PGE2 hyperpolarizes gallbladder neurons and inhibits synaptic potentials in gallbladder ganglia

Lee J. Jennings and Gary M. Mawe

Department of Anatomy and Neurobiology, The University of Vermont, Burlington, Vermont 05405

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Gallbladder prostaglandin E2 (PGE2) levels are significantly elevated in pathophysiological conditions, resulting in changes in gallbladder motility or secretion that may involve actions of the prostanoid in intramural ganglia. This study was undertaken to examine the effects of PGE2 on neurons of the intramural ganglia of the guinea pig gallbladder. Application of PGE2 by microejection or superfusion elicited a complex triphasic change in the resting membrane potential (RMP). For example, application of PGE2 by microejection (100 µM) resulted in a brief hyperpolarization (mean duration 11.1 ± 1.3 s), followed by a mid-phase repolarization toward or above RMP (mean duration 50.7 ± 8.1 s), and finally a long-lasting hyperpolarization (mean duration 157.3 ± 36.7 s). Associated with these PGE2-evoked alterations in RMP were changes in input resistance measured via injection of hyperpolarizing current pulses. An examination of the action potential afterhyperpolarization (AHP) during the PGE2-evoked response revealed an attenuation of both the amplitude and duration of the AHP. However, only a slight increase in excitability of gallbladder neurons in the presence of PGE2 was evident in response to depolarizing current pulses, and PGE2 did not cause the cells to fire spontaneous action potentials. Application of PGE2 reduced the amplitudes of both fast and slow excitatory synaptic potentials. These results suggest that increased prostaglandin production may decrease ganglionic output and therefore contribute to gallbladder stasis.

neuroimmune interactions; innervation; myenteric; presynaptic modulation; enteric nervous system

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

PROSTAGLANDINS, particularly prostaglandin E2 (PGE2), have been shown to be intimately associated with cholecystitis (27). Early studies (40) employing diseased human gallbladders demonstrated that both the mucosa and the muscularis of the organ produce high levels of PGE2. Furthermore, a correlation between severity of inflammation and PGE concentrations has been observed (16). Clinical studies using indomethacin, an inhibitor of cyclooxygenase activity, have shown that symptoms of acute cholecystitis can be significantly reduced, indicating a potential role for prostaglandins in the inflammatory pathophysiology (34). In animal model studies, PGE2 has been shown to have two major effects: a dose-dependent contraction of the tissue (39) and a significant reversal in net fluid movement from absorption to secretion, including an increase in mucin secretion (18, 37). These two observations may reflect a cytoprotective role for PGE2 in the gallbladder, at least in acute inflammation, by expelling gallbladder contents and preserving mucosal integrity. In several investigations (13, 14, 28, 29) it has been noted indirectly that PGE2 probably exerts its effects, at least in part, through the neural network resident within the organ. This is not surprising considering that PGE2 has been demonstrated to have significant effects on the release of acetylcholine (ACh) from the enteric nervous system (4, 11, 15).

It has previously been demonstrated from intracellular recording studies that the ganglia of the gallbladder are a target for several neural and hormonal signals that influence this organ (21). For example, at picomolar concentrations, cholecystokinin (CCK) acts to facilitate the release of ACh from vagal terminals via CCK-A receptors (23, 25). Conversely, norepinephrine released from sympathetic postganglionic nerve terminals acts to suppress vagal ACh release through the presynaptic alpha 2-adrenoceptor (22, 25). In addition, sensory afferent neurotransmitters that can be released by capsaicin (19) mediate slow excitatory postsynaptic potentials (EPSPs), recorded from gallbladder neurons, and enhance neuron excitability, possibly through a local axon reflex (9, 24).

The aim of this study was to characterize the effects of PGE2 on the electrical properties of gallbladder neurons. To achieve this, PGE2 was applied to gallbladder whole mount preparations, while the activities of individual neurons were recorded with intracellular microelectrodes.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

The methods that were employed in this study have been described previously in considerable detail (21, 23). Adult guinea pigs of either sex, weighing 250-350 g, were used for this study. Animals were anesthetized with isoflurane until no hindlimb reflex was evident and then exsanguinated. This method was reviewed and approved by the Institutional Animal Care and Use Committee of the University of Vermont. The gallbladder was immediately removed and opened with a single incision from the end of the cystic duct to the base of the gallbladder. It was then pinned flat, mucosal side up, under recirculating ice-cold Krebs solution in a dish lined with Sylgard 184 elastomer (Dow Corning, Midland, MI). The mucosal layer and underlying connective tissue were gently removed with forceps under microscopic observation. The preparations were then pinned out in a Sylgard-lined tissue chamber and placed on the stage of an inverted microscope (Diaphot, Nikon). Individual ganglia were visualized at ×200 with Hoffman modulation contrast optics (Modulation Optics, Greenvale, NY).

The preparations were continuously perfused at a rate of 10-12 ml/min with a modified Krebs solution that was aerated with 95% O2-5% CO2, and the temperature was 36-37°C at the recording site. The solution contained (in mM) 121 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 25 NaHCO3, 1.2 NaH2PO4, and 8 glucose. Nifedipine (0.5 µM) was added to the Krebs solution to minimize smooth muscle contractions.

Glass microelectrodes used for intracellular recording were filled with 2.0 M potassium chloride and had resistances in the range of 50-110 MOmega . A negative-capacity compensation amplifier (Axoclamp 2A; Axon Instruments, Foster City, CA) with bridge circuitry for injecting positive and negative current pulses (0.1-0.5 nA, 0.5-500 ms, 0.5-1 Hz) was used to record membrane potentials. Synaptic inputs were elicited using monopolar extracellular electrodes made from Teflon-insulated platinum wire (25-µm diameter) to apply 0.2- to 0.5-ms duration stimuli (0.2-20 Hz frequency) to the interganglionic connectives.

