Abstract
The contribution of 5-hydroxytryptamine (serotonin; 5-HT) acting at 5-HT3 receptors to fast excitatory postsynaptic potentials (fEPSPs) and the properties of 5-HT3 receptors in the guinea pig small intestinal myenteric plexus were investigated using electrophysiological methods. In 11% of neurons studied in the acutely isolated myenteric plexus, ondansetron (1 μM) inhibited hexamethonium (100 μM)-resistant fEPSPs. 5-HT elicited an inward current in neurons maintained in primary culture. The peak current reached maximum in <150 ms and desensitized with a double exponential time course (τ1 = 1.1 ± 0.1 s; τ2 = 6.9 ± 0.9 s). The whole-cell current/voltage relationship was linear, with a reversal potential of 2.7 ± 1.5 mV. The rapidly activating and desensitizing current was completely blocked by ondansetron (1 μM) and partly inhibited by d-tubocurare (1 μM). The 5-HT3-receptor agonist, 2-methyl-5-HT (100 μM), caused a peak current that was 18% of the peak current caused by 5-HT in the same cells; 2-methyl-5-HT (1 μM) inhibited currents caused by 5-HT. 5-HT-activated single-channel currents in outside-out patches; this response was blocked by ondansetron. The single-channel conductance was 17 ± 1 pS. The single-channel current/voltage relationship was linear between −110 and 70 mV and had a reversal potential near 0 mV. These data indicate that 5-HT contributes to fEPSPs in the myenteric plexus. The 5-HT3 receptor expressed by guinea pig myenteric neurons has pharmacological and electrophysiological properties that distinguish it from 5-HT3 receptors expressed by other autonomic neurons and neurons in the central nervous system.
5-Hydroxytryptamine (serotonin; 5-HT) is a neurotransmitter and paracrine messenger substance in the gastrointestinal tract. In the gastrointestinal tract, 5-HT can act on at least four different receptors to alter neuronal activity. 5-HT acting at 5-HT1Preceptors is a mediator of some slow excitatory synaptic potentials in the myenteric and submucosal plexuses (Gershon, 1995). The 5-HT1P receptor is a G protein-linked receptor that couples to one or more intracellular signaling pathways, resulting in inhibition of resting potassium channels and membrane depolarization (Pan et al., 1997). 5-HT also acts at 5-HT1Areceptors, which couple via an unidentified signaling mechanism to activation of a potassium channel and neuronal inhibition (Galligan et al., 1988; Pan and Galligan, 1994; Galligan, 1996). 5-HT4 receptors are localized on cholinergic nerves, and activation of 5-HT4 receptors causes facilitation of acetylcholine (ACh) release (Pan and Galligan, 1994;Tonini and De Ponti, 1995). 5-HT1A and 5-HT4 receptors are G protein-linked receptors.
5-HT3 receptors are ligand-gated cation channels (Derkach et al., 1989; Fletcher and Barnes, 1998). In the gastrointestinal tract, 5-HT3 receptors are localized to enteric sensory nerve endings in the mucosal layer (Foxx-Orenstein et al., 1996; Bertrand et al., 1998). 5-HT released from enterochromaffin cells in response to stimulation of the mucosa acts on the nerve terminal 5-HT3 receptors to initiate motor reflexes (Foxx-Orenstein et al., 1996). 5-HT3 receptors are also localized to the nerve cell body of many enteric neurons, where they mediate rapidly developing and desensitizing depolarizations (Galligan, 1995, 1996). 5-HT3-mediated responses have a reversal potential near 0 mV and are due to activation of a nonspecific cation conductance (Yakel and Jackson, 1988; Derkach et al., 1989). Although most enteric nerves express 5-HT3 receptors, their contribution to synaptic excitation is unclear.
Although the macroscopic response caused by 5-HT3-receptor activation in the myenteric plexus is similar to that mediated at nicotinic ACh receptors (Zhou and Galligan, 1996a), the pharmacological or single-channel properties of 5-HT3 receptors in myenteric neurons have not been characterized. The purpose of our study was to determine whether 5-HT3 receptors contribute to synaptic excitation in acutely isolated myenteric plexus preparations and to examine in detail the properties of whole-cell and single-channel currents caused by 5-HT3-receptor activation in myenteric neurons maintained in primary culture.
Materials and Methods
Conventional Intracellular Electrophysiological Recording.
