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Non-competitive inhibition of 5-HT3 receptor-mediated currents by progesterone in rat nodose ganglion neurons
Neuroscience Letters 278 (2000) 37±40 www.elsevier.com/locate/neulet
Non-competitive inhibition of 5-HT3 receptor-mediated currents by progesterone in rat nodose ganglion neurons Fong-Sen Wu*, Cheng-Pou Lai, Bie-Ching Liu Department of Physiology, College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan Received 2 July 1999; received in revised form 1 November 1999; accepted 2 November 1999
Abstract The effect of progesterone on the serotonin type 3 (5-HT3) receptor-mediated response was studied in acutely dissociated rat nodose ganglion neurons by using the whole-cell voltage-clamp technique. Progesterone rapidly and reversibly inhibited 5-HT-induced currents in a dose-dependent manner, with an EC50 of 31 mM and a maximal inhibition of 75%. Neither the 5-HT response nor inhibition of the 5-HT response by extracellularly applied progesterone was signi®cantly affected by inclusion of a saturating concentration of progesterone in the electrode buffer, arguing that progesterone acted at the extracellular surface of the membrane. Progesterone also inhibited the 5-HT response noncompetitively by a voltage- and agonist-independent mechanism that was distinct from that of open-channel blockers. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Neurosteroid; Progesterone; 5-HT3 receptor; Nodose ganglion neurons; Whole-cell recordings; Non-competitive inhibition
The serotonin type 3 (5-HT3) receptor is a ligand-gated ion channel that primarily gates monovalent cations such as Na 1 and K 1 [4], and mediates rapid excitatory synaptic transmission [12]. 5-HT3 receptors have attracted particular attention by virtue of their proposed role in emesis, nociception, and psychiatric disorders such as anxiety and depression [7]. On the other hand, steroid hormones are known to in¯uence profoundly the neuronal excitability [11]. In particular, the neurosteroid progesterone has been proposed as a positive modulator of the g-aminobutyric acid type A (GABAA) receptor and a negative modulator of the glycine receptor [15]. Progesterone also potentiates N-methyl-daspartate (NMDA), kainate, and a-amino-3-hydroxy-5methyl-4-isoxazolepropionate (AMPA) responses [16]. In the present study, we examine the effect of progesterone on the 5-HT3 receptor-mediated current in acutely dissociated rat nodose ganglion neurons. Nodose ganglion neurons from Sprague±Dawley male rats (150±300 g) were used 1±10 h after plating, as described previously [14]. Nodose ganglion neurons also were taken from female rats of similar body weight. Whole-cell currents were recorded by the whole-cell variant of the patch clamp technique [8]. Electrode resistance was * Corresponding author. Tel.: 1886-6-235-3535 ext. 5455; fax: 1886-6-236-2780. E-mail address: [email protected] (F.-S. Wu)
3±5 MV when ®lled with an intracellular solution. The electrode solution contained (in mM): 140 KCl, 1 CaCl2, 2 MgCl2, 11 EGTA, 10 HEPES, and 2 MgATP (pH adjusted to 7.2 with KOH). Sucrose was added to make the osmolarity 310 mosM. The osmolarity of the solution was measured by an osmometer (Osmomat 030, Gonotech GmbH, Germany). In some experiments such as determination of voltage dependency of progesterone effect on the 5-HTinduced current, a Cs 1-containing intracellular solution (in mM, 140 CsCl, 1 CaCl2, 2 MgCl2, 11 EGTA, 10 HEPES, and 2 MgATP, pH adjusted to 7.2 with CsOH) was used. The bath solution contained (in mM): 150 NaCl, 5 KCl, 2.5 CaCl2, 1 MgCl2, 10 HEPES, and 10 d-glucose (pH adjusted to 7.2 with NaOH and osmolarity to 340 mosM with sucrose). All experiments were performed at room temperature (23±258). Recordings were made using an Axopatch-1D patch clamp ampli®er (Axon Instruments, Foster City, CA). Cells with series resistance greater than 10 MV were rejected. Only cells with resting membrane potential more negative than 240 mV and input resistance in excess of 150 MV were used. Rat nodose ganglion neurons had resting membrane potentials of 254 ^ 0.8 mV (n 182). Unless otherwise indicated, all recordings were made with the cell membrane potential clamped at 270 mV. Currents were ®ltered at 1 kHz using an eight-pole Bessel ®lter and digi-
