ATP is a mediator of the fast inhibitory junction potential in human jejunal circular smooth muscle

L. Xue1, G. Farrugia1,2, M. G. Sarr3, and J. H. Szurszewski1,2

1 Department of Physiology and Biophysics, 2 Division of Gastroenterology and Hepatology, and 3 Department of Surgery, Mayo Clinic, Rochester, Minnesota 55905


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The neurotransmitter(s) that generates the fast component of the inhibitory junction potential (IJP-F) in human jejunal circular smooth muscle is not known. The aim of this study was to determine the role of ATP and purinergic receptors in the generation of the IJP-F in human jejunal circular smooth muscle strips. The P2-receptor antagonist suramin (100 µM) reduced the IJP-F by 28%. Apamin (1 µM) reduced the IJP-F by 25%. Desensitization of muscle strips with the putative P2x-receptor agonist alpha ,beta -methylene ATP (alpha ,beta -MeATP, 100 µM) decreased the IJP-F by 44%, and desensitization with the putative P2y-receptor agonist adenosine 5'-O-2-thiodiphosphate (ADPbeta S) completely abolished the IJP-F. Desensitization with the putative P2y-receptor agonist 2-methylthioATP had no effect on the IJP-F. Exogenous ATP evoked a hyperpolarization with a time course that matched the IJP-F. The ATP-evoked hyperpolarization was reduced by apamin and suramin, reduced by desensitization with alpha ,beta -MeATP (69% decrease), and abolished by desensitization with ADPbeta S. These data suggest that the IJP-F in human jejunal circular smooth muscle is mediated in part by ATP through an ADPbeta S-sensitive P2 receptor.

neurotransmission; microelectrodes; purinergic receptors


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ELECTRICAL FIELD stimulation (EFS) of enteric nerves in the presence of blockers of both cholinergic and adrenergic receptors results in a nonadrenergic, noncholinergic (NANC) inhibitory junction potential (IJP) in mammalian gastrointestinal smooth muscle accompanied by smooth muscle relaxation (3). The NANC IJP can alter gastrointestinal motility by directly inducing smooth muscle relaxation as a result of membrane hyperpolarization or indirectly by inhibiting action potentials and hence decreasing contractility. The shape of the NANC IJP and the neurotransmitter(s) that mediates the NANC IJP have been studied extensively in various animal species. In the canine small intestine, the NANC IJP evoked by EFS consists primarily of a fast monophasic hyperpolarization, the amplitude of which is frequency dependent (21). The duration of the fast hyperpolarization evoked by EFS at 30 Hz is on the order of 2.5 s (21). The NANC IJP in the canine jejunum can be abolished by inhibitors of nitric oxide synthase (NOS) such as NG-nitro-L-arginine methyl ester (L-NAME) and NG-nitro-L-arginine (L-NNA); moreover, the change in membrane voltage evoked by exogenous application of nitric oxide (NO) mimics the IJP. These observations suggest that the IJP in canine small intestine is mediated by NO (21). In contrast, in human and guinea pig gastrointestinal circular smooth muscle, the NANC IJP consists of an initial fast hyperpolarization (IJP-F), similar to the IJP in the canine smooth muscle, followed by a slower, longer-lasting hyperpolarization of smaller amplitude (IJP-S). (7, 18-20). In human jejunal circular smooth muscle, EFS at 30 Hz evokes an IJP-F with a duration of 2 s followed by the IJP-S with a duration of about 13 s (20). A similar but less pronounced biphasic NANC IJP is also seen in guinea pig circular smooth muscle (7, 18-19). In both human and guinea pig, the NANC IJP-S can be abolished by pretreatment with L-NAME and L-NNA and mimicked by exogenous application of NO (13, 20). However, in both human and guinea pig, inhibitors of NOS have no effect on the IJP-F, suggesting it is not mediated by NO.