Guinea pigs (300–400 g) were obtained from the Michigan Department of Public Health (Lansing, MI). The animals were sacrificed by a blow to the head and bleeding through the major neck vessels after halothane inhalation anesthesia. This procedure was approved by the All University Animal Use and Care Committee at Michigan State University. A segment of ileum taken 10 cm proximal to the ileal-cecal junction was removed and placed in oxygenated (95% O2/5% CO2) Krebs’ solution in the following composition: 117 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 25 mM NaHCO3, 1.2 mM NaH2PO4, and 11 mM glucose. The solution contained nifedipine (1 μM) and scopolamine (1 μM) to block contractions of the muscle layer during the intracellular recordings. A segment of ileum (5 cm) was cut open along the mesenteric attachment and pinned flat in a silastic elastomer-lined Petri dish. The mucosa and submucosa were carefully peeled away, and a 5-mm2 section of longitudinal muscle myenteric plexus was cut out with fine scissors and forceps. The longitudinal muscle myenteric plexus preparation was transferred to a silastic elastomer-lined recording chamber and pinned flat. The chamber was then mounted on the stage of an inverted microscope (Nikon Diaphot), and the chamber was superfused continuously with warm (36°C) Krebs’ solution at a flow rate of 3.5 ml/min. Neurons were impaled with 2 M KCl-containing electrodes (tip resistance, 80–100 MΩ), and membrane potential was recorded with an Axoclamp 2A amplifier (Axon Instruments, Foster City, CA). Synaptic potentials were evoked by use of a focal electrode to stimulate the interganglionic connectives entering the ganglion containing the impaled neuron. The focal stimulating electrode was a glass pipette (tip diameter, 60 μm) filled with Krebs’ solution. Single stimuli (0.5 ms duration) were used to evoke fast excitatory postsynaptic potentials (fEPSPs). Stimuli were provided by a pulse generator (Master 8, A.M.P.I.) and a constant-current stimulus isolation unit. A maximal amplitude stimulus (4–9 mA) was used to elicit fEPSPs. The initial membrane potential was adjusted to approximately −90 mV to prevent the fEPSP (or drug responses, see below) from reaching action-potential threshold. This permitted a more accurate measurement of the amplitude of the fEPSP and the effect of drugs on fEPSP amplitude. Data were acquired with a Labmaster 125 analog-digital converter, a personal computer, and Axotape 2.0 software (Axon Instruments). Membrane-potential changes were sampled at 2 kHz and were filtered at 1 kHZ (4-pole Bessel filter; Warner Instruments, New Haven, CT). A digital average of six fEPSPs was used to measure fEPSP amplitude under control conditions and after drug treatments.
Tissue Culture.
Myenteric neurons were cultured via techniques described previously (Zhou and Galligan, 1998). Newborn guinea pigs (<36 h old) were sacrificed by severing the major neck blood vessels and spinal cord after deep halothane anesthesia. These procedures were also approved by the All University Committee on Animal Use and Care at Michigan State University. The small intestine was placed in cold (4°C) sterile-filtered Krebs’ solution of the following composition: 117 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 1.2 mM NaH2PO4, 25 mM NaHCO3, and 11 mM glucose. The longitudinal muscle myenteric plexus was removed from the entire length of small intestine and cut into 5-mm-long pieces. The dissected tissues were divided into four aliquots and placed in 1 ml of Krebs’ solution containing 1600 U of trypsin (Sigma Chemical Co., St. Louis, MO) for 25 to 30 min at 37°C. After trypsin incubation, the tissues were triturated 30 times and then centrifuged at 900g for 5 min with a bench-top centrifuge. The supernatant was discarded, and the pellet was resuspended in sterile Krebs’ solution and incubated (25–30 min, 37°C) in Krebs’ solution containing 2000 U crab hepatopancreas collagenase (Calbiochem-Novabiochem, Corp., La Jolla, CA). The suspension was triturated and then centrifuged for 5 min. The pellet was resuspended in Eagle’s minimum essential medium containing 10% fetal calf serum, gentamicin (10 μg/ml), penicillin (100 U/ml), and streptomycin (50 μg/ml) (all from Sigma). Cells were plated on plastic dishes coated with poly-l-lysine and maintained in an incubator at 37°C in an atmosphere of 5% CO2 for up to 2 weeks. After 2 days in culture, 10 μM cytosine arabinoside was added to the minimum essential medium to limit smooth muscle and fibroblast proliferation, and the medium was changed twice weekly thereafter.