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 88 3- 6
38
F.-S. Wu et al. / Neuroscience Letters 278 (2000) 37±40
tized (4 ms/point) using an on-line data acquisition system (pClamp, Axon Instruments). Drug solutions were applied to single neurons by pressure ejection (15 psi) from seven-barrel pipettes [1,2]. All drugs were obtained from Sigma, with the exception of 5-HT hydrochloride, MDL 72222, tropisetron, and m-chlorophenylbiguanide (Research Biochemicals). Stock solutions of steroids were prepared in dimethyl sulfoxide (®nal concentration, 0.5%, v/v). To obviate the possible effect of dimethyl sulfoxide on the 5-HT-induced currents, all other drug solutions, including 5-HT and external buffer (in the pressure pipette), also contained 0.5% dimethyl sulfoxide. In all experiments, neurons received a 10-s prepulse of either external buffer or steroid solution, followed by a 10-s application of 5-HT or 5-HT plus steroid, and further by a 20-s pulse of external buffer solution. A period of 3±4 min was allowed between successive applications of 5-HT. At a holding potential of 270 mV, 5-HT produced inward currents, which reversed at or near 0 mV in standard recording solutions, as expected for Na 1 and K 1-mediated currents (data not shown). The current induced by 3 mM 5-HT was completely blocked by 100 nM MDL 72222 (n 3) or 100 nM tropisetron (n 3), consistent with the activation of 5-HT3 receptors. In agreement with the previous work [6], the current induced by 3 mM 5-HT quickly desensitized to a steady level. The selective 5-HT3 agonist m-chlorophenylbiguanide (5 mM) induced a current with similar kinetics (data not shown). The effect of progesterone on the current induced by 3 mM 5-HT in male rat neurons is illustrated in Fig. 1. Pressure application of 100 mM progesterone rapidly and reversibly inhibited the 5-HT response (Fig. 1A). Progesterone alone produced little or no direct response. The magnitude of the 5-HT response reduction (57 ^ 2.9%, n 10) by progesterone in female rat neurons was similar to that (64 ^ 3.6%, n 5) observed in male rat neurons, indicating that the effect of progesterone is sex-independent. The current induced by 5 mM mchlorophenylbiguanide was also reduced (84 ^ 6.4%, n 4) by 100 mM progesterone, suggesting that progesterone acts on 5-HT3 receptors or related areas. Not all steroids inhibit the 5-HT response. The progesterone reduced metabolite 5a-pregnan-3a-ol-20-one at 50 mM (which represents its maximal solubility in the external buffer) did not exert any signi®cant effect on the 3 mM 5-HT-induced current (2 ^ 3.0% potentiation, n 5). To quantitatively evaluate the potency and ef®cacy of progesterone for 5-HT3 receptors, pooled data were used to construct the dose-response curve for inhibition of the 3 mM 5-HT response by progesterone. Progesterone inhibited the 5-HT-induced current in a dose-dependent manner, with an EC50 of 31 mM, a maximal inhibition of 75%, and a Hill coef®cient of 1.27 (Fig. 1B). The threshold concentration for an effect of progesterone on the 5-HT-induced current was 10 mM. The maximal effect on the 5-HT response was achieved at approximately 200 mM. To determine whether progesterone acts intracellularly to
Fig. 1. Progesterone inhibits the 5-HT response. (A) 100 mM progesterone (P) inhibits rapidly and reversibly the current induced by 3 mM 5-HT. Horizontal bar above each trace, period of drug application. (B) Dose-response curve for inhibition of the 5-HT (3 mM) response by progesterone. Data points, percentage change in peak current in the presence of progesterone (mean of four to eight experiments). Error bars, standard errors. Progesterone dose-response curve is ®tted with the logistic equation [3]. (% inhibition)/(% inhibitionmax P nH = P nH 1 EC50 nH ) where [P] is the concentration of progesterone, and nH is the Hill coef®cient.