ATP was proposed by Burnstock and colleagues (4) in 1970 as a NANC-inhibitory neurotransmitter in gastrointestinal smooth muscle. Since then, convincing evidence supports this hypothesis (2, 12). In guinea pig ileal circular smooth muscle, ATP mediates the apamin-sensitive NANC IJP-F (9). Also in guinea pig ileal circular smooth muscle, the NANC IJP-F appears to be mediated by ATP because it is antagonized by alpha ,beta -methylene ATP (alpha ,beta -MeATP) desensitization and by reactive blue 2, a purinergic receptor agonist and antagonist, respectively, and it is blocked by apamin, a blocker of a class of small and intermediate conductance calcium-activated potassium channels (9). ATP, acting through P2 receptors, is also implicated in the NANC IJP-F in guinea pig colonic circular smooth muscle (16, 24).

The identity of the neurotransmitter mediating the NANC IJP-F in the human small intestine is, however, not known. The aim of the present study was to determine whether ATP mediates the IJP-F in human jejunal circular smooth muscle.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Smooth muscle of the human jejunum was obtained as surgical waste tissue from 29 patients undergoing gastric bypass surgery for morbid obesity after approval by the Institutional Review Board at the Mayo Clinic. The segment of jejunum was taken ~30-45 cm from the duodenojejunal junction and placed immediately in iced, preoxygenated Krebs solution and transported to the laboratory consistently within 30 min. The jejunal segments were opened along their antimesenteric borders and transferred to fresh oxygenated Krebs solution. After removal of the mucosal using a binocular microscope, full-thickness muscle strips (1 × 10 mm) were prepared with the long axis cut parallel to the circular muscle. Muscle strips were then placed in a 3-ml recording chamber with the circular muscle facing up. One end of the muscle strip was pinned to a Sylgard-coated (Dow Corning, Midland, MI) floor of the chamber to record intracellular electrical activity, whereas the other end was attached to an isometric force transducer to record mechanical activity. The chamber was perfused with prewarmed (37°C) and preoxygenated Krebs solution at a constant rate of 3 ml/min. After an equilibration period of at least 2 h, the muscle strips were stretched to an initial tension of 250 mg. Intracellular electrical recordings were obtained from circular smooth muscle cells using glass capillary microelectrodes filled with 3 M KCl and with resistances ranging from 30 to 80 MOmega . Intracellular potentials were amplified using a WPI M-707 amplifier (WPI, New Haven, CT) and displayed on an oscilloscope (Tektronix 5113, Tektronix, Beaverton, OR). Force was measured isometrically and amplified with a bridge circuit amplifier. Both electrical signals and mechanical activity were recorded on chart paper (Gould 220, Gould, Cleveland, OH) and also with an FM tape recorder (Hewlett-Packard 3964A, Hewlett-Packard, San Diego, CA). Two platinum wires parallel to the long axis of the preparation and connected through a square-wave stimulator (Grass 588, Grass, Quincy, MA) and a stimulus isolation unit (Grass SIU 5A) were used to apply EFS. Individual electrical pulses were of 0.35-ms duration, and 100- to 150-V intensity. The range of frequencies evaluated was 1-30 Hz with six pulses per application. ATP was administered by a pressure application device (picospritzer, General Valve Company, East Hanover, NJ). A micropipette (10 µm diam) filled with 0.1 M ATP was placed as close as possible to the recording electrode. Pressure pulses at 12 psi and 60-ms duration were used to deliver ATP.

Krebs solution had the following ionic composition (in mM): 127.4 Na+, 5.9 K+, 2.5 Ca2+, 1.2 Mg2+, 134 Cl-, 15.5 HCO-3, 1.2 H2PO-4, and 11.5 glucose. The solution was aerated with 97% oxygen-3% CO2, and maintained at pH 7.4. Atropine, propranolol, and phentolamine (1 µM each) were present in all solutions used in the study. These drugs and apamin, suramin, TTX, L-NNA, ATP, alpha ,beta -MeATP, adenosine 5'-O-2-thiodiphosphate (ADPbeta S), and 2-methylthioATP (2-MeSATP) were obtained from Sigma Chemical (St. Louis, MO).