Whole-Cell and Single-Channel Patch-Clamp Recording.
Whole-cell and outside-out patch-clamp recordings were obtained via standard methods. Recordings were carried out at room temperature with patch pipettes with tip resistances of 3 to 5 MΩ for whole-cell and 5 to 10 MΩ for single-channel currents in outside-out patches; seal resistances were >5 GΩ. The tips of pipettes used for single-channel recordings were coated with Sylgard (Dow Corning, Midland, MI). The pipette solution contained the following: 160 mM CsCl, 2 mM MgCl2, 1 mM EGTA, 10 mM HEPES, 1 mM ATP, and 0.25 mM GTP, the pH and osmolarity were adjusted to 7.4 (with CsOH) and 315 mosmol/kg (with CsCl), respectively. All recordings were made with an Axopatch 200A amplifier. Data were acquired with Axotape 2.0 and pClamp 6.0 software. Currents were sampled at 2 kHz and were filtered at 1 kHz (4-pole Bessel filter, Warner Instruments, Hamden, CT) and stored on a computer hard drive.
Current-voltage relationships for agonist-induced currents were obtained by measuring the amplitude of agonist-induced currents at holding potentials between −100 and 60 mV during a 2-s application of agonist with 2 min between successive agonist applications.
Drug Application.
Drugs were applied in three ways in studies with conventional intracellular electrophysiological methods to record from neurons in the intact myenteric plexus. 5-HT was applied from a fine-tipped (<10 μm) pipette positioned near the neuron. The concentration of 5-HT in the pipette was 1 mM and was ejected from the pipette using a brief nitrogen pulse (Picospritzer, General Valve Inc., Fairfield, NJ). The amplitude of single 5-HT responses under control conditions and in the presence of antagonists was measured. ACh was applied by iontophoresis onto the impaled neuron. The concentration of ACh in the iontophoretic electrode was 1 M, and ACh was ejected from the electrode via positive current (50–190 nA). A holding current of −6 nA was used to minimize leak of ACh from the iontophoretic electrode. A digital average of six ACh responses was used to measure the amplitude of the ACh response under control conditions and in the presence of antagonists. Antagonists were applied in known concentrations by adding the drugs to the superfusing Krebs’ solution.
Drugs were applied in two ways when using patch-clamp methods to study myenteric neurons maintained in primary culture. When constructing steady-state 5-HT concentration-response curves, 5-HT was applied onto individual neurons by gravity flow from a linear array of quartz tubes (320 μM i.d. and 450 μM o.d., Polymicron Technologies). The distance from the mouth of the tubes to the cell examined was ∼200 μM, and the position of the tubes containing different drug concentrations was controlled manually with a micromanipulator. In experiments requiring precise timing of the onset and offset of drug application, computer-controlled solenoid valves (General Valve Co.) were used to gate solution flow through the tubes. When using solenoid-controlled flow, the time required for the drug to reach steady-state concentration was estimated by measuring open-tip currents produced when the solution flowing onto the tip of a patch pipette was switched from one containing normal Krebs’ solution (117 mM NaCl) to one containing 58.5 mM NaCl. In some experiments, 5 to 10 mM additional NaCl was added to the Krebs-containing 5-HT solutions in the flow tube. The rate of solution exchange was verified by measuring the time course of open-tip currents after the neuron was expelled from the pipette tip with positive pressure. In these experiments, the time to the steady-state junction current was 9 ± 1 ms.
Statistics.
Data are expressed as means ± S.E. Student’s t test for paired data or ANOVA were used to establish significant differences between control and treatment groups. Agonist concentration-response curves obtained from individual neurons were fit with the following logistic function:
Results
Conventional Intracellular Recordings.
Recordings of fEPSPs were obtained from 100 neurons. At an initial membrane potential of −96 ± 2 mV, the average amplitude of the fEPSP in these neurons was 23 ± 1 mV. In 37 of these neurons, hexamethonium (C6) (100 μM) reduced the fEPSP to 4.0 ± 0.4 mV, or by 83 ± 1%. These neurons were not studied further. In 67 of these neurons, C6 reduced the fEPSP to 15 ± 1 mV, or by 34 ± 2%. The contribution of nerve-released 5-HT to the noncholinergic fEPSP was investigated in the latter group of neurons.