inhibit the 5-HT response, we tested the effect of a saturating concentration (200 mM) of intracellular progesterone on inhibition of the 5-HT response by extracellularly applied progesterone (100 mM). Inclusion of progesterone in the electrode buffer had no signi®cant effect on the 5-HTinduced current at 18 min after the cell was ruptured and did not block the effect of extracellular progesterone (Fig. 2A). In ®ve cells, average currents induced by 3 mM 5-HT at 6, 12, and 18 min after cell rupture were, respectively 2462 ^ 514.8, 2322 ^ 468.9, and 2287 ^ 475.3 pA, which did not differ signi®cantly from that (2505 ^ 541.5 pA) immediately (0 min) after cell rupture (P . 0:05, paired t-test). In addition, the average inhibition produced by extracellular progesterone with progesterone inside was 59 ^ 4.1% (n 5), which was not signi®cantly different from that (64 ^ 3.6%, n 5) measured without intracellular progesterone (P . 0:05, unpaired t-test) (Fig. 2B). This result indicates that progesterone acts at the extracellular surface of the membrane. To investigate whether inhibition of the 5-HT response by progesterone is competitive, pooled data were used to construct dose-response curves for 5-HT in the presence and absence of 100 mM progesterone. All responses were normalized to the peak current induced by 3 mM 5-HT to
F.-S. Wu et al. / Neuroscience Letters 278 (2000) 37±40
Fig. 2. Progesterone acts extracellularly to inhibit the 5-HT response. (A) Dialyzing the inside of the cell with a saturating concentration (200 mM) of progesterone in¯uences neither the 5HT (3 mM)-induced current nor inhibition by extracellularly applied progesterone (100 mM) of the 5-HT-induced current. Time after cell rupture is shown above each tracing. (B) Averaged data for the effect of extracellularly applied progesterone (100 mM) on the 5-HT-induced current in the absence (control) and presence (P inside) of intracellular progesterone (200 mM). Number of cells is indicated in parentheses.
obviate cell-to-cell variability with respect to the maximal current induced by 5-HT. Progesterone decreased the 5-HT maximal response with little effect on the 5-HT EC50 (Fig. 3), suggesting that the blocking action of progesterone on the 5-HT response was non-competitive. To determine whether block of the 5-HT response by progesterone is voltage-dependent, we tested the effect of 100 mM progesterone on the current induced by 3 mM 5-HT at two different holding potentials. In ®ve cells, the average inhibition produced by progesterone at 150 mV (57 ^ 1.9%) was not signi®cantly different from that at 270 mV (59 ^ 1.7%) (P . 0:05, paired t-test), indicating that the blockade by progesterone of the 5-HT response was not voltage-dependent. The blockade by progesterone was immediate and affected peak and plateau currents equally, and the recovery of the 5-HT response after washout of progesterone was rapid, suggesting that progesterone inhibition of the 5-HT response was not agonist-dependent and did not require opening of the 5-HT-gated channels. In the present study, the neurosteroid progesterone inhibited 5-HT3 receptor-mediated currents. The high lipophilicity of the steroids raised the possibility that the effect of progesterone was due to a non-speci®c mechanism of action, such as perturbation of the membrane lipids surrounding the 5-HT3 receptor protein. However, 5a-pregnan-3a-ol-20-one, a reduced metabolite of progesterone,
39
produced no signi®cant effect on the 5-HT response. This ®nding argued that inhibition of the 5-HT-induced current by progesterone was a speci®c effect. The ineffectiveness of 5a-pregnan-3a-ol-20-one on the 5-HT response indicated that reduction converted an active steroid to an inactive steroid inhibitor, suggesting that 5a-reductase and 3ahydroxysteroid oxidoreductase could play an important role in regulating 5-HT3 receptor activity in the mammalian nervous system. Recent studies on human embryonic kidney cells (HEK 293 cells) stably expressing 5-HT3 receptors from mouse neuroblastoma cells have shown that both progesterone and 5a-pregnan-3a-ol-20-one inhibit the 5-HT-induced current [13]. This is contrary to our observation that progesterone, but not 5a-pregnan-3a-ol-20-one, antagonizes the 5HT response in acutely dissociated rat nodose ganglion neurons. It may be that rat nodose ganglion neurons are less sensitive than HEK 293 cells to the modulatory effect of 5a-pregnan-3a-ol-20-one at the 5-HT3 receptor due to possible differences in subunit composition of the 5-HT3 receptors expressed in these systems. Intracellular progesterone affects neither the 5-HT response nor inhibition by extracellularly applied progesterone of the 5-HT response (Fig. 2), indicating that the progesterone modulatory site is located externally. There are a number of potential sites at which progesterone could exert its blocking action including: (1) competitive inhibition at the 5-HT binding site; (2) blockade of the 5-HT3 receptor channel; or (3) non-competitive inhibition or allosteric modulation at a distinct site. First, progesterone inhi-
Fig. 3. Antagonism of the 5-HT response by progesterone is noncompetitive. Dose-response curves for 5-HT in the presence and absence of 100 mM progesterone. All responses are normalized to the peak current (*) induced by 3 mM 5-HT. Data points, normalized peak currents (mean of four to eleven experiments). Error bars, standard errors. Error bars are not indicated when smaller than the size of the circle. Each set of data points is ®tted with the nH logistic equation [3]. I=I max 5±HT nH = 5±HT nH 1 EC50 ) where Imax is the maximal normalized current, [5±HT] is the concentration of 5-HT, and nH is the Hill coef®cient. In the absence of progesterone, EC50 5:0 mM, I max 2:68, and nH 1:03. In the presence of progesterone, EC50 4:0 mM, I max 1:19, and nH 1:66.