All observed values are expressed as the means ± SE. Statistical significance was determined using paired and nonpaired Student's t-test. P < 0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

General observations. The mean resting membrane potential recorded from circular smooth muscle cells of the human jejunum was -59.9 ± 0.4 mV (n = 108 cells from 25 preparations). EFS elicited IJPs and smooth muscle relaxation in all preparations. No excitatory junction potentials were observed. The NANC IJPs evoked by an EFS stimulus of six pulses, each 0.35 ms at 30 Hz, consisted of a fast hyperpolarization (IJP-F: amplitude 26.7 ± 0.7 mV, duration 1.55 ± 0.1 s, n = 96 from 25 preparations) followed by a slow hyperpolarization (IJP-S: amplitude 4.8 ± 0.2 mV, duration 5.5 ± 2.3 s, n = 96 from 25 preparations). The amplitude and duration of both the IJP-F and IJP-S increased in a frequency-dependent manner. A simultaneous recording of electrical and mechanical activity showing the response to several different frequencies of EFS is shown in Fig. 1. The characteristics of IJPs and smooth muscle relaxation in the human jejunum were similar to those previously reported for the canine small intestine (21).


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Fig. 1.   Simultaneous recordings of mechanical (top trace in each panel) and intracellular electrical activity (bottom trace in each panel) in response to electrical field stimulation (EFS; delivered at , 6 pulses each 0.35 ms at 30 Hz) in presence of atropine, phentolamine, and propranolol (each 1 µM) in human jejunal circular smooth muscle. Nonadrenergic, noncholinergic (NANC) inhibitory junction potential (IJP) consisted of initial fast (IJP-F) followed by slow hyperpolarization (IJP-S) accompanied by muscle relaxation. Amplitudes of IJP-F and IJP-S were frequency dependent.

Effect of NOS inhibition on NANC IJP. L-NNA (100 µM), an inhibitor of NOS, had no effect on the IJP-F (26.2 ± 2.4 mV in Krebs solution compared with 26.6 ± 2.2 mV in presence of L-NNA, P > 0.05, n = 5 from 5 preparations, Fig. 2A) but completely abolished the IJP-S (5.5 ± 0.6 mV in Krebs solution compared with 0 ± 0 mV in presence of L-NNA, n = 5 from 5 preparations, P < 0.05, Fig. 2A).


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Fig. 2.   Effect of NG-nitro-L-arginine (L-NNA), inhibitor of nitric oxide synthase, of suramin, P2-purinoceptor antagonist, and of apamin on IJP-F and IJP-S evoked by EFS (6 pulses each 0.35 ms, 30 Hz, ). A: L-NNA (100 µM) had no effect on IJP-F but completely inhibited IJP-S. B: suramin (100 µM) inhibited IJP-F but had no effect on IJP-S even after 36-min exposure. C: apamin (1 µM, 16 min) decreased amplitude of both IJP-F and IJP-S.

Effect of purinergic receptor agonists and antagonists on NANC IJP induced by EFS. Suramin (100 µM), a nonspecific P2-purinoceptor antagonist, was used to determine whether ATP, acting through P2 purinoceptors, mediated the NANC IJP-F and IJP-S. Suramin significantly reduced IJP-F (26.8 ± 1.2 mV in Krebs solution compared with 19.3 ± 1.8 mV in the presence of suramin, n = 5 from 3 preparations, P < 0.05, Fig. 2B) but had no significant effect on IJP-S (3.25 ± 0.8 mV in Krebs solution compared with 4.25 ± 1.8 mV in the presence of suramin, n = 5 from 3 preparations, P > 0.05, Fig. 2A).

In gut smooth muscle, the apamin-sensitive component of the NANC IJP is thought to be mediated by ATP (26). Apamin (1 µM) partially inhibited both the IJP-F and IJP-S. The amplitude of IJP-F evoked by EFS (6 pulses each of 0.35 ms at 30 Hz) was significantly reduced (27%) by apamin (28.5 ± 1.2 mV in Krebs solution compared with 20.5 ± 0.9 mV in the presence of apamin, n = 7 from 4 preparations, P < 0.001, Fig. 2C). The amplitude of the IJP-S was reduced by 58% (5.2 ± 0.5 mV in Krebs solution compared with 2.2 ± 0.3 mV in the presence of apamin, n = 7 from 4 preparations, P < 0.001, Fig. 2C). Increasing the concentration of apamin tenfold (10 µM) failed to further reduce the amplitude of either the IJP-F or IJP-S (data not shown).