In 11 of 67 neurons, ondansetron inhibited the C6-resistant component of the fEPSP (Fig.1A). In this group of neurons, C6 (100 μM) inhibited the fEPSP by 37 ± 6% (P < .01; Fig. 2A). The C6-resistant component of the fEPSP was completely blocked by tetrodotoxin (Fig. 2A). C6inhibited responses caused by ACh iontophoresis by 95 ± 2% (Fig. 1B; P < .01) but did not affect responses caused by pressure-applied 5-HT. In two neurons, the control 5-HT responses were 19 and 30 mV, whereas, in the presence of C6, these responses were 24 and 26 mV, respectively. Ondansetron produced a concentration-dependent (0.1, 0.3, and 1 μM) inhibition of the C6-resistant fEPSP (Fig. 2B) but did not inhibit the response to iontophoretically applied ACh. The control ACh response was 25 ± 3 mV, whereas, in the presence of ondansetron (1 μM), the amplitude of the ACh response was 25 ± 2 mV (n = 4; P > .05). Similar data were obtained with the 5-HT3/5-HT4-receptor antagonist tropisetron. In seven neurons in which C6 produced an incomplete inhibition of the fEPSP, tropisetron (1 μM) inhibited the residual synaptic response by 34 ± 4% (P < .05).
These data suggest that 5-HT is a fast synaptic transmitter at some synapses in the guinea pig ileum myenteric plexus. There are differences in the electrophysiological and pharmacological properties of 5-HT3 receptors in different neurons. Therefore, the properties of guinea pig myenteric plexus 5-HT3 receptors were studied in detail with patch-clamp methods to record from myenteric neurons maintained in primary culture.
Whole-Cell Currents Caused by 5-HT and 2-Me-5-HT.
5-HT-induced currents were studied with whole-cell patch-clamp methods to record from myenteric neurons maintained in primary culture for between 7 and 20 days. 5-HT (30 μM) caused an inward current in 95 of 120 (79%) cells tested. The 5-HT-induced current was biphasic, with an initial rapidly developing peak that desensitized in the presence of agonist and a slower developing, sustained inward current (Fig.3A). At −70 mV, the peak amplitude of the 5-HT-induced current was −517 ± 28 pA (Fig. 3A). Complete concentration-response curves for 5-HT were obtained in seven neurons in which the EC50 for 5-HT was 8 ± 2 μM (Fig. 3B).
2-Me-5-HT (100 μM) caused a peak inward current of 88 ± 12 pA (n = 7); this was only 18 ± 1% of the maximum response caused by 5-HT in the same neurons (Fig. 3, A and B). The EC50 value for 2-Me-5-HT was 14 ± 3 μM. These data suggest that 2-Me-5-HT is a partial agonist at 5-HT receptors expressed by myenteric neurons. To test this possibility directly, 5-HT concentration-response curves were obtained in the absence and presence of 10 μM 2-Me-5HT, a concentration that produced little or no response itself. 2-Me-5-HT produced a 4-fold rightward shift in the 5-HT concentration-response curve (Fig. 3C). In these cells, the control 5-HT EC50 was 10 ± 1 μM, whereas, in the presence of 2-Me-5-HT, the 5-HT EC50 was 39 ± 5 μM (n = 4; P < .05).
Inward currents caused by 5-HT and 2-Me-5-HT were inhibited by ondansetron (1 μM). Ondansetron completely blocked the rapidly developing inward current caused by 5-HT (30 μM), leaving a sustained inward current (Fig. 4, A and B). Ondansetron also blocked the peak of the rapidly developing and desensitizing inward current caused by 2-Me-5-HT (30 μM), leaving a sustained inward current (Fig. 4, C and D). Taken together, these data indicate that myenteric neurons express 5-HT3receptors that mediate a desensitizing inward current. An ondansetron-insensitive 5-HT receptor mediates a sustained inward current.
The current-voltage relationship for 5-HT-induced currents was determined by measuring the peak amplitude of currents caused by 5-HT at holding potentials between −110 and 70 mV (Fig.5A). The current-voltage relationship was linear in this range of membrane potentials; chord conductances at −70 and 70 mV were 7.9 ± 1 and 7.2 ± 1.4 nS, respectively (Fig.5B). The reversal potential for the 5-HT-induced current determined from the current-voltage plots was 2.7 ± 1.5 mV (n = 6).