In some experiments, the composition of the Krebs solution was altered to facilitate the examination of a particular response. In these instances, molarity was compensated for by adjustment of NaCl within the solution. For example, when the K+ concentration was raised from 5.9 to 20 mM, the NaCl concentration was reduced from 121 to 107 mM.

Compounds were applied by pressure microejection from glass micropipettes (0.01-1.0 mM in Krebs solution; 15- to 20-µM tip diameter) by pulses of nitrogen gas (300 kg/cm2; 10-1,000 ms in duration) or by addition to the circulating Krebs solution. The distance between the tip of the spritz micropipette and the impaled neuron was maintained between 50 and 100 µm. PGE2 was purchased from Sigma Chemical (St. Louis, MO). For stock solutions, PGE2 was dissolved in dimethyl sulfoxide (DMSO) and diluted at least 1,000 times before tissue application. Microejection or superfusion of the drug vehicle (DMSO) at relevant concentrations did not have any measurable effect on the active or passive properties of gallbladder neurons.

Averaged numerical values are presented as means ± SE, and P < 0.05 (Student's t-test) was considered significant.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Data were obtained from 187 gallbladder neurons in 141 different guinea pig gallbladders. The passive and active membrane characteristics of these cells were similar to those reported previously for guinea pig gallbladder neurons (21). The cells usually generated only one action potential at the onset of a depolarizing current pulse, regardless of the duration or amplitude of the pulse, and anodal break action potentials were rarely observed in control conditions. The cells studied here had a mean resting membrane potential (RMP) of -52.3 ± 2.1 mV and a membrane input resistance of 75.8 ± 6.2 MOmega .

PGE2 elicits a triphasic change of the RMP. Application of PGE2 to gallbladder neurons by either microejection or superfusion elicited a complex triphasic change of the RMP. The characteristic PGE2-evoked response was triphasic, with three distinct components: early, mid-, and late phases.

The early phase consisted of a membrane hyperpolarization whose magnitude was measured from the RMP to the peak of the hyperpolarization. The mid-phase of the PGE2-evoked response was characterized by a membrane repolarization back toward the initial RMP, which in the majority of neurons led to a membrane depolarization. The amplitude of this mid-phase was calculated from the peak of the preceding membrane hyperpolarization to the peak of the repolarization/depolarization. The late phase of the response consisted of a second membrane hyperpolarization whose amplitude was measured from RMP to the peak of the hyperpolarization.

The duration of the early phase hyperpolarization was calculated by measuring the time from the onset of the response to the point that the cell repolarized to the RMP. In cells that did not return to the RMP, the slope of the repolarization was extrapolated to the RMP and this provided the second time point. The duration of the mid-phase repolarization was taken from the point that the cell returned to the RMP (or from slope extrapolation) after the initial hyperpolarization to the point at which the late phase hyperpolarization started. The duration of the late phase hyperpolarization was measured from the end of the preceding repolarization/depolarization to a return to the RMP or until the impalement was lost. It was impossible to separate these phases and examine them individually, and as a consequence it is likely that each phase could have had an effect on the amplitude and/or the duration of the preceding phase.

Microejection of PGE2. Application of PGE2 by pressure microejection (1 µM-1 mM; 0.1-3 s) elicited a complex change of the RMP of gallbladder neurons as mentioned above (Fig. 1A). All three phases were seen at all concentrations applied. For example, at a concentration of 100 µM (n = 12), the early phase consisted of membrane hyperpolarization having a mean magnitude of 9.3 ± 1.6 mV (range: 3.4-18.9 mV) and a mean duration of 11.1 ± 1.3 s (range: 5.4-21.4 s). A decrease in membrane input resistance (16.6 ± 7.8%), measured by changes in response to injection of hyperpolarizing current pulses, was associated with this early hyperpolarization.


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Fig. 1.   A: application of prostaglandin E2 (PGE2) (100 µM, 400 ms) evoked a characteristic triphasic change in resting membrane potential (RMP) consisting of an early hyperpolarization, a repolarization toward or above RMP, and a long-lasting late hyperpolarization. Associated with these events were fluctuations in input resistance as measured by changes in electrotonic potentials produced by injecting hyperpolarizing current pulses. B: the response of these neurons to PGE2 (100 µM, 200 ms) was not altered by the presence of TTX (0.5 µM), indicating that PGE2 was probably having a direct effect on the neurons. RMP (indicated by dashed line) for cells were -52 mV (A) and -41 mV (B).

The mean peak amplitude of the mid-phase repolarization (at 100 µM) was 14.2 ± 1.3 mV (range: 5.4-21.4 mV) with a mean duration of 50.7 ± 8.1 s (range: 17.8-100 s). A decrease in membrane input resistance (58.6 ± 4.7%) was evident during the mid-phase membrane repolarization.

The late phase of the PGE2-induced triphasic response consisted of a long-lasting hyperpolarization with a mean peak amplitude of 4.3 ± 0.7 mV (range: 1.2-8.7 mV). Of the cells exposed to 100 µM PGE2 by microejection only 5 of the 12 returned to the RMP. In these cells, the mean duration of the late phase was 157.3 ± 36.7 s (range: 92.9-220 s). An increase in membrane input resistance (18.5 ± 7.4%) accompanied the late phase hyperpolarization.