Effect of P2x- and P2y-receptor agonists and antagonists on NANC IJP. To determine which subtype of P2 receptors mediated the effects of suramin and apamin on the IJP-F, the putative P2x-receptor agonist alpha ,beta -MeATP was used to desensitize the P2x receptor. Tissues were incubated for 30 min in alpha ,beta -MeATP (100 µM), and the amplitude and duration of the IJP were recorded. A transient hyperpolarization (6.6 ± 0.4 mV, n = 9 from 4 preparations) lasting 9.5 ± 1 min occurred on adding alpha ,beta -MeATP to the bath after which the membrane potential repolarized back to the baseline value. After desensitization with alpha ,beta -MeATP, the amplitude of IJP-F decreased (35.5 ± 1.4 mV in Krebs solution compared with 20.0 ± 1.1 mV in the presence of alpha ,beta -MeATP, n = 9 from 4 preparations, P < 0.05, Fig. 3A), whereas the amplitude of IJP-S was unchanged (4.5 ± 0.4 mV in Krebs solution compared with 5.4 ± 0.3 mV in the presence of alpha ,beta -MeATP desensitization, n = 9 from 4 preparations, P > 0.05, Fig. 3A). Desensitization (30 min, n = 3 from 2 preparations) with alpha ,beta -MeATP (100 µM) in the presence of apamin (1 µM) did not further inhibit the IJP-F (Fig. 3B).


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Fig. 3.   Effect of desensitization of purinergic receptors by P2x-receptor agonist alpha ,beta -methylene ATP (alpha ,beta -MeATP) and by P2y-receptor agonist 2-methylthio-ATP (2-MeSATP) on IJP-F and IJP-S evoked by EFS (6 pulses each 0.35 ms, 30 Hz). A: alpha ,beta -MeATP desensitization (100 µM for 30 min) decreased amplitude of IJP-F with no effect on IJP-S. B: in presence of apamin (1 µM, 20-min exposure), alpha ,beta -MeATP desensitization had no effect on either IJP-F or IJP-S. C: desensitization to 2-MeSATP (50 µM for 30 min) had no effect on either IJP-F or IJP-S evoked by EFS (delivered at ).

Desensitization of P2y receptors with 2-MeSATP and ADPbeta S, both putative P2y agonists, was also tested. 2-MeSATP had no significant effect on the amplitude of IJP-F (22.2 ± 1.9 mV in Krebs solution compared with 19.1 ± 2.4 mV in the presence of 2-MeSATP, n = 3 from 3 preparations, P > 0.05, Fig. 3C) nor did it affect the amplitude of IJP-S (3.2 ± 0.54 mV in Krebs solution compared with 2.5 ± 0.4 mV in the presence of 2-MeSATP, n = 3 from 3 preparations, P > 0.05, Fig. 3C). In contrast, desensitization for 20 min with ADPbeta S (100 µM, n = 4 from 2 preparations) completely abolished the IJP-F (21.8 ± 2 mV in Krebs solution compared with 0 ± 0 mV in the presence of ADPbeta S; P < 0.001, Fig. 4) without affecting the IJP-S (4.8 ± 0.6 mV in Krebs solution compared with 5.5 ± 1.3 mV in the presence of ADPbeta S, P > 0.05). A transient hyperpolarization occurred on adding ADPbeta S to the bath after which the membrane potential repolarized back to the baseline value. The transient hyperpolarization was not blocked by TTX (1 µM, n = 2, data not shown), suggesting that it was due to a postjunctional effect of ADPbeta S. In tissue strips pretreated with L-NNA (100 µM, n = 2 from 1 preparation), addition of ADPbeta S blocked completely both the IJP-F and the IJP-S (Fig. 4B). The effects of apamin, suramin, alpha ,beta -MeATP, 2-MeSATP, ADPbeta S, and L-NNA on the IJP-F and the IJP-S are summarized in Fig. 5, A and B, respectively.