Kinetics of 5-HT3-Mediated Whole-Cell Currents.
The rate of solution exchange measured by the time to steady state of open-tip junction currents recorded after expelling the neuron from the pipette was 9 ± 1 ms (n = 5) (Fig.6A). In these neurons, the 10 to 90% rise time of the 5-HT-induced current was 127 ± 19 ms (n = 5). The data described above indicate that 5-HT was acting at two receptors to cause an inward current: a 5-HT3 receptor and an ondansetron-insensitive receptor. To study the rate of 5-HT3-receptor desensitization, the response mediated at this receptor needs to be isolated. This was accomplished by recording 5-HT responses in the absence and presence of 1 μM ondansetron (Fig. 6B). Responses recorded in the presence of ondansetron were subtracted from control responses to yield the ondansetron-sensitive current (Fig. 6B). These studies revealed that the 5-HT3-mediated inward current desensitized with double exponential time course (τ1 = 1.1 ± 0.1 s; τ2 = 6.9 ± 0.9 s; n = 5).
5-HT-Induced Single-Channel Currents in Outside-Out Patches.
5-HT (10–30 μM) activated single-channel currents in 18 of 24 (75%) patches tested at a patch potential of −110 mV. Single-channel currents in the absence of 5-HT were rarely observed. When recordings were obtained with ATP and GTP omitted from the pipette solution, the single-channel amplitude at −110 mV was 1.9 ± 0.1 pA (n = 4 patches; Fig. 7, A and B). This value was derived from Gaussian fits of the amplitude/frequency histograms constructed from recordings from four patches and yielded a single-channel conductance of 17 ± 1 pS. A smaller amplitude event of 0.9 ± 0.03 pA (8.2 pS) was observed in some recordings (Fig. 7A); however, this conductance occurred too infrequently to alter the overall amplitude/frequency histogram (Fig.7A). 5-HT-induced single-channel currents were blocked completely by ondansetron (1 μM) in three patches tested (Fig.8, A and B). Under control conditions, the open probability in the presence of 10 μM 5-HT was 0.04 ± 0.001, whereas, in the presence of ondansetron, the open probability was 0.0003 ± 0.0003 (P < .05).
The current-voltage relationship for the single-channel current was constructed from cursor measurements of single-channel amplitudes at patch potentials between −110 and −50 mV and between 30 and 70 mV. Single-channel currents between −50 and 20 mV were too small in amplitude to measure reliably. The single-channel current-voltage relationship was linear, with the cord conductance at −70 mV of 17 ± 1 pS and at 70 mV of 19 ± 1 pS (n = 10).
Discussion
Drugs acting at 5-HT3 receptors can alter gastrointestinal motility (Tonini and De Ponti, 1995; Sanger, 1996). Studies in vivo showed that 5-HT3-receptor antagonists delay gastrointestinal transit in mice and rats (Nagakura et al., 1996; Ito et al., 1997), and ondansetron, may be effective in treating gastrointestinal motility disorders in humans (Sanger, 1996;Wilde and Markham, 1996). 5-HT can also affect peristalsis in vitro (Bulbring and Crema, 1958), and initiation of peristalsis in guinea pig colon involves stimulation of sensory nerves by endogenous 5-HT acting at 5-HT3 receptors (Foxx-Orenstein et al., 1996). Terminals of intestinal sensory nerves express 5-HT3 receptors (Hillsley and Grundy, 1998;Bertrand et al., 1998) that are activated by 5-HT released from enterochromaffin cells in response to mucosal stimulation (Gershon, 1995). 5-HT may also contribute to ganglionic transmission responsible for peristalsis. In rabbit colon, ondansetron inhibits descending relaxation of circular muscle caused by distention oral to the recording site (Messori et al., 1995). In dog and guinea pig intestine, 5-HT3 receptors participate in neurotransmission mediating ascending excitation (Neya et al., 1993; Yuan et al., 1994). However, there has been no direct demonstration of synaptic responses mediated by 5-HT3 receptors in enteric nerves.