The possibility that any component of the PGE2-induced potential change was a secondary effect due to PGE2-induced release of neurotransmitters from nerve terminals was tested by application of PGE2 in the presence and absence of tetrodotoxin (TTX). In three cells, the presence of 0.5 µM TTX (Fig. 1B) did not significantly alter the early phase amplitude or duration (P = 0.21 and 0.18, respectively), the mid-phase amplitude or duration (P = 0.34 and 0.29, respectively), or the late phase amplitude or duration (P = 0.48 and 0.51, respectively). In addition, we examined the response of gallbladder neurons to PGE2 (1 mM, 400 ms) in a Ca2+-free Krebs solution (3.7 mM MgCl) and compared these with control responses. On examination (n = 3), neither the early phase amplitude or duration (P = 0.51 and 0.43, respectively), the mid-phase amplitude or duration (P = 0.14 and 0.8, respectively), nor the late phase amplitude or duration (P = 0.18 and 0.47, respectively) was significantly altered compared with control responses.

The magnitude of the response to PGE2 was dependent on the duration of PGE2 microejection. The minimal pulse duration that resulted in a detectable response varied among the cells, but was usually in the range of 50 to 100 ms. Progressive increase in duration of PGE2 application resulted in slightly larger responses, with a maximal response usually occurring with application durations in the range of 1-3 s. The maximal response in a given cell was quite reproducible when the preparation was rinsed for periods of 5 min between applications.

Superfusion of PGE2. Superfusion of PGE2 (10 pM-10 µM) evoked a concentration-dependent triphasic change in the RMP with characteristics similar to those seen when PGE2 was applied by microejection (Fig. 2). The peak amplitude (11.3 ± 1.9 mV; range: 6.1-19.2 mV) of the early hyperpolarization was seen at a concentration of 1 µM. Of the eight cells studied at this concentration, two cells were lost immediately after the initial hyperpolarization, four cells had a mean duration of 22.1 ± 3.9 s (range: 13.9-2.5 s), and two cells remained hyperpolarized for longer than 4 min. The maximum amplitude of the mid-phase repolarization (6.8 ± 0.7 mV; range: 2.5-9.5 mV) occurred at 10 µM and had a duration of 62.5 ± 11.5 s (range: 22.3-112.1 s; n = 6). The amplitude of the late phase hyperpolarization also was greatest at a concentration of 10 µM (8.6 ± 1.9 mV; range: 3.3-16.1 mV), and of the six cells studied only two returned to the RMP (duration >2 min). The other cells remained hyperpolarized throughout the recording. Superfusion of the drug vehicle (DMSO) at relevant concentrations did not have any measurable effect on the membrane potential of gallbladder neurons.


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Fig. 2.   Application of PGE2 elicited a concentration-dependent (10 pM-10 µM) triphasic change in RMP of gallbladder neurons consisting of an early hyperpolarization phase, followed by a mid-phase repolarization toward or above RMP, and finally a late long-lasting hyperpolarization phase. Maximal responses were seen at a concentration of 1 µM for the early phase and at 10 µM for the mid- and late phases. Nos. in parentheses indicate no. of cells studied at each concentration, and the RMP (indicated by the dashed line) of the cell shown here was -48 mV.

Ionic currents that underlie the PGE2-evoked responses. To examine the ionic currents potentially involved in the complex PGE2-evoked voltage response, PGE2 was applied to cells that were current clamped at potentials more positive or more negative than the RMP (Fig. 3).

In eight cells, the amplitude of the early phase hyperpolarization decreased at more negative potentials and also decreased at membrane potentials more positive than RMP. Unfortunately, the current-voltage relationship of gallbladder neurons rectifies significantly in the depolarizing direction (21). Therefore, the estimated reversal potential was plotted using responses obtained at RMP and at membrane potentials more negative than RMP. The mean estimated reversal potential for the early phase was -81.9 ± 11.6 mV, a value close to the predicted (Nernst equation) K+ equilibrium potential (-92.8 mV assuming that the internal K+ concentration is equal to 190 mM) for these cells (21).

The amplitude of the mid-phase repolarization increased at more negative potentials and decreased when the RMP was current clamped toward 0 mV. Because of the rectification behavior of these neurons, as mentioned above, it was difficult to depolarize the neurons to levels that were sufficiently positive to detect a true reversal of the mid-phase response. However, estimated reversal potentials were obtained by plotting the membrane potentials against the responses obtained and using a line of best fit (assuming linearity) to derive the estimated reversal potentials. The mean estimated reversal potential for the mid-phase of the PGE2-evoked response was -3.2 ± 11.6 mV.

The amplitude of the late phase hyperpolarization became larger at more negative membrane potentials, but the response could often be reversed when the cells were current clamped at or close to 0 mV. The mean estimated reversal potential for this late phase hyperpolarization was -30.9 ± 7.8 mV.

Given the reversal potential data and decrease in input resistance, we hypothesize that the PGE2-evoked early hyperpolarization may involve the activation of a K+ conductance. To test this, reversal potential measurements were made using Krebs solution containing either 5.9, 12, or 20 mM KCl. When the KCl concentration was increased from 5.9 to 12 mM the mean reversal potential was shifted from -81.9 ± 11.6 to -77.8 ± 4.3 mV (n = 3). The predicted K+ equilibrium with 12 mM KCl equaled -73.8 mV. In Krebs solution with 20 mM KCl the mean reversal potential was estimated to be -68.2 ± 6.5 (n = 5), whereas the predicted K+ equilibrium potential was -60.2 mV (Fig. 4). In addition, the amplitude of the early phase of hyperpolarization was decreased as the KCl concentration was increased. Also, a true reversal of the hyperpolarization could be elicited at the higher KCl concentrations. The mid- and late phases of the PGE2-evoked potential change were not significantly altered by the presence of the higher KCl concentrations. The reversal potential estimated for the mid-phase of the PGE2 response was -9.9 ± 1.7 mV (n = 3) in 12 mM KCl and was -8.0 ± 4.7 mV (n = 4) in 20 mM KCl. The mean estimated reversal potential for the late phase was -22.4 ± 2.2 mV (n = 3) in 12 mM KCl and was -25.1 ± 7.2 mV (n = 4) in 20 mM KCl.