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Fig. 4.   Effect of desensitization of purinergic receptors by P2y-receptor agonist adenosine 5'-O-2-thiodiphosphate (ADPbeta S) on IJP evoked by EFS (delivered at , 6 pulses each 0.35 ms, 30 Hz). A: ADPbeta S desensitization (100 µM for 20 min) abolished IJP-F with no effect on IJP-S. B: preincubation with L-NNA (100 µM for 20 min) followed by ADPbeta S desensitization (100 µM for 20 min) completely abolished both IJP-F and IJP-S.




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Fig. 5.   Summary of effects of apamin, suramin, alpha ,beta -MeATP, 2-MeSATP, ADPbeta S, and L-NNA on IJP-F (A) and IJP-S (B). Numbers in bars reflect number of cells tested. Control, response to EFS; * P < 0.05 compared with control.

Effect of exogenous ATP. The partial inhibition of the IJP-F by the P2-purinoceptor antagonist suramin and by alpha ,beta -MeATP desensitization, and the complete block of the IJP-F by ADPbeta S suggested that ATP, acting through P2 purinoceptors, was involved in the generation of IJP-F. Therefore, the effect of exogenous ATP was tested to determine if exogenous ATP, applied via a picospritzer, would mimic the IJP-F. ATP (0.1 M) evoked a membrane hyperpolarization of 7.1 ± 0.6 mV with a duration of 16,571.4 ± 1,435 ms (n = 43 from 19 preparations, Fig. 6). The time to 50% maximum amplitude and to peak hyperpolarization were 1,200 ± 114.6 ms and 2,657.1 ± 193.6 ms, respectively (n = 33 from 14 preparations). The voltage trajectory of the initial hyperpolarizing response evoked by exogenous ATP was similar to that of an IJP-F evoked by EFS. The time to 50% maximum amplitude and to peak hyperpolarization of the IJP-F were 760 ± 161 ms and 1,700 ± 220 ms, respectively (n = 9 from 5 preparations). Unlike the initial hyperpolarizing response, the duration of the hyperpolarizing response to exogenous ATP varied from preparation to preparation (Fig. 6, A and B). Superimposition of the time course of an ATP-evoked hyperpolarization and an IJP evoked by EFS is shown in Fig. 6.


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Fig. 6.   Effect of ATP on membrane potential of human jejunal circular smooth muscle cells. A and B: typical hyperpolarizations evoked by exogenous ATP (0.1 M, via picospritzer at ). Note initial hyperpolarization is similar in both, whereas duration of hyperpolarization differs markedly. Similar responses, with an initial fast hyperpolarization sustained for variable time period, were obtained in 42 other cells. C: comparison of time course of ATP-evoked hyperpolarization and of IJP evoked by EFS (0.35-ms pulse, 30 Hz, 100 V). Both recordings were made from same cell. Note that slope of ATP-evoked hyperpolarization was similar to that of IJP-F. EFS applied at .

In three other cells tested, exogenously applied ATP evoked a different response consisting of a biphasic change in membrane potential. After the initial hyperpolarization, a depolarization was noted (5.5 ± 0.8 mV, duration of 22,666.7 ± 1,453.0 ms, data not shown). In one cell, ATP evoked a brief depolarization followed by hyperpolarization (data not shown).

The effect of ATP was also examined in the presence of apamin, suramin, alpha ,beta -MeATP, 2-MeSATP, or ADPbeta S. Apamin (1 µM, Fig. 7A) abolished the hyperpolarization evoked by ATP (0.1 M). Suramin (100 µM) reduced the ATP-evoked hyperpolarization (10.1 ± 1.2 mV in Krebs solution compared with 4.8 ± 0.7 mV in the presence of suramin, n = 3 from 3 preparations, P < 0.05, Fig. 7B). Desensitization for 15 min with alpha ,beta -MeATP (100 µM) inhibited the ATP-evoked hyperpolarization (8 ± 0.8 mV in Krebs solution compared with 2.5 ± 0.7 mV in the presence of alpha ,beta -MeATP, n = 3 from 3 preparations, P < 0.05, Fig. 7C). Desensitization for 15 min with ADPbeta S (100 µM) completely abolished the effect of ATP (10 ± 1 mV in Krebs solution compared with 0 ± 0 mV in the presence of ADPbeta S, n = 2 from 2 preparations, P < 0.05, Fig. 7E). Desensitization for 30 min with 2-MeSATP (100 µM) had no effect on the ATP-evoked hyperpolarization (8.3 ± 1.5 mV in Krebs solution compared with 7.9 ± 1.0 mV in the presence of 2-MeSATP, n = 2 from 2 preparations, P > 0.05, Fig. 7D). L-NNA applied to the bath for 30 min had no effect on the ATP-evoked hyperpolarization (Fig. 7F). The effects of apamin, suramin, alpha ,beta -MeATP, 2-MeSATP, ADPbeta S, and L-NNA on the ATP-evoked hyperpolarization are summarized in Fig. 8.