FEPSPs recorded from myenteric neurons are caused by ACh acting at nicotinic receptors and by other transmitters. The noncholinergic component of the fEPSP is mediated largely by ATP acting at P2X receptors, but some fEPSPs persist in the presence of C6 and P2X-receptor antagonists (Galligan and Bertrand, 1994; LePard et al., 1997; LePard and Galligan, 1999). In 11% of neurons, fEPSPs are inhibited by C6 and ondansetron or tropisetron. C6 blocked responses to ACh, whereas neither ondansetron nor tropisetron affected ACh responses. Therefore, C6-resistant fEPSPs are not due to incomplete block of nicotinic receptors, and ondansetron or tropisetron do not block nicotinic receptors. ACh and 5-HT must mediate fEPSPs recorded from some myenteric neurons.
It is unclear whether ACh and 5-HT are released from the same nerves. 5-HT-containing neurons contain choline acetyltransferase, suggesting that 5-HT and ACh could be cotransmitters (Steele et al., 1991; Young and Furness, 1995). In addition, there are neurons that accumulate 5-HT that also contain choline acetyltransferase (Steele et al., 1991;Meedeniya et al., 1998). The 5-HT/ choline acetyltransferase-containing neurons have long (up to 100 mm), aborally directed projections and comprise 11% of all aborally projecting neurons (Meedeniya et al., 1998). The small number of 5-HT-containing neurons would account for the infrequent observation of 5-HT3-mediated fEPSPs detected in this study.
5-HT may be released by descending interneurons. Therefore, 5-HT3-mediated fEPSPs would be recorded from descending interneurons or inhibitory motorneurons receiving input from descending interneurons. 5-HT-containing terminals rarely contact inhibitory motor neurons (Young and Furness, 1995), so it is likely that 5-HT3-receptor-mediated fEPSPs were recorded from descending interneurons. However, 5-HT3-receptor antagonists do not block the descending inhibitory component of the peristaltic reflex (Yuan et al., 1994) but do inhibit the ascending excitatory component in the same preparations. It is possible that 5-HT3-mediated fEPSPs contribute to descending motor responses other than those involved in peristalsis. Alternatively, there may be an unidentified ligand released from ascending interneurons that is an agonist at 5-HT3 receptors (Yuan et al., 1994).
Properties of 5-HT3-Mediated Whole-Cell Currents.
5-HT elicited a biphasic inward current in most neurons. This response was characterized by a rapidly developing and desensitizing current and a small, sustained current. Similar results were obtained with the 5-HT3-receptor agonist 2-Me-5-HT; however, the peak response caused by 2-Me-5-HT was only 20% of that caused by 5-HT. These data indicate that 2-Me-5-HT is a partial agonist at guinea pig enteric 5-HT3 receptors as in other cells (Butler et al., 1990; Hussy et al., 1994; Niemeyer and Lummis, 1998), and 2-Me-5-HT should block the 5-HT3 receptor. We found that a low concentration of 2-Me-5-HT caused a rightward shift in the 5-HT concentration-response curve, indicating that 2-Me-5-HT is a 5-HT3-receptor antagonist in enteric neurons.d-Tubocurare (d-TC) is an antagonist with low nanomolar affinity for 5-HT3 receptors (Hussy et al., 1994; Jones and Surprenant, 1994). However, the 5-HT3 receptor in myenteric neurons is resistant to antagonism by d-TC. The mechanism of block of 5-HT3 receptor by d-TC is unclear, but the structural features that provide d-TC sensitivity must be absent in guinea pig myenteric plexus 5-HT3receptors. Ondansetron inhibited peak currents elicited by 5-HT and 2-Me-5-HT. However, in the presence of ondansetron, both agonists induced a sustained inward current. The 5-HT receptor mediating the sustained current was not identified, but the time course of this response is consistent with it being mediated by 5-HT1P receptors (Gershon, 1995).
Current-Voltage Relationship.
Currents activated by 5-HT reversed near 0 mV, consistent with this response being mediated by an increase in a nonspecific cation conductance (Yakel and Jackson, 1988;Derkach et al., 1989). The current-voltage relationship was linear and similar to that obtained from heterologously expressed guinea pig enteric 5-HT3 receptors (Lankiewicz et al., 1998). However, 5-HT3 receptors from human, rat, and mouse tissues exhibit marked inward rectification (Hussy et al., 1994; Fletcher and Barnes, 1998; Lankiewicz et al., 1998). This may be because of intrinsic differences in the conducting properties of the 5-HT3-receptor subunit(s) expressed by different cells or modifications caused by unidentified 5-HT3-receptor subunits (Fletcher and Barnes, 1998).
