 374. 153-164. CASTLE, N. A., HAYLETT, D. G. & JENKINSON, D. H. (1989). Toxins in the characterization of potassium channels. Trends in Neurosciences 12, 59-65. COCKS, T. & BURNSTOCK, G. (1979). Effects of neuronal polypeptides on intestinal smooth muscle; a comparison with non-adrenergic, non-cholinergic nerve stimulation and ATP. European Journal of Pharmacology 54, 251-259. COSTA, M. & FURNESS, J. B. (1976). The peristaltic reflex: an analysis of the nerve pathways and their pharmacology. Naunyn-Schmiedeberg's Archives of Pharmacology 294, 47-60. COSTA, M. & FURNESS, J. B. (1983). The origins, pathways and terminations of neurons with VIPlike immunoreactivity in the guinea-pig small intestine. Neuroscience 8, 665-676. COSTA, M., FURNESS, J. B. & HUMPHREYS, C. M. (1986). Apamin distinguishes two types of relaxation mediated by enteric nerves in the guinea-pig gastrointestinal tract. NaunynSchmiedeberg's Archives of Pharmacology 332, 79-88. CRIST, J. R. & HE, X. D. (1991). Non-cholinergic membrane potential responses to transmural nerve stimulation in the guinea-pig ileum. American Journal of Physiology 260, G240-249. CRIST, J. R., HE, X. D. & GOYAL, R. K. (1991a). The nature of non-cholinergic membrane potential responses to transmural stimulation in guinea-pig ileum. Gastroenterology 100, 1006-1015. CRIST, J. R., HE, X. D. & GOYAL, R. K. (1991 b). Chloride mediated inhibitory junction potentials in circular muscles of guinea pig ileum. American Journal of Physiology 261, G742-751. DANIEL, E. E., HELMY-ELKHOLY, A., JAGER, L. P. & KANNAN, M. S. (1983). Neither a purine nor VIP is the mediator of inhibitory nerves of opossum oesophageal smooth muscle. Journal of

Physiology 336, 243-260. FREW, R. & LUNDY, P. M. (1982). Evidence against ATP being the nonadrenergic, noncholinergic inhibitory transmitter in guinea pig stomach. European Journal of Pharmacology 81, 333-336. FURNESS, J. B. & COSTA, M. (1987). The Enteric.Nervous System, pp. 44-89. Churchill-Livingstone, Edinburgh. GRIDER, J. R. (1989). Regulation of intestinal peristalsis by neuropeptides. Regulatory Peptide Letter 1, 1-5. GRIDER, J. R., CABLE, M. B., SAID, S. I. & MAKHLOUF, G. M. (1985). Vasoactive intestinal peptide as a neural mediator of gastric relaxation. American Journal of Physiology 248, G73-78. GRIDER, J. R. & MAKHLOUF, G. M. (1986). Colonic peristaltic reflex: identification of vasoactive intestinal peptide as mediator of descending relaxation. American Journal of Physiology 251, G40-45. GRIDER, J. R. & MAKHLOUF, G. M. (1987). Prejunctional inhibition of vasoactive intestinal peptide release. American Journal of Physiology 253, G7-12. GRIDER, J. R. & MAKHLOUF, G. M. (1988). Vasoactive intestinal peptide: Transmitter of inhibitory motor neurons of the gut. Annals of the New York Academy of Sciences 527, 369-377. GRIDER, J. R. & RIVIER, J. R. (1990). Vasoactive intestinal peptide (VIP) as transmitter of inhibitory motor neurons of the gut: evidence from the use of selective VIP antagonists and VIP antiserum. Journal of Pharmacology and Experimental Therapeutics 253, 738-742. HILLS, J. M., COLLIS, C. S. & BURNSTOCK, G. (1983). The effects of vasoactive intestinal polypeptide on the electrical activity of guinea-pig intestinal smooth muscle. European Journal of Pharmacology 88, 371-376. HOYLE, C. H. V. & BURNSTOCK, G. (1989). Neuromuscular transmission in the gastrointestinal tract. In Handbook of Physiology: The Gastrointestinal System, vol. 1: Motility and Circulation, ed. WOOD, J. D., pp. 435-464. American Physiological Society, Bethesda, MD, USA. KUMAMOTO, E. & KUBA, K. (1985). Effects of K'-channel blockers on transmitter release in bullfrog sympathetic ganglia. Journal of Pharmacology and Experimental Therapeutics 235, 241-247.

INHIBITORY ATEUROTRANSMITTERS IN THE ILEUM

131

MAAS, A. J. (1981). The effects of apamin on responses evoked by field stimulation in guinea-pig taenia caeci. European Journal of Pharmacology 73, 1-9. MAAS, A. J. & DEN HERTOG, A. (1979). The effect of apamin on the smooth muscle cells of the guinea-pig taenia coli. European Journal of Pharmacology 58, 151-156. MAAS, A. J., DEN HERTOG, A., RAS, R. & VAN DEN AKKER, J. (1980). The action of apamin on guinea-pig taenia caeci. European Journal of Pharmacology 67 (2-3), 265-274. MACKENZIE, I. & BURNSTOCK, G. (1980). Evidence against vasoactive intestinal polypeptide being the non-adrenergic, non-cholinergic inhibitory transmitter released from nerves supplying the smooth muscle of the guinea-pig taenia coli. European Journal of Pharmacology 67, 255-264. MOLGO, J., LEMEIGNAM, M. & LECHAT, P. (1977). Effects of 4-aminopyridine at the frog neuromuscular junction. Journal of Pharmacology and Experimental Therapeutics 203, 653-663. NIEL, J. P., BYWATER, R. A. & TAYLOR, G. S. (1983). Apamin-resistant post-stimulus hyperpolarization in the circular muscle of the guinea-pig ileum. Journal of the Autonomic Nervous System 9, 565-569. SMITH, T. K., BORNSTEIN, J. C. & FURNESS, J. B. 1990. Distension-evoked ascending and descending reflexes in circular muscle of guinea-pig ileum: an intracellular study. Journal of the Autonomic Nervous System 29, 203-218. SMITH, T. K. & FURNESS, J. B. (1988). Reflex changes in circular muscle activity elicited by stroking the mucosa: an electrophysiological analysis in the isolated guinea-pig ileum. Journal of the Autonomic Nervous System 25, 205-218. SMITH, T. K., FURNESS, J. B., COSTA, M. & BORNSTEIN, J. C. (1988). An electrophysiological study of the projections of motor neurones that mediate non-cholinergic excitation in the circular muscle of the guinea-pig small intestine. Journal of the Autonomic Nervous System 22, 115-128. TAYLOR, G. S. & BYWATER, R. A. (1986). Antagonism of non-cholinergic excitatory junction potentials in the guinea-pig ileum by a substance P analogue antagonist. Neuroscience Letters 63, 23-26. TODA, N., BABA, H. & OKAMURA, T. (1990). Role of nitric oxide in non-adrenergic, non-cholinergic nerve-mediated relaxation in dog duodenal longitudinal muscle strips. Japanese Journal of Pharmacology 53, 281-284. WOOD, J. D. (1989). Electrical and synaptic behaviour of enteric neurons. In Handbook of Physiology: The Gastrointestinal System, vol. 1: Motility and Circulation, ed. WOOD, J. D., pp. 465-517. American Physiological Society, Bethesda, MD, USA.

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Both ATP and the peptide VIP are inhibitory neurotransmitters in guinea-pig ileum circular muscle.

1. Intracellular membrane potential recordings were made from circular smooth muscle cells of the guinea-pig ileum in the presence of atropine (1 micr...
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