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F.-S. Wu et al. / Neuroscience Letters 278 (2000) 37±40
bits the 5-HT maximal response with little effect on the 5HT EC50 (Fig. 3), demonstrating that the action of progesterone is non-competitive. This contrasts with the competitive mechanism of action of tetraethylammonium [10], cocaine [6], and morphine [5] on the 5-HT3 receptor and suggests that progesterone and these three drugs act through different sites to inhibit the 5-HT response. Second, inhibition of the 5-HT-induced current by progesterone is neither voltage- nor agonist-dependent, arguing against the possibility that progesterone acts as an open-channel blocker. The highest reported concentration of progesterone in rat plasma is about 20 mM in late pregnancy [9], which is close to our EC50 value for progesterone effect on the 5-HT response (Fig. 1). Due to the highly lipophilic nature of progesterone and its possible localized accumulation in the peripheral and central nervous system, it is likely that progesterone under normal physiological conditions may modulate the 5-HT3 receptor in the mammalian nervous system. The study was supported by the National Science Council (NSC 88±2314-B006±135) of Taiwan. [1] Chan, C.Y. and Farb, D.H., Modulation of neurotransmitter action: control of the g-aminobutyric acid response through the benzodiazepine receptor. J. Neurosci., 5 (1985) 2365± 2373. [2] Choi, D.W. and Fischbach, G.D., GABA conductance of chick spinal cord and dorsal root ganglion neurons in cell culture. J. Neurophysiol., 45 (1981) 605±620. [3] De Lean, A.P., Munson, P.J. and Rodbard, D., Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay, and physiological doseresponse curves. Am. J. Physiol., 235 (1978) E97±E102. [4] Derkach, V., Surprenant, A. and North, R.A., 5-HT3 receptors are membrane ion channels. Nature, 339 (1989) 706±709. [5] Fan, P., Nonopioid mechanism of morphine modulation of
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[15] [16]
the activation of 5-hydroxytryptamine type 3 receptors. Mol. Pharmacol., 47 (1995) 491±495. Fan, P., Visentin, S. and Weight, F.F., Effects of cocaine on the serotonin-induced inward current in rat nodose ganglion neurons. J. Pharmacol. Exp. Ther., 271 (1994) 262±266. Greenshaw, A.J., Behavioural pharmacology of 5-HT3 receptor antagonists: a critical update on therapeutic potential. Trends Pharmacol. Sci., 14 (1993) 265±270. Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.J., Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. P¯uÈgers Arch., 391 (1981) 85±100. Ichikawa, S., Sawada, T., Nakamura, Y. and Morioka, H., Ovarian secretion of pregnane compounds during the estrous cycle and pregnancy in rats. Endocrinology, 94 (1974) 1615±1620. Kooyman, A.R., Zwart, R. and Vijverberg, H.P., Tetraethylammonium ions block 5-HT3 receptor-mediated ion current at the agonist recognition site and prevent desensitization in cultured mouse neuroblastoma cells. Eur. J. Pharmacol., 246 (1993) 247±254. Majewska, M.D., Steroids and brain activity: essential dialogue between body and mind. Biochem. Pharmacol., 36 (1987) 3781±3788. Sugita, S., Shen, K.-Z. and North, R.A., 5-Hydroxytryptamine is a fast excitatory transmitter at 5-HT3 receptors in rat amygdala. Neuron, 8 (1992) 199±203. Wetzel, C.H.R., Hermann, B., Behl, C., Pestel, E., Rammes, G., Zieglgansberger, W., Holsboer, F. and Rupprecht, R., Functional antagonism of gonadal steroids at the 5-hydroxytryptamine type 3 receptor. Mol. Endocrinol., 12 (1998) 1441±1451. Wiley, J.W., Gross, R.A. and MacDonald, R.L., Agonists for neuropeptide Y receptor subtypes NPY-1 and NPY-2 have opposite actions on rat nodose neuron calcium currents. J. Neurophysiol., 70 (1993) 324±330. Wu, F.-S., Gibbs, T.T. and Farb, D.H., Inverse modulation of g-aminobutyric acid- and glycine-induced currents by progesterone. Mol. Pharmacol., 37 (1990) 597±602. Wu, F.-S., Yu, H.-M. and Tsai, J.-J., Mechanism underlying potentiation by progesterone of the kainate-induced current in cultured neurons. Brain Res., 779 (1998) 354±358.