Kinetics of 5-HT3-Receptor-Mediated Current.
To study the properties of currents caused by 5-HT3-receptor activation, the contribution of the sustained current to the total current needed to be removed. Because the receptor mediating the sustained current was not identified, it was not possible to block this response pharmacologically. However, the contribution of the sustained current to the total 5-HT-induced current was removed by subtracting ondansetron-insensitive currents. The 5-HT3-mediated inward current reached a peak in <150 ms, similar to that for long- and short-form guinea pig enteric 5-HT3 receptors expressed in HEK293 cells (Lankiewicz et al., 1998). The response mediated at native 5-HT3 receptors in myenteric neurons desensitized with a double exponential time course, whereas desensitization of responses mediated at heterologously expressed guinea pig enteric 5-HT3 receptors decayed with a linear time course. There are several explanations for the difference in desensitization kinetics between native and heterologously expressed 5-HT3 receptors. Desensitization of 5-HT3 receptors may be regulated by receptor phosphorylation (Yakel and Jackson, 1988; Boddeke et al., 1996), and intracellular regulatory mechanisms in HEK293 cells may differ from those in myenteric neurons. In addition, the subunit composition of native 5-HT3 receptors may be different from receptors composed of single subunits expressed heterologously (van Hooft et al., 1997). Native receptors may have regulatory sites absent from expressed receptors. Finally, simultaneous activation of other 5-HT receptors expressed by myenteric neurons could activate intracellular mechanisms that regulate 5-HT3-receptor function.
Single-Channel Currents.
5-HT activated single-channels in outside-out patches whose properties were unaffected by the absence or presence of ATP and GTP in the recording pipette solution. In addition, channel activation caused by 5-HT was blocked by ondansetron. These data indicate that 5-HT-activated single-channel currents are mediated by directly-gated 5-HT3 receptors.
Previous studies of myenteric and submucosal neurons from guinea pig intestine showed that the pharmacological properties of the 5-HT3 receptors in these two plexuses were similar (Vanner and Surprenant, 1990). However, patch-clamp studies of submucosal neurons showed that 5-HT activated either a single channel with two conductance levels or two channels with conductances of 14 and 9 pS (Derkach et al., 1989). Only one prominent conductance level of 17 pS was identified in myenteric neurons, although rare events of smaller amplitude were observed. Variations in tissue-culture conditions could account for the apparent differences in the single-channel properties of 5-HT3 receptors in myenteric and submucosal neurons. However, it is also possible that the structure of the 5-HT3 receptor in submucosal neurons differs from that in myenteric neurons. Thus far, only one 5-HT3-receptor subunit has been identified in guinea pig intestine (Lankiewicz et al., 1998), but a second 5-HT3-receptor subunit (5-HT3B) was cloned recently (Davies et al., 1999). When 5-HT3B subunits are coexpressed with 5-HT3A receptors, the resulting heteromeric receptor exhibits the properties of native neuronal 5-HT3 receptors. There may be other 5-HT3-receptor subunits that contribute to the unique properties of submucosal and myenteric neuronal 5-HT3 receptors.
Conclusions.
These studies showed that 5-HT3 receptors contribute to fEPSPs at some synapses in guinea pig small intestinal myenteric plexus. Based on the known aboral projection of 5-HT-containing neurons in the myenteric plexus, the 5-HT3 synapses are likely to play a role in descending motor or secretomotor responses. The properties of myenteric 5-HT3 receptors differ in their rate of desensitization, current-voltage relationship, and gating properties from nicotinic and P2X receptors (Zhou and Galligan, 1996b). P2X and nicotinic receptors also mediate fEPSPs in the myenteric plexus. These different properties may be important during bursts of fEPSPs or during coactivation of fast and slow excitatory synaptic pathways.
Footnotes
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Send reprint requests to: James J. Galligan, Ph.D., Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI 48824. E-mail: galliga1{at}pilot.msu.edu
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↵1 This study was supported by National Institutes of Health Grants NS-33289 and NS-01738.
- Abbreviations:
- ACh
- acetylcholine
- C6
- hexamethonium
- d-TC
- d-tubocurare
- fEPSP
- fast excitatory postsynaptic potential
- 2-Me-5-HT
- 2-methyl-5-hydroxytryptamine
- Received January 5, 1999.
- Accepted April 9, 1999.
- The American Society for Pharmacology and Experimental Therapeutics