Role of GABAA and GABAC Receptors in the Biphasic GABA Responses in Neurons of the Rat Major Pelvic Ganglia

Takashi Akasu,1 Yoshikazu Munakata,1,2 Masashi Tsurusaki,1 and Hiroshi Hasuo1

 1Department of Physiology and  2Department of Urology, Kurume University School of Medicine, Kurume 830-0011, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Akasu, Takashi, Yoshikazu Munakata, Masashi Tsurusaki, and Hiroshi Hasuo. Role of GABAA and GABAC Receptors in the Biphasic GABA Responses in Neurons of the Rat Major Pelvic Ganglia. J. Neurophysiol. 82: 1489-1496, 1999. The role of gamma -aminobutyric acid-A (GABAA) and GABAC receptors in the GABA-induced biphasic response in neurons of the rat major pelvic ganglia (MPG) were examined in vitro. Application of GABA (100 µM) to MPG neurons produced a biphasic response, an initial depolarization (GABAd) followed by a hyperpolarization (GABAh). The input resistance of the MPG neurons was decreased during the GABAd, whereas it was increased during the GABAh. The GABAd could be further separated into the early component (early GABAd) with a duration of 27 ± 5 s (mean ± SE; n = 11) and the late component (late GABAd) with a duration of 109 ± 11 s (n = 11). The duration of the GABAh was 516 ± 64 s (n = 11). The effects of GABA (5-500 µM) in producing the depolarization and the hyperpolarization were concentration-dependent. GABA (5-30 µM) induced only late depolarizations. The early component of the depolarization appeared when the concentration of GABA was >50 µM. Muscimol produced only early depolarizing responses. Baclofen (100 µM) had no effect on the membrane potential and input resistance of MPG neurons. Bicuculline (60 µM) blocked the early GABAd but not the late GABAd and the GABAh. Application of picrotoxin (100 µM) with bicuculline (60 µM) blocked both the late GABAd and the GABAh. CGP55845A (3 µM), a selective GABAB receptor antagonist, did not affect the GABA-induced responses. cis-4-Aminocrotonic acid (CACA, 1 mM) and trans-4-aminocrotonic acid (TACA, 1 mM), selective GABAC receptor agonists, produced late biphasic responses in the MPG neurons. The duration of the CACA responses was almost the same as those of the late GABAd and GABAh obtained in the presence of bicuculline. Imidazole-4-acetic acid (I4AA, 100 µM), a GABAC receptor antagonist, depressed the late GABAd and the GABAh but not the early GABAd. I4AA (100 µM) and picrotoxin (100 µM) also suppressed the biphasic response to CACA. The early GABAd and the late GABAd were reversed in polarity at -32 ± 3 mV (n = 7) and -38 ± 2 mV (n = 4), respectively, in the Krebs solution. The reversal potential of the GABAh was -34 ± 2 mV (n = 4) in the Krebs solution. The reversal potentials of the late GABAd and the GABAh shifted to -20 ± 3 mV (n = 5) and -22 ± 3 mV (n = 5), respectively, in 85 mM Cl- solution. These results indicate that the late GABAd and the GABAh are mediated predominantly by bicuculline-insensitive, picrotoxin-sensitive GABA receptors, GABAC (or GABAAOr) receptors, in neurons of the rat MPG.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The pelvic parasympathetic ganglion is not only a relay station that distributes excitatory signals from the CNS to the urogenital organs but also a modulatory site for the neuronal information (Akasu and Nishimura 1995; de Groat and Booth 1980; Keast 1995). Neurons in the pelvic ganglion receive cholinergic input from the S2-S4 sacral region of the spinal cord via the pelvic nerve and adrenergic input from the inferior mesenteric (sympathetic) ganglia via the hypogastric nerve (Akasu and Nishimura 1995; de Groat et al. 1993; Keast 1995). Stimulation of the pelvic nerve evoked an excitatory postsynaptic potential mediated by the nicotinic actions of acetylcholine (Akasu and Nishimura 1995; de Groat and Booth 1980; Gallagher et al. 1982). gamma -Aminobutyric acid (GABA), a typical inhibitory transmitter in the CNS, may play a role in the lower urinary tract (Maggi et al. 1983, 1985a-c). GABA inhibited bladder contractions evoked by stimulation of preganglionic nerve fibers in the rat major pelvic ganglia (MPG). Neurons in the rat MPG contain GABA, which is released by stimulation of the pelvic nerve (de Groat 1970; Kusunoki et al. 1984). Binding sites and synthesizing enzymes for GABA exist in the neurons of the rat MPG (de Groat 1970; Kusunoki et al. 1984). In the pelvic ganglia of the cat urinary bladder, GABA induced a biphasic response, a depolarization followed by an afterhyperpolarization, which were mediated by changes of Cl- conductance (Mayer et al. 1983).