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Fig. 7.   Effects of apamin, suramin, alpha ,beta -MeATP, 2-MeSATP, ADPbeta S, and L-NNA on hyperpolarizating response evoked by exogenous ATP (0.1 M, via picospritzer at black-down-triangle ). A: apamin (1 µM for 20 min) abolished hyperpolarization evoked by ATP. B: suramin (100 µM for 28 min) decreased amplitude of ATP-evoked hyperpolarization. C: desensitization by alpha ,beta -MeATP (100 µM for 30 min) inhibited hyperpolarization evoked by ATP. D: desensitization by 2-MeSATP (100 µM for 30 min) had no effect on ATP-evoked hyperpolarization. E: ADPbeta S desensitization (100 µM for 20 min) completely abolished hyperpolarization evoked by ATP. F: L-NNA (100 µM for 20 min) had no effect on hyperpolarization evoked by ATP.



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Fig. 8.   Summary of effects of apamin, suramin, alpha ,beta -MeATP, 2-MeSATP, ADPbeta S, and L-NNA on hyperpolarization evoked by ATP (0.1 M, via picospritzer). Numbers in bars reflect number of cells tested. Control, response to ATP; * P < 0.05 compared with control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This report provides data supporting the hypothesis that ATP is one of the neurotransmitters that mediates the IJP-F in human jejunum circular smooth muscle. Three lines of evidence support this conclusion: 1) purinergic receptor block by the P2-purinoceptor antagonist suramin or desensitization of purinergic receptors inhibited the IJP-F evoked by EFS, 2) application of ATP via a picospritzer mimicked closely the IJP-F, and 3) desensitization of purinergic receptors or P2-purinoceptor blockade by suramin markedly decreased the effects of exogenous ATP.

In this study suramin decreased the amplitude of the IJP evoked by EFS and blocked the effect of exogenous ATP on membrane voltage. These data suggest that the effect of ATP was mediated by a P2 receptor. The P2 receptor subtypes are a family of ion channels that conduct Na+, K+, and rarely Ca2+. The P2 receptors identified so far are P2x, P2y, and P2z receptors, each having several subtypes (11). P2x receptors are ligand-gated ion channels that conduct K+, Na+, and rarely Ca2+. P2y receptors are coupled to intracellular second-messenger systems. Unequivocal identification of the subtype that mediates the EFS-evoked IJP-F in human jejunal circular smooth muscle and the response to exogenous ATP is not possible for two reasons. First, currently available P2-receptor agonists and antagonists do not allow absolute and confident discrimination between P2 receptor subtypes (cf. Ref. 25). For example, alpha ,beta -MeATP, a putative selective P2x agonist, acts as a P2y agonist in guinea pig taenia coli (1), and ADPbeta S, a putative P2y receptor agonist, acts on P2x receptors in rat urinary bladder (22). Second, several P2 receptor types are expressed in the same tissue. In a recent study (25), the effect of ADPbeta S was carefully examined in circular muscle of guinea pig colon. At least three types of P2 receptors were found, including inhibitory P2 receptors activated by alpha ,beta -MeATP and endogenous purines and blocked by suramin and pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), inhibitory P2 receptors activated by ADPbeta S but resistant to suramin and PPADS, and excitatory P2 receptors activated by ADPbeta S and blocked by suramin and PPADS. In addition, there was evidence for a pool of specialized junctional P2 receptors mediating the NANC IJP (25).