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Fig. 3.   A: the PGE2-evoked (100 µM, 400 ms) response of a single gallbladder neuron, current clamped at levels more positive and more negative than RMP (-42 mV), differed dependent on the membrane potential of the cell. B: scatter graphs demonstrating the relationship between amplitude of response and membrane potential in 8 neurons. Mean estimated reversal potential of each phase of the response is indicated by the filled symbol. The amplitude of the early phase hyperpolarization became smaller at membrane potentials more negative and more positive than the RMP and had a mean estimated reversal potential of -82 mV (calculated from cells current clamped to potentials more negative than RMP). The amplitude of the mid-phase repolarization became larger at membrane potentials more negative than RMP and smaller at membrane potentials more positive than RMP (mean estimated reversal potential = -3 mV). The amplitude of the late long-lasting hyperpolarization became larger at membrane potentials more negative than RMP and smaller or reversed at membrane potentials more positive than RMP. The mean estimated reversal potential for this late phase was -30.9 mV.


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Fig. 4.   Increasing the external K+ concentration decreased the amplitude of the early hyperpolarization, reversed the response at membrane potentials more negative than the RMP, and shifted the reversal potential to the right in close agreement with the predicted K+ equilibrium potential.

To test further the hypothesis that the early hyperpolarization phase was due to the activation of a K+ conductance, we determined the effect of barium chloride (BaCl), a nonspecific blocker of K+ channels (32), on the PGE2 response. Barium chloride, at a concentration of 2 mM, was applied to the whole mount preparation by superfusion (n = 8). PGE2 was then applied (100 µM, 200 ms), and the PGE2 response in BaCl was compared with control responses. BaCl depolarized the gallbladder neurons (mean, 11.1 ± 2.7 mV; range: 3.9-29.1 mV). In addition, BaCl increased excitability of neurons as demonstrated by the development of anodal break excitation at the offset of a hyperpolarizing current pulse (Fig. 5). To compare the PGE2 response in BaCl with that obtained in control conditions, the neurons were current clamped back to the RMP to negate the BaCl-induced depolarization. In the presence of BaCl, the mean early phase hyperpolarization elicited by PGE2 was decreased by ~50% (control, 5.0 ± 1.6 mV; range: -12.3 to -1.2 mV; BaCl, 2.7 ± 0.9 mV; range: -6.2 to 0 mV; P < 0.05; Fig. 5). The mid- and late phases of the PGE2-evoked response were not significantly affected by the presence of BaCl.


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Fig. 5.   A: superfusion of BaCl (2 mM) depolarized gallbladder neurons and increased excitability as measured by production of anodal break action potentials at the offset of a hyperpolarizing current pulse. B: an example of the anodal break excitation produced during BaCl superfusion from the cell shown in A at a faster sweep speed. C: the early hyperpolarization evoked by PGE2 (100 µM, 200 ms) was examined before (control) BaCl application, during BaCl application (the neuron was current clamped back to the RMP), and finally after washout of BaCl. In all cells, BaCl reduced the amplitude of the early hyperpolarization. RMP: A, -53 mV; C, -48 mV.

PGE2 attenuates action potential afterhyperpolarization. The action potential afterhyperpolarization (AHP) of gallbladder neurons typically has a magnitude of ~15 mV and a duration of ~170 ms (21). Previous work from our laboratory (21, 26) has demonstrated that the AHP involves at least two Ca2+-activated K+ conductances, an early conductance that is tetraethylammonium sensitive and a later conductance that is apamin sensitive. In this study, we tested whether PGE2 influenced AHP parameters. To analyze the AHP, single action potentials were generated using stimulation pulse width durations that terminated on the upstroke of the action potential (4-7 ms). In 15 cells, AHP control parameters were similar to those previously reported. Measurements were also taken of the magnitude of the AHP at a point 80 ms from the start of the AHP to detect any changes in the apamin-sensitive K+ conductance.

Application of PGE2 by either superfusion or picospritz reduced all parameters of the AHP that were studied (Fig. 6). The magnitude of the AHP was reduced by 30% (control, 15.6 ± 0.8 mV; PGE2, 10.9 ± 1.4 mV; P < 0.001), the duration was reduced by 27.6% (control, 209.1 ± 11.0 ms; PGE2, 151.3 ± 17.9 ms; P < 0.003), and the amplitude of the AHP measured at 80 ms was reduced by 42.3% during PGE2 application (control, 7.8 ± 1.0 mV; PGE2, 4.5 ± 1.1 ms; P < 0.01). In 8 of the 15 cells, the parameters of the AHP fully recovered to their control values after washout of the PGE2 application.


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Fig. 6.   Gallbladder neuron action potential afterhyperpolarizations (AHP) are mediated by 2 K+ conductances that control the magnitude and the duration of the AHP. Application of PGE2 consistently reduced both the magnitude and the duration of the AHP in these cells. Recordings shown here are 5 overlapping traces of AHPs from the same cell taken before PGE2 application (Control), during PGE2 application (1 µM superfusion), and after recovery from the PGE2-evoked response (Wash). Cells were current clamped back to the RMP during the late phase hyperpolarization to obtain this data. RMP for this cell was -47 mV.