Recently, it has been reported that GABA receptors can be classified as GABAA and GABAC receptors, which are ionotropic receptors, or as GABAB receptors, which are metabotropic receptors coupled to the GTP-binding protein. GABAA receptors have several subunits (alpha 1-6, beta 1-3, gamma 1-3, delta ), which form a pentameric chloride channel (Macdonald and Olsen 1994). As a subtype of the GABAA receptor, GABAC receptors are probably pentameric Cl- channels composed of the recently discovered rho  subunits (rho 1-3) (Barnard et al. 1998; Cutting et al. 1991; Ogurusu and Shingai 1996). Patch-clamp studies have shown that the single-channel conductance of the GABAA receptors was larger than that of the GABAC-receptor channels in retinal cells (Feigenspan and Bormann 1994). The ion channel of the GABAC receptors opened for a longer time and was less liable to desensitization than most GABAA receptors. GABAC receptors are not blocked by bicuculline and are not modulated by barbiturates, benzodiazepines, or neuroactive steroids (Bormann and Feigenspan 1995; Dong et al. 1994; Feigenspan et al. 1993; Johnston 1997; Lukasiewicz 1996; Lukasiewicz et al. 1994; Polenzani et al. 1991; Qian and Dowling 1993, 1994; Wang et al. 1994).

The aim of the present study is to examine the contribution of GABAA and GABAC receptors to the biphasic response in the neurons of the rat MPG. Application of GABA induced a complex response that consisted of early and late depolarizations and a hyperpolarization. It is suggested that GABAA receptors mediate the early GABA-induced depolarization (early GABAd), whereas GABAC or GABAAOr receptors (Barnard et al. 1998) mediate the late GABA-induced depolarization (late GABAd) and the hyperpolarization (GABAh). A preliminary account of some of this work has been published previously (Tsurusaki et al. 1998).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Male Wistar rats weighing 200 g were killed by decapitation. The MPG located on a lateral site of the prostate were dissociated and then pinned onto silicone elastomer (Sylgard) at the bottom of a superfusion chamber (0.5 ml total volume). The MPG were continuously superfused with Krebs solution (3 ml/min) with the following composition (in mM): 117 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 25 NaHCO3, and 11 glucose (295-305 mOsm). The Krebs solution was gassed with 95% O2-5% CO2 and preheated to 32°C at the recording site. In some experiments, MPG neurons were superfused with a modified Krebs solution containing 85 mM Cl- (low Cl- solution), where 40.4 mM NaCl was replaced with Na-isethionate. The pH of the Krebs solution was adjusted to 7.4. Intracellular microelectrodes filled with 3 M K-acetate had tip resistances of 80-120 MOmega . Membrane potential and current were recorded with an Axoclamp 2A (Axon Instruments). The MacLab system software program (AD Instruments) operating on Apple Computers (Macintosh 8100/100AV and PowerBook 5300CS) was used to continuously record the membrane potentials. The membrane potential and membrane current were continuously monitored with a memory oscilloscope (Nihon-Kohden, RTA-1100). The voltage and current were also digitized and stored in the computer (Power Mac 8500: Apple Computer) with a data-acquisition system (AxoData: Axon Instruments) for later analysis. The voltage-current (V-I) relationship was obtained by applying cathodal and anodal current pulses with a duration of 300 ms. The following drugs were used: GABA, muscimol, imidazole-4-acetic acid (I4AA) hydrochloride, and baclofen were purchased from SIGMA; (-)-bicuculline methiodide from Nacalai Tesque; picrotoxin from Wako Pure Chemical Industries; and cis-4-aminocrotonic acid (CACA) and trans-4-aminocrotonic acid (TACA) from TOCRIS. CGP55845A was a gift from CIBA-GEIGY. All drugs were dissolved directly in the Krebs solution. The data were expressed as mean ± SE.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of GABA on the membrane potential of MPG neurons