In the present study on human jejunal circular smooth muscle, prolonged application of alpha ,beta -MeATP, a putative selective P2x agonist, decreased the amplitude of the IJP-F evoked by EFS and blocked the effect of exogenous ATP, effects presumably mediated by receptor desensitization. Similar attempts at desensitization of the P2y receptor by prolonged application of the putative P2y agonist 2-MeSATP had no effect on the IJP. However, the putative P2y agonist ADPbeta S completely abolished the IJP-F and blocked the effect of exogenous ATP. These data suggest that the IJP-F and the effect of ATP on membrane voltage in human jejunal circular smooth muscle cells were mediated through an ADPbeta S-sensitive P2 receptor that was also partly sensitive to alpha ,beta -MeATP. This receptor appears to be different from the receptor thought to mediate the NANC IJP in guinea pig colonic circular smooth muscle as the pharmacology of the P2 receptor activated by alpha ,beta -MeATP in guinea pig matched the pharmacology of the IJP, whereas the receptor activated by ADPbeta S did not (25).

Apamin, a blocker of subtypes of small and intermediate conductance calcium-activated potassium channels, inhibited a component of the human jejunal IJP-F. Also, receptor desensitization with alpha ,beta -MeATP following preincubation with apamin did not further block the IJP-F, suggesting that ATP mediated its effects through an apamin-sensitive pathway. Because apamin also blocked part of the slow component of the IJP mediated by NO (20), it appears that the effects of apamin in human jejunal circular smooth muscle cannot be used as a selective marker of purinergic involvement in inhibitory neurotransmission by ATP.

The single channel ionic conductances that mediate the effects of ATP on human gastrointestinal smooth muscle membrane voltage are unknown. Recently, small conductance (5-10 pS) and intermediate conductance (~39 pS) apamin-sensitive potassium channels have been described in murine ileal and colonic smooth muscle (14, 23). P2y-purinoceptor agonists activated both the small and intermediate conductance potassium channels, suggesting that these channels may mediate membrane hyperpolarization evoked by ATP in the mouse. Whether similar channels are also present in human jejunal circular smooth muscle is unknown. Together with potassium channels, other channels may also be involved in the generation of the IJP. In the guinea pig ileum, chloride channels have been suggested to play a role in the generation of excitatory junction potential as well as NANC IJPs (8).

In 3 of 15 preparations studied in the present report, exogenous ATP evoked a biphasic change in the membrane potential, which consisted of an initial hyperpolarization followed by a long-lasting depolarization. Similar effects of local application of ATP have been reported in chicken rectum and in guinea pig urinary bladder, suggesting that ATP may function not only as an inhibitory neurotransmitter but also may have a role as an excitatory neurotransmitter (5, 15). Purine-related rebound excitation may be another possible explanation for the depolarization noted in the small number of preparations in this report. The messengers involved in purine-related rebound excitation are not fully understood, but in the guinea pig taenia coli (6), rat duodenum (17), and mouse colon (10) they appear to depend on prostaglandin synthesis because indomethacin attenuates the rebound excitation.

In summary, exogenous application of ATP in human jejunal circular smooth muscle evokes a membrane hyperpolarization accompanied by smooth muscle relaxation. The effect of ATP appears to be mediated via an ADPbeta S-sensitive P2 receptor. ATP appears to be involved in the generation of the IJP-F because exogenous ATP closely mimicked the IJP-F, and desensitization of purinergic receptors or use of the P2-receptor antagonist suramin attenuated the IJP-F. These data suggest that the IJP in human jejunal circular smooth muscle appears to be mediated by several neurotransmitters. ATP appears to be one of the mediators of the IJP-F, whereas NO mediates the IJP-S.


    ACKNOWLEDGEMENTS

We thank Gary Stoltz for technical assistance and Kristy Zodrow for secretarial assistance.


    FOOTNOTES

This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-17238, DK-52766, and DK-39337.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: G. Farrugia, Mayo Clinic, Guggenheim 8, 200 First St. SW, Rochester, MN 55905.

Received 29 September 1998; accepted in final form 23 February 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastroint Liver Physiol 276(6):G1373-G1379
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