When the gallbladder neuron AHP is attenuated by apamin (20) or substance P (23), an increase in excitability has been detected as an increase in the number of spikes generated during a depolarizing current pulse. As noted above, the neurons of the guinea pig gallbladder are relatively inexcitable under normal conditions; they typically generate only one or two action potentials at the onset of a prolonged depolarizing current pulse, and they rarely exhibit anodal break activity. To determine whether PGE2 altered the excitability of gallbladder neurons, action potential generation was evaluated before and after application of PGE2. During these experiments, the cells were maintained electrotonically at their RMP during the late phase hyperpolarization of the PGE2 response to negate any change in driving forces.

Only a minimal increase in excitability was detected in gallbladder neurons on application of PGE2 by either superfusion or microejection. Application of PGE2 did not initiate spontaneous action potential generation or result in anodal break activity at the offset of a hyperpolarizing current pulse. In 7 of 13 cells tested, an increase in the number of action potentials during a depolarizing current pulse was noted. However, the increase in action potential activity that was noted was slight compared with increased excitability in response to apamin or substance P.

PGE2 inhibits fast synaptic transmission. All neurons in guinea pig gallbladder ganglia exhibit nicotinic fast EPSPs in response to stimulation of interganglionic fiber bundles (21). The vagus nerves are a major source of these fast excitatory synaptic inputs to gallbladder neurons (25). Experiments were conducted to determine whether PGE2 affected fast EPSPs in gallbladder ganglia. Fast synaptic events were elicited by stimulating interganglionic nerve bundles (0.5 Hz, 0.5 ms, 1-10 V) before and during PGE2 application. Data were obtained by measuring fast EPSP amplitudes from signal averages of five consecutive events. PGE2 caused a 67% reduction in the fast EPSP amplitude (control, 8.1 ± 3.4 mV; range: 4.7-13.4 mV; PGE2, 2.7 ± 3.6 mV; range: 0-8.7 mV; P < 0.05; Fig. 7). In three of the six cells, PGE2 reversibly eliminated the fast EPSP.


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Fig. 7.   Subthreshold synaptic events (5 overlapping traces) were elicited by fiber tract stimulation before (Control), during PGE2 superfusion (100 nM), and after washout (Wash) of the prostanoid. The amplitude of the fast EPSP was significantly reduced by the application of PGE2 in all cells studied. RMP was -39 mV.

To determine whether PGE2 was acting presynaptically to suppress the fast EPSP, the effect of PGE2 on the responsiveness of neurons to ACh (0.1 mM, 100-ms microejection) was tested. If PGE2 was acting postsynaptically, we would expect a reduction of the ACh response after PGE2 application. The responsiveness of gallbladder neurons to ACh was not significantly altered by PGE2 (control, 9.2 ± 1.5 mV; PGE2, 8.7 ± 2.9 mV; n = 4).

PGE2 attenuates slow synaptic transmission. Slow EPSPs can be elicited in ~40% of gallbladder neurons (21). These long, slow depolarizations can be blocked by omission of Ca2+ in the superfusing solution, but not by atropine. Results obtained in previous studies (9, 19, 24) suggest that the mediators of this response include substance P and calcitonin gene-related peptide. Because changes in both prostaglandin levels and sensory afferent fiber plasticity have been implicated in inflammation, we examined the effects of PGE2 on slow EPSPs. Slow EPSPs were elicited in eight cells by stimulating interganglionic nerve bundles supramaximally at a high rate (20 Hz, 5-s train, 0.5-ms pulse width). Electrical stimulation was repeated during the PGE2-induced late phase hyperpolarization, with the cell current clamped to the RMP. Because the slow EPSP is subject to desensitization, sEPSPs were elicited in 15-min intervals. PGE2 reduced the amplitude of the slow EPSP by 42% (control, 5.2 ± 1.5 mV; range, 2.8-7.5 mV; PGE2, 3.0 ± 1.1 mV; range: 0-9.2 mV; P < 0.05; Fig. 8). After a 30-min washout period the magnitude of the slow EPSP was still reduced (27.3%) compared with control.


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Fig. 8.   Slow excitatory postsynaptic potentials (sEPSPs) were elicited by high frequency nerve tract stimulation in the absence and presence of PGE2 (1 µM superfusion). In all cells studied, both the magnitude and the duration of the sEPSP was reduced in the presence of PGE2. Full recovery of the sEPSP could not be obtained but partial recovery was evident in most cells. RMP for this cell was -48 mV as indicated by the dashed line. FTS, fiber tract stimulation.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The aim of this study was to investigate the effects of PGE2 on neurons within the wall of the guinea pig gallbladder. The data presented here demonstrate that PGE2 acts directly on gallbladder neurons to elicit a complex triphasic change in the RMP and a decrease in the duration and amplitude of the AHP. Each component of the triphasic response was concentration dependent, associated with a change in input resistance, and changed in amplitude when the membrane potential was electrotonically increased or decreased. In addition to the direct effects of PGE2 on gallbladder neurons, PGE2 acted presynaptically to attenuate both fast and slow excitatory synaptic responses.

The actions of PGE2 have been studied directly in the myenteric ganglia and indirectly in the submucosal ganglia preparation of the guinea pig bowel (5, 6, 10). In myenteric neurons, PGE2 causes a monophasic, long-lasting depolarization and the activation of action potentials in myenteric type 1/S cells. In the myenteric plexus preparation, no hyperpolarizing responses were reported and the mechanism of the depolarizing response was not investigated. The effect of PGE2 on the membrane potential of gallbladder neurons is relatively complex, consisting of an early hyperpolarization, a mid-phase repolarization, and a long-lasting late hyperpolarization. The application of PGE2 did not induce action potential generation in gallbladder neurons. A response to PGE2, reminiscent of that observed in gallbladder neurons, has been observed in canine renal epithelioid cells, which undergo a similar triphasic change in membrane potential in the presence of either PGE2 or arachidonic acid (33). Steidl et al. (33) suggest that the electrical changes observed in their cells were the result of an increased K+ conductance followed by an increase in Cl- conductance and that a return to the hyperpolarization state was due to a decrease in the chloride conductance.