The resting membrane potential and input resistance of rat MPG neurons were -62 ± 2 mV (n = 159) and 70 ± 5 MOmega (n = 52), respectively. The action potential had an afterhyperpolarization with an amplitude of 14 ± 1 mV (n = 11) and a duration of 333 ± 52 ms (n = 11). Application of GABA (300 µM) to the external solution for 1 min induced a biphasic response, an initial depolarization (GABAd) followed by a hyperpolarization (GABAh) in 88 (75%) of 117 MPG neurons (Fig. 1A). The remaining 29 (25%) neurons did not respond to GABA. The input resistance of the rat MPG neurons decreased during the GABAd and increased during the GABAh (Fig. 1A). The falling phase of the GABA-induced depolarization consisted of early and late components (Table 1). When the GABAd reached the maximum amplitude, it initially declined rapidly toward the resting membrane potential and then subsequently entered a slow decay phase. Thus the GABA-induced depolarization sometimes had a plateau on the falling phase (Fig. 1B). The early GABAd measured at the half-maximum amplitude had a duration of 27 ± 5 s (n = 11). The late GABAd lasted for 60-140 s; the mean duration of the late GABAd was 129 ± 11 s (n = 11). The duration of the late GABAh ranged from 300 to 600 s with a mean duration of 516 ± 64 s (n = 11) at the resting membrane potential. The effect of GABA in producing the depolarization was concentration dependent (Fig. 2). At a concentration of 5 µM, GABA depolarized the neurons by 4 ± 1 mV (n = 6). Only a slow depolarizing response was produced by 5-30 µM GABA. GABA (500 µM) produced a maximal response of 22 ± 1 mV (n = 30). The fast depolarization was seen at concentrations of GABA >50 µM (Fig. 2). The GABAh was also concentration dependent. The minimum effective concentration of GABA was 100 µM that produced a hyperpolarization with an amplitude of 3 ± 1 mV (n = 14). GABA (500 µM) produced the maximal hyperpolarization with an amplitude of 7 ± 2 mV (n = 30).



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Fig. 1. GABA-induced responses in a major pelvic ganglion (MPG) neuron. A, top: continuous pen record of the GABA-induced depolarization (GABAd) and the following hyperpolarization (GABAh). Resting membrane potential was -55 mV. Period of the bath-application of GABA (300 µM) is indicated by a horizontal bar. Downward deflections on the trace indicate hyperpolarizing electrotonic potentials evoked by anodal current pulses with an amplitude of 0.3 nA and a duration of 200 ms. Bottom: expanded records of the electrotonic potentials. a-d were obtained at the times marked by the respective letters in the top trace. B: measurements of the amplitude and duration of the GABA-induced responses. a and b indicate the amplitude of the early GABAd and the GABAh. Durations of the late GABAd and GABAh are shown by c and d. Inset: expanded record of the GABAd. Duration of the early GABAd (e) was measured at the half-maximum amplitude. R.M.P, resting membrane potential.


                              
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Table 1. Duration of responses induced by GABA, muscimol, and CACA in MPG neurons



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Fig. 2. Concentration-dependent properties of the GABA responses. A: responses were induced by bath-application of GABA (10-500 µM) in the Krebs solution. - - -, original membrane potential. B: concentration-response curves for the GABA-induced depolarizations () and hyperpolarizations (open circle ), ± SE of the mean. Number of experiments is shown in parentheses.

Effects of agonists and antagonists for GABA receptors

The effects of GABA-receptor agonists on the membrane potential were examined in rat MPG neurons. Figure 3 shows the effects of GABA (300 µM), muscimol (100 µM) and baclofen (100 µM) on a single MPG neuron. GABA produced a typical biphasic response in this neuron (Fig. 3A). Muscimol (100 µM) produced only a depolarizing response that was similar to the early GABAd (Fig. 3B and Table 1). The muscimol-induced depolarization had an amplitude of 22 ± 4 mV (n = 4) and was associated with a decreased membrane resistance. The muscimol-induced depolarization was not followed by an obvious plateau potential on the falling phase or by an afterhyperpolarization. The duration of the muscimol-induced depolarization measured at the half-maximum amplitude was 22 ± 3 s (n = 6). Baclofen (10 µM), a selective GABAB-receptor agonist (Bowery 1989), produced no changes in the membrane potential or resistance (Fig. 3C). The effects of the GABAA-receptor antagonists on the GABA-induced responses were examined in the MPG neurons (Fig. 4). Bicuculline (60 µM), a GABAA-receptor antagonist, reduced the amplitude of the early GABAd by 48 ± 3% (n = 8). In the neurons treated with bicuculline (60 µM), the amplitude and duration of the slow depolarization induced by GABA (300 µM) were 9 ± 2 mV (n = 12) and 98 ± 16 s (n = 12), respectively. The GABA-induced hyperpolarization obtained in the presence of bicuculline (100 µM) had a duration of 403 ± 41 s (n = 8) and an amplitude of 4 ± 1 mV (n = 8). The biphasic GABA responses obtained in the presence of bicuculline (100 µM) were identical to the late GABAd and GABAh in normal Krebs solution (Table 1). The application of picrotoxin (100 µM) to a Krebs solution containing bicuculline (60 µM) abolished the residual GABA responses (Fig. 4A). CGP55845A (3 µM), a selective GABAB-receptor antagonist (Bowery 1997), had no significant effect on the GABA-induced responses in the rat MPG neurons (n = 4).



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Fig. 3. Effects of GABA (A), muscimol (B) and baclofen (C) on a rat MPG neuron. Drugs were applied in the Krebs solution at the time indicated by the horizontal bars.



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Fig. 4. Effects of bicuculline, picrotoxin and imidazole-4-acetic acid (I4AA) on the GABA-induced biphasic response. A, top: typical GABA-induced response obtained from an MPG neuron in Krebs solution. Middle: GABA-induced response in the presence of bicuculline (60 µM). Bottom: picrotoxin (100 µM) was added to the Krebs solution containing bicuculline (100 µM). All records were from the same cell. Note the bicuculline-resistant, picrotoxin-sensitive component of the GABA response. B: effect of I4AA (100 µM) on the GABA-induced responses. Top: typical response to GABA (1 mM) in an MPG neuron. Bottom: obtained 5 min after the application of I4AA (100 µM). These records were obtained from the same cell.

Properties of a bicuculline-insensitive, picrotoxin-sensitive GABA response

Recent studies have demonstrated a bicuculline-insensitive, picrotoxin-sensitive GABA response mediated by GABAC receptors in retinal bipolar cells (Bormann and Feigenspan 1995; Feigenspan and Bormann 1994; Feigenspan et al. 1993; Johnston 1996; Qian and Dawling 1995). The possibility that the GABA responses are mediated by GABAC receptors in rat MPG neurons was examined. It has been reported that I4AA (100 µM) acts as an antagonist at GABAC receptors (Bormann and Feigenspan 1995; Kusama et al. 1993; Pan and Lipton 1995; Qian and Dowling 1994, 1995). Figure 4B shows the effect of I4AA (100 µM) on the GABA-induced responses in MPG neurons. I4AA (100 µM) did not block the early GABAd but markedly depressed the late GABAd and the GABAh. Pooled data from these studies are shown in Table 2. The effects of GABAC-receptor agonists on the membrane potential were compared with those of GABA on the same MPG neurons (Fig. 5). In this neuron, GABA (300 µM) produced a typical biphasic response. CACA (1 mM), a selective agonist for GABAC receptors (Bormann and Feigenspan 1995; Johnston 1996, 1997), also produced a biphasic response composed of a depolarization followed by a hyperpolarization in the same MPG neuron (Fig. 5B). There was no obvious early component in the CACA-induced biphasic response. The amplitudes of the depolarization and hyperpolarization induced by CACA (1 mM) were 12 ± 1 mV (n = 8) and 4 ± 1 mV (n = 12), respectively. The input resistance of the MPG neurons was decreased during the CACA-induced depolarization and was increased during the hyperpolarization. The durations of the CACA-induced responses were similar to those of GABA-induced responses obtained in the presence of bicuculline (Table 1). The CACA-induced depolarization and hyperpolarization lasted for 118 ± 4 s (n = 8) and 463 ± 23 s (n = 8), respectively. TACA (1 mM), another GABAC-receptor agonist (Bormann and Feigenspan 1995; Johnston 1996), also produced depolarizing responses with a plateau on the falling phase (Fig. 5C). The amplitude of the TACA (1 mM)-induced depolarization was 7 ± 1 mV (n = 8). A hyperpolarizing response with an amplitude of 3.0 ± 0.8 mV (n = 8) followed the TACA-induced depolarization. The effects of GABA-receptor antagonists on the CACA-induced biphasic response were examined in MPG neurons (Fig. 6A). Bicuculline (100 µM) produced only a small depression of the CACA-induced responses (Table 2). Picrotoxin (100 µM) blocked the biphasic response produced by CACA (1 mM). The magnitude of depression of the CACA-induced responses by picrotoxin is shown in Table 2. Figure 6B shows the effect of I4AA on the CACA-induced depolarization and hyperpolarization. The CACA (100 µM)-induced biphasic response was markedly depressed by I4AA (100 µM). Statistical data for the I4AA-induced depression of the CACA responses are shown in Table 2.