Several lines of evidence suggest that the early hyperpolarization detected in gallbladder neurons involves the activation of a K+ conductance. This component of the response is characterized by decreased input resistance, a reversal potential that correlates with EK, and sensitivity to Ba+. The conductances responsible for the mid-phase repolarization and the late phase hyperpolarization are not resolved, but they probably are not due to movement of K+. The reversal potentials of the mid- and late phases of the PGE2 response in gallbladder neurons did not correlate with predicted EK in normal or elevated K+ conditions. The mid-phase repolarization may involve the activation of a nonselective cation conductance, because it is associated with a decrease in input resistance and its reversal potential is near 0 mV. From the data obtained, it is difficult to make any assumptions concerning the identity of the ionic current(s) involved in the late phase hyperpolarization except to suggest that it probably does not involve the movement of K+.

Results reported here, related to changes in the AHP and evoked synaptic potentials, indicate that Ca2+ and Ca2+-activated K+ conductances may be suppressed by PGE2 in gallbladder ganglia. The AHP of gallbladder neurons is composed of two consecutive Ca2+-activated K+ conductances that are sensitive to tetraethylammonium and apamin, respectively (21). The amplitude of the AHP is dependent on the first conductance, and the duration is related to the second conductance. In the presence of PGE2, the AHP of gallbladder neurons was reduced in amplitude and duration, indicating that both K+ conductances were reduced either indirectly or directly by PGE2. Attenuation of the AHP by prostaglandins has also been observed in enteric neurons of the guinea pig submucosal and myenteric plexi (5, 8) as well as in rabbit visceral afferent neurons (7, 38). In rabbit sensory neurons, the AHP was inhibited by PGE2 in the presence of a Ca2+ ionophore, indicating that a reduction in Ca2+ influx was not responsible for the PGE2-induced inhibition of the AHP.

In guinea pig gallbladder ganglia, vagal efferent fibers provide the principal source of nicotinic fast synaptic input (25). Because PGE2 reduces the amplitude of EPSPs but does not alter the responsiveness of gallbladder neurons to exogenously applied ACh, PGE2 is likely to attenuate the release of ACh from preganglionic vagal terminals. This is a significant observation, because it provides another example of the importance of vagal efferent terminals as a target of modulatory inputs in this system. Other examples include CCK, which acts at physiological concentrations on vagal terminals to increase ACh release, and activation of sympathetic nerves, which decreases ACh release from vagal terminals (21, 22).

The slow EPSP observed in the gallbladder is mediated primarily by substance P via an NK3 receptor (24), although calcitonin gene-related peptide may provide a minor component of the response (9). We have previously hypothesized that the sEPSP may be a local axonal reflex response and could act to increase ganglionic output in an effort to increase gallbladder luminal pressure and expel any obstructions (9). The data presented here show that PGE2 significantly decreases the amplitude of the sEPSP. In a similar study in the myenteric plexus of the guinea pig, PGE2 did not decrease the amplitude of sEPSPs (5), but, in the submucosal plexus, the related prostanoid PGD2 did decrease the amplitude of these events (8).

Prostaglandins have been shown to inhibit transmitter release in several systems. For example, release of norepinephrine is attenuated by PGE2 in the colon (41), in the heart (20), and at the iris-ciliary body (30), and PGE2 decreases nonadrenergic, noncholinergic transmitter release in the rabbit stomach (1). In the gut, PGE2 decreases ACh release (26, 34) and suppresses fast EPSPs in myenteric ganglia (5). In some cases, a prostaglandin induced an increase in ACh output in the presence of PGE2 (4, 11, 15), but this may be due to a direct action of PGE2 on enteric neurons.

It is possible that, in the gallbladder and other systems, prostaglandins act through an inhibitory effect on Ca2+ channels to decrease transmitter release. In rat sympathetic ganglia, PGE2 has been shown to cause a rapid reduction of the Ca2+ current, which is due to a depolarizing shift in the activation of N-type Ca2+ channels (12).

When considering the overall actions of prostaglandins in the biliary tract, a pertinent consideration is that treatment with cyclooxygenase inhibitors relieves biliary tract pain (35, 36). As prostaglandins are known to contract gallbladder muscle, this might suggest that a reduction in prostaglandin production may diminish gallbladder contractility, hence creating a decrease in gallbladder luminal pressure and leading to biliary pain relief. However, it is more likely that the reduction in prostaglandin production acts to prevent an increase in the sensitivity of primary afferent nerves within the organ. Prostaglandins have the ability to increase the sensitivity of primary afferents to noxious stimulation (31, 42). A subset of the sensory nerve fibers that innervate the gallbladder are selectively sensitive to noxious stimulation, indicating that pure visceral pain afferent fibers exist in the gallbladder (2). Taken together, these observations indicate that a decrease in prostaglandin production by cyclooxygenase inhibitors may prevent sensitization of the primary afferent fibers and lead to biliary pain relief despite any direct effects of prostaglandins on muscle or ganglionic output. In support of this hypothesis, it has been observed that in human tissue (17, 39) as well as in some animal models (3), gallbladder muscle becomes desensitized to prostaglandins. With the contractile effect of prostaglandin diminished with time, chronic prostaglandin production may contribute to the gallbladder stasis by effectively denervating the tissue. Long-lasting hyperpolarization of the neurons and suppression of fast and slow excitatory input would significantly decrease ganglionic output since gallbladder neurons need to be synaptically driven to generate action potentials.