                              
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Table 2. Depression of CACA-induced slow responses by GABA antagonists in rat MPG neurons



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Fig. 5. Biphasic responses induced by GABA and GABAC-receptor agonists, cis- and trans-4-aminocrotonic acid (CACA and TACA). A: typical GABA-induced biphasic response in an MPG neuron, which had a resting membrane potential of -65 mV. B: CACA (100 µM)-induced slow biphasic response. C: TACA (1 mM)-induced slow biphasic response in an MPG neuron. All records were obtained from the same cell. · · · · , original resting membrane potential.



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Fig. 6. Effects of bicuculline (A) and I4AA (B) on CACA-induced responses. A, top: CACA response obtained from an MPG neuron superfused with Krebs solution. CACA (1 mM) was applied to the MPG neurons during the period indicated (). Bottom: effect of bicuculline on the CACA-induced response. CACA (1 mM) was applied to the MPG neuron 1 min after the application of bicuculline (100 µM). , time of the application of bicuculline (100 µM). B: effect of I4AA (100 µM) on the response to CACA (1 mM). I4AA (100 µM) was applied in the superfusing solution 5 min before the application of CACA. Top and bottom: responses to CACA taken before and during the application of I4AA (100 µM), respectively. , time of application of I4AA (100 µM). , times for the application of CACA (1 mM).

Reversal potentials of GABA-induced responses

It has been reported that GABAA and GABAC receptors are ionotropic receptors which include a Cl- selective channel (Bormann 1988; Feigenspan and Bormann 1994; Feigenspan et al. 1993; Macdonald and Olsen 1994). In the vesical pelvic ganglia of the cat urinary bladder wall, the GABA-induced depolarization and afterhyperpolarization were produced by activation and inhibition of Cl- channels, respectively (Mayer et al. 1983). Therefore the contributions of Cl- to the GABA-induced responses were examined in rat MPG neurons. Direct application of GABA (1 mM) to MPG neurons by pressure pulses (70 kPa for 600 ms) induced fast depolarizations with durations of 8-12 s followed by neither a slow depolarization nor a hyperpolarization (Fig. 7). Bicuculline (20 µM) blocked the fast depolarization from pressure application of GABA (n = 5), indicating that it was a GABAA-receptor-mediated response. The fast depolarization was also decreased in amplitude, when MPG neurons were depolarized. Figure 7 shows a linear relationship between the membrane potential and the amplitude of the early GABAd. The reversal potential of the early GABAd obtained by extrapolation of this curve was -32 ± 3 mV (n = 7). Reversal potentials for the late GABAd and the GABAh were also examined in MPG neurons. Electrotonic potentials were produced by applying cathodal and anodal current pulses with a duration of 300 ms. Figure 8 shows examples of the voltage-current relationships (V-I curves). The intersection of the V-I curves obtained before and during the late GABAd yielded a reversal potential of -43 mV in this particular neuron. For the same cell, V-I curves taken before and during the GABAh yielded a reversal of potential -34 mV. From four other experiments, the reversal potentials of the late GABAd and GABAh were -38 ± 2 mV and -34 ± 2 mV, respectively. To examine the Cl- dependency of the late GABAd and GABAh, MPG neurons were superfused with a low-Cl- solution containing 85 mM Cl-. In this solution, the reversal potentials of late GABAd and the GABAh were shifted to -17 ± 3 mV (n = 5) and -18 ± 3 mV (n = 5), respectively. Examples of these study are shown in Fig. 9.



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Fig. 7. Reversal potentials of the early GABAd in MPG neurons. Amplitudes of the early GABAd produced by pressure (70 kPa for 600 ms) application of GABA (1 mM) through a micropipette were plotted against the membrane potential. Vertical lines indicate the SE of the means obtained from 7 cells. Inset: example of the early GABAd. Open triangles indicate the times of the pressure applications of GABA.



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Fig. 8. Reversal potential of the early GABAd and the GABAh in an MPG neuron. A, top and bottom: current injected through a microelectrode and the membrane potential changes, respectively. Anodal and cathodal current pulses with a duration of 300 ms were applied to MPG neurons to evoke electrotonic potentials. B: voltage-current (V-I) relationships obtained before and during the GABA responses. open circle , control V-I curve obtained before the application of GABA. triangle  and , obtained during the late GABAd and the GABAh, respectively.



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Fig. 9. Effects of lowering extracellular Cl- on the late GABAd (A and B) and the GABAh (C and D). In A and C, neurons were superfused with the Krebs solution (Cl-: 124.4 mM). In B and D, the concentration of external Cl- was reduced to 85 mM. open circle  and triangle  and  and black-triangle, taken in the Krebs solution and low Cl- solution, respectively. up-arrow , reversal potential of GABA-induced responses.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study demonstrated that GABA induced a depolarization (GABAd) followed by a hyperpolarization (GABAh) in neurons of the rat MPG. The initial GABAd was associated with a decreased membrane resistance, whereas the GABAh was associated with an increased membrane resistance. Such a biphasic GABA response resembles that observed in neurons of the vesical pelvic ganglion of the cat urinary bladder (Mayer et al. 1983). The GABAh was sometimes followed by an additional slow depolarizing response associated with a decreased membrane resistance in rat MPG neurons; however, the properties of the late depolarization remain to be investigated. The GABAd in rat MPG neurons had two components, the early GABAd and the late GABAd, based on their decay time course. Muscimol, a classic GABAA-receptor agonist, recently has been shown to be a partial agonist at GABAC receptors (Johnston 1997). Application of muscimol to MPG neurons, however, produced only a fast depolarization with a duration that was similar to the early GABAd. Bicuculline blocked the early GABAd but not the late GABAd or the GABAh. These results indicate that the early GABAd is mediated by GABAA receptors in rat MPG neurons.

Recently, the GABA receptors responsible for the bicuculline-insensitive, picrotoxin-sensitive GABA response were described as "GABAC receptors" in vertebrate retina (Bormann and Feigenspan 1995; Feigenspan and Bormann 1994; Johnston 1996; Qian and Dowling 1993, 1995; Wellis and Werblin 1995). GABAC receptors are members of the GABA-gated chloride ion-channel superfamily of receptors as are GABAA receptors. Recent classification by international union of pharmacology (IUPHA) suggest that GABAC receptors appear to be a subtype of GABA ionotropic receptors, the GABAA receptor (Barnard et al. 1998). However, there are some differences between these two receptor subtypes (Dong et al. 1994; Feigenspan et al. 1993; Qian and Dowling 1993; Wang et al. 1994). Molecular biological studies have indicated that GABAC receptors are homooligomeric protein complexes formed by rho  subunits and are distinct from the heterooligomeric GABAA-receptor complexes formed by alpha , beta , gamma  (or delta ) subunits (Cutting et al. 1991; Macdonald and Olsen 1994; Ogurusu and Shingai 1996). Patch-clamp studies have shown that the single-channel conductance of the GABAC receptor is approximately four times larger than that of GABAA-receptor channels in retinal cells (Feigenspan and Bormann 1994). GABAC receptors are more sensitive to GABA than are GABAA receptors (Feigenspan and Bormann 1994; Kusama et al. 1993). The ion channel of the GABAC receptor remains open for a longer time and is less liable to desensitization than most GABAA receptors (Feigenspan et al. 1993). GABAC receptors are not modulated by barbiturates, benzodiazepines, or neuroactive steroids (Dong et al. 1994; Feigenspan et al. 1993; Lukasiewicz et al. 1994; Polenzani et al. 1991; Qian and Dowling 1993, 1994; Wang et al. 1994). GABAC receptors have been demonstrated to be localized in the CNS (Johnston 1997). Other bicuculline-insensitive GABA receptors that fall outside the GABAABC classification were described in embryonic brain-stem neurons (Momose-Sato et al. 1995) and rat retinal bipolar cells (Pan and Lipton 1995). They closely resemble vertebrate GABAC receptors; but these receptors, called GABAD receptors, were insensitive to both bicuculline and picrotoxin (Momose-Sato et al. 1995; Pan and Lipton 1995). Another bicuculline-insensitive, picrotoxin-sensitive GABA current (mediated by Cl-) has been observed in the hippocampus of 0- to 10-day-old rats (Martina et al. 1995).