In conclusion, PGE2 elicits a complex triphasic effect on the RMP of gallbladder neurons with the predominant effect being a long-lasting hyperpolarization. In addition, PGE2 attenuates synaptic input to the gallbladder neurons, an important site of neuromodulation in this organ. Our results suggest that PGE2 may be involved in a reduction in contractility at the level of the intramural ganglia and over the long term may contribute to the gallbladder stasis seen in pathophysiological conditions.

    ACKNOWLEDGEMENTS

We thank Dr. Rodney Parsons for valuable discussion and for reviewing the manuscript.

    FOOTNOTES

This work was supported by National Institutes of Health Grants NS-26995 and DK-45410.

Address for reprint requests: G. M. Mawe, C-423 Given Bldg., Univ. of Vermont, Burlington, VT 05405 (E-mail: gmawe{at}zoo.uvm.edu).

Received 24 September 1997; accepted in final form 2 December 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Baccari, M. C., F. Calamai, and G. Staderini. Prostaglandin E2 modulates neurally induced nonadrenergic noncholinergic gastric relaxations in the rabbit in vivo. Gastroenterology 110: 129-138, 1996[Medline].

2.   Cervero, F. Afferent activity evoked by natural stimulation of the biliary system in the ferret. Pain 13: 137-151, 1982[Medline].

3.   Chapman, W. C., G. A. Peterkin, W. W. LaMorte, and L. F. Williams. Alterations in biliary motility correlate with increased gallbladder prostaglandin synthesis in early cholelithiasis in prairie dog. Dig. Dis. Sci. 34: 1420-1424, 1989[Medline].

4.   Das, M., and D. K. Ganguly. Effect of prostaglandin E2 on acetylcholine release from some peripheral cholinergic nerve terminals. Eur. J. Pharmacol. 100: 41-46, 1984[Medline].

5.   Dekkers, J. A. J. M., L. M. A. Akkermans, and A. B. A. Kroese. Effects of the inflammatory mediator prostaglandin E2 on myenteric neurons in guinea pig ileum. Am. J. Physiol. 272 (Gastrointest. Liver Physiol. 35): G1451-G1456, 1997[Abstract/Free Full Text].

6.   Diener, M., R. J. Bridges, S. F. Knobloch, and W. Rummel. Neuronally mediated and direct effects of prostaglandins on ion transport in rat colon descendens. Naunyn Schmiedebergs Arch. Pharmacol. 337: 74-78, 1988[Medline].

7.   Fowler, J. C., R. Greene, and D. Weinreich. Two calcium-sensitive spike after-hyperpolarizations in visceral sensory neurones of the rabbit. J. Physiol. (Lond.) 365: 59-75, 1985[Abstract].

8.   Frieling, T., C. Rupprecht, A. B. Kroese, and M. Schemann. Effects of the inflammatory mediator prostaglandin D2 on submucosal neurons and secretion in guinea pig colon. Am. J. Physiol. 266 (Gastrointest. Liver Physiol. 29): G132-G139, 1994[Abstract/Free Full Text].

9.   Gokin, A. P., L. J. Jennings, and G. M. Mawe. Actions of calcitonin gene-related peptide in guinea pig gallbladder ganglia. Am. J. Physiol. 271 (Gastrointest. Liver Physiol. 34): G876-G883, 1996[Abstract/Free Full Text].

10.   Goldhill, J. M., R. Burakoff, V. Donovan, K. Rose, and W. H. Percy. Defective modulation of colonic secretomotor neurons in a rabbit model of colitis. Am. J. Physiol. 264 (Gastrointest. Liver Physiol. 27): G671-G677, 1993[Abstract/Free Full Text].

11.   Hedqvist, P., L. Gustafsson, P. Hjemdahl, and K. Svanborg. Aspects of prostaglandin action on autonomic neuroeffector transmission. Adv. Prostaglandin Thromboxane Leukot. Res. 8: 1245-1248, 1980.

12.   Ikeda, S. R. Prostaglandin modulation of Ca2+ channels in rat sympathetic neurones is mediated by guanine nucleotide binding proteins. J. Physiol. (Lond.) 458: 339-359, 1992[Abstract].

13.   Jivegard, L., E. Thornell, and J. Svanvik. Fluid secretion by gallbladder mucosa in experimental cholecystitis is influenced by intramural nerves. Dig. Dis. Sci. 32: 1389-1394, 1987[Medline].

14.   Jivegard, L., A. Thune, and J. Svanvik. Intraluminal prostaglandin E2 affects gallbladder function by activation of intramural nerves in the anaesthetized cat. Acta Physiol. Scand. 132: 549-555, 1988[Medline].

15.   Kadlec, O., I. Seferna, and K. Masek. Modulatory role of prostaglandins on cholinergic neurotransmission in the guinea pig ileum. Adv. Prostaglandin Thromboxane Leukot. Res. 8: 1255-1257, 1980.

16.   Kaminski, D. L., Y. Deshpande, L. Thomas, and W. Blank. Evaluation of the role of prostaglandins E and F in human cholecystitis. Prostaglandins Leukot. Med. 16: 109-120, 1984[Medline].

17.   Kotwall, C. A., A. S. Clanachan, H. P. Baer, and G. W. Scott. Effects of prostaglandins on motility of gallbladders removed from patients with gallstones. Arch. Surg. 119: 709-712, 1984[Abstract].

18.   Lee, S. P., J. T. LaMont, and M. C. Carey. Role of gallbladder mucus hypersecretion in the evolution of cholesterol gallstones: studies in the prairie dog. J. Clin. Invest. 67: 1712-1723, 1981[Medline].