The present study also describes bicuculline-insensitive GABA responses, the late GABAd and the GABAh, that were completely blocked by picrotoxin in rat MPG neurons. CACA and TACA, selective and potent GABAC-receptor agonists (Bormann and Feigenspan 1995; Johnston 1996), produced a slow depolarizing response associated with a decreased membrane resistance followed by a slow hyperpolarizing response associated with an increased membrane resistance in rat MPG neurons. The time courses of the CACA-induced responses were almost the same as those of the GABA-induced slow responses. The CACA-induced response was blocked by picrotoxin but not by bicuculline. I4AA, a GABAC-receptor antagonist (Bormann and Feigenspan 1995; Kusama et al. 1993; Qian and Dowling 1994), selectively depressed the late GABAd and the GABAh but not the early GABAd. I4AA also depressed the CACA-induced responses. These pharmacological properties indicate that GABA receptors that mediate the late GABAd and the GABAh in rat MPG neurons belong to the GABAC (or GABAAOr) (Barnard et al. 1998) receptor type.

Previously, it was demonstrated that the GABA-induced depolarization is produced by the activation of Cl- conductance in vesical pelvic ganglia (VPG) neurons of the cat urinary bladder (Mayer et al. 1983). The reversal potential of the early GABAd in rat VPG neurons was -32 ± 2 mV, which was comparable with that for the GABA-induced depolarization in cat pelvic neurons. Mayer et al. (1983) have also reported that the GABA-induced afterhyperpolarization is produced by a depression of the Cl- conductance in neurons of the cat VPG. In rat MPG, the GABAh was associated with an increased membrane resistance. The reversal potentials of the depolarization and the hyperpolarization induced by GABA in rat MPG neurons are comparable with those observed in cat VPG neurons (Mayer et al. 1983). When the concentration of extracellular Cl- was reduced from 125.4 to 85 mM, the reversal potentials of the late GABAd and the GABAh were shifted to a depolarizing potential according to the Nernst equation of Cl-. These results suggest that the Cl- conductance is also responsible for the GABA-induced biphasic responses in rat MPG.

GABA is known to downregulate the function of the lower urinary tract and is a putative inhibitory transmitter in the brain, spinal cord (Desarmenien et al. 1984; Kontani et al. 1988; Maggi et al. 1987; Sillén et al. 1980, 1985), pelvic ganglia (de Groat 1970; Maggi et al. 1983, 1985a) and neuron-smooth muscle junction (Chen et al. 1992; Kusunoki et al. 1984; Maggi et al. 1985a,c). It has been shown that neurons in the rat MPG contain GABA and its synthesizing enzyme (de Groat 1970; Kusunoki et al. 1984). Binding sites for GABA have also been demonstrated in rat MPG neurons (Kusunoki et al. 1984). The results of the present study together with previous studies indicate that the hyperpolarization induces a long-lasting inhibition of urinary bladder activity.


    ACKNOWLEDGMENTS

This work was supported by a Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Science, Sports and Culture of Japan (08457017).


    FOOTNOTES

Address for reprint requests: T. Akasu, Dept. of Physiology, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan

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. Section 1734 solely to indicate this fact.

Received 18 December 1998; accepted in final form 25 May 1999.


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