19.   Maggi, C. A., P. Santicioli, D. Renzi, R. Patacchini, C. Surrenti, and A. Meli. Release of substance P- and calcitonin gene-related peptide-like immunoreactivity and motor response of the isolated guinea pig gallbladder to capsaicin. Gastroenterology 96: 1093-1101, 1989[Medline].

20.   Mantelli, L., S. Amerini, A. Rubino, and F. Ledda. Prejunctional prostanoid receptors on cardiac adrenergic terminals belong to the EP3 subtype. Br. J. Pharmacol. 102: 573-576, 1991[Abstract].

21.   Mawe, G. M. Intracellular recording from neurones of the guinea-pig gall-bladder. J. Physiol. (Lond.) 429: 323-338, 1990[Abstract].

22.   Mawe, G. M. Noradrenaline as a presynaptic inhibitory neurotransmitter in ganglia of the guinea-pig gall-bladder. J. Physiol. (Lond.) 461: 387-402, 1993[Abstract].

23.   Mawe, G. M. The role of cholecystokinin in ganglionic transmission in the guinea-pig gall-bladder. J. Physiol. (Lond.) 439: 89-102, 1991[Abstract].

24.   Mawe, G. M. Tachykinins as mediators of slow EPSPs in guinea-pig gall-bladder ganglia: involvement of neurokinin-3 receptors. J. Physiol. (Lond.) 485: 513-524, 1995[Abstract].

25.   Mawe, G. M., A. P. Gokin, and D. G. Wells. Actions of cholecystokinin and norepinephrine on vagal inputs to ganglion cells in guinea pig gallbladder. Am. J. Physiol. 267 (Gastrointest. Liver Physiol. 30): G1146-G1151, 1994[Abstract/Free Full Text].

26.   Mawe, G. M., E. K. Talmage, E. B. Cornbrooks, A. P. Gokin, L. Zhang, and L. J. Jennings. Innervation of the gallbladder: structure, neurochemical coding, and physiological properties of guinea pig gallbladder ganglia. Microsc. Res. Tech. 38: 1-13, 1997.

27.   Myers, S. I., and L. Bartula. Human cholecystitis is associated with increased gallbladder prostaglandin I2 and prostaglandin E2 synthesis. Hepatology 16: 1176-1179, 1992[Medline].

28.   Nakata, K., K. Ashida, K. Nakazawa, and M. Fugiwara. Effects of indomethacin on prostaglandin synthesis and on contractile response of the guinea pig gallbladder. Pharmacology 23: 95-101, 1981[Medline].

29.   Nakata, K., Y. Osumi, and M. Fujiwara. Prostaglandins and the contractility of the guinea pig biliary system. Pharmacology 22: 24-30, 1981[Medline].

30.   Ohia, S. E., and J. E. Jumblatt. Prejunctional inhibitory effects of prostanoids on sympathetic neurotransmission in the rabbit iris-ciliary body. J. Pharmacol. Exp. Ther. 255: 11-16, 1990[Abstract].

31.   Pitchford, S., and J. D. Levine. Prostaglandins sensitize nociceptors in cell culture. Neurosci. Lett. 132: 105-108, 1991[Medline].

32.   Rudy, B. Diversity and ubiquity of K channels. Neuroscience 25: 729-749, 1988[Medline].

33.   Steidl, M., M. Ritter, and F. Lang. Regulation of potassium conductance by prostaglandins in cultured renal epithelioid (Madin-Darby canine kidney) cells. Pflügers Arch. 418: 431-436, 1991[Medline].

34.   Thornell, E. Mechanisms in the development of acute cholecystitis and biliary pain. A study on the role of prostaglandins and effects of indomethacin. Scand. J. Gastroenterol. 76: 1-31, 1982.

35.   Thornell, E., R. Jansson, and J. Svanik. Indomethacin intravenously-a new way for effective relief of biliary pain: a double blind study in man. Surgery 90: 468-472, 1981[Medline].

36.   Thornell, E., B. Nilsson, R. Jansson, and J. Svanik. Effect of short term indomethacin treatment on the clinical course of acute obstructive cholecystitis. Eur. J. Surg. 157: 127-130, 1991[Medline].

37.   Thornell, E., J. Svanvik, and J. R. Wood. Effects of intra-arterial prostaglandin E2 on gallbladder fluid transport, motility, and hepatic bile in the cat. Scand. J. Gastroenterol. 16: 1083-1088, 1981[Medline].

38.   Weinreich, D., and W. F. Wonderlin. Inhibition of calcium-dependent spike after-hyperpolarization increases excitability of rabbit visceral sensory neurons. J. Physiol. (Lond.) 394: 415-427, 1987[Abstract].

39.   Wood, J. R., S. H. Saverymuttu, A. B. Ashbrooke, and I. F. Stamford. Effects of various prostanoids on gallbladder muscle. Adv. Prostaglandin Thromboxane Leukot. Res. 8: 1569-1571, 1980.

40.   Wood, J. R., and I. F. Stamford. Prostaglandins in chronic cholecystitis. Prostaglandins 13: 97-106, 1977[Medline].

41.   Wu, Z. C., and T. S. Gaginella. Release of [3H]norepinephrine from nerves in rat colonic mucosa: effects of norepinephrine and prostaglandin E2. Am. J. Physiol. 241 (Gastrointest. Liver Physiol. 4): G416-G421, 1981[Abstract/Free Full Text].

42.   Yanagisawa, M., M. Otsuka, and J. E. Garcia-Arraras. E-type prostaglandins depolarize primary afferent neurons of the neonatal rat. Neurosci. Lett. 68: 351-355, 1986[Medline].


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