Non-competitive inhibition of 5-HT3 receptor-mediated currents by progesterone in rat nodose ganglion neurons Fong-Sen Wu*, Cheng-Pou Lai, Bie-Ching Liu Department of Physiology, College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan Received 2 July 1999; received in revised form 1 November 1999; accepted 2 November 1999
Abstract The effect of progesterone on the serotonin type 3 (5-HT3) receptor-mediated response was studied in acutely dissociated rat nodose ganglion neurons by using the whole-cell voltage-clamp technique. Progesterone rapidly and reversibly inhibited 5-HT-induced currents in a dose-dependent manner, with an EC50 of 31 mM and a maximal inhibition of 75%. Neither the 5-HT response nor inhibition of the 5-HT response by extracellularly applied progesterone was signi®cantly affected by inclusion of a saturating concentration of progesterone in the electrode buffer, arguing that progesterone acted at the extracellular surface of the membrane. Progesterone also inhibited the 5-HT response noncompetitively by a voltage- and agonist-independent mechanism that was distinct from that of open-channel blockers. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Neurosteroid; Progesterone; 5-HT3 receptor; Nodose ganglion neurons; Whole-cell recordings; Non-competitive inhibition
The serotonin type 3 (5-HT3) receptor is a ligand-gated ion channel that primarily gates monovalent cations such as Na 1 and K 1 [4], and mediates rapid excitatory synaptic transmission [12]. 5-HT3 receptors have attracted particular attention by virtue of their proposed role in emesis, nociception, and psychiatric disorders such as anxiety and depression [7]. On the other hand, steroid hormones are known to in¯uence profoundly the neuronal excitability [11]. In particular, the neurosteroid progesterone has been proposed as a positive modulator of the g-aminobutyric acid type A (GABAA) receptor and a negative modulator of the glycine receptor [15]. Progesterone also potentiates N-methyl-daspartate (NMDA), kainate, and a-amino-3-hydroxy-5methyl-4-isoxazolepropionate (AMPA) responses [16]. In the present study, we examine the effect of progesterone on the 5-HT3 receptor-mediated current in acutely dissociated rat nodose ganglion neurons. Nodose ganglion neurons from Sprague±Dawley male rats (150±300 g) were used 1±10 h after plating, as described previously [14]. Nodose ganglion neurons also were taken from female rats of similar body weight. Whole-cell currents were recorded by the whole-cell variant of the patch clamp technique [8]. Electrode resistance was * Corresponding author. Tel.: 1886-6-235-3535 ext. 5455; fax: 1886-6-236-2780. E-mail address: [email protected] (F.-S. Wu)
3±5 MV when ®lled with an intracellular solution. The electrode solution contained (in mM): 140 KCl, 1 CaCl2, 2 MgCl2, 11 EGTA, 10 HEPES, and 2 MgATP (pH adjusted to 7.2 with KOH). Sucrose was added to make the osmolarity 310 mosM. The osmolarity of the solution was measured by an osmometer (Osmomat 030, Gonotech GmbH, Germany). In some experiments such as determination of voltage dependency of progesterone effect on the 5-HTinduced current, a Cs 1-containing intracellular solution (in mM, 140 CsCl, 1 CaCl2, 2 MgCl2, 11 EGTA, 10 HEPES, and 2 MgATP, pH adjusted to 7.2 with CsOH) was used. The bath solution contained (in mM): 150 NaCl, 5 KCl, 2.5 CaCl2, 1 MgCl2, 10 HEPES, and 10 d-glucose (pH adjusted to 7.2 with NaOH and osmolarity to 340 mosM with sucrose). All experiments were performed at room temperature (23±258). Recordings were made using an Axopatch-1D patch clamp ampli®er (Axon Instruments, Foster City, CA). Cells with series resistance greater than 10 MV were rejected. Only cells with resting membrane potential more negative than 240 mV and input resistance in excess of 150 MV were used. Rat nodose ganglion neurons had resting membrane potentials of 254 ^ 0.8 mV (n 182). Unless otherwise indicated, all recordings were made with the cell membrane potential clamped at 270 mV. Currents were ®ltered at 1 kHz using an eight-pole Bessel ®lter and digi-
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 88 3- 6
38
F.-S. Wu et al. / Neuroscience Letters 278 (2000) 37±40
tized (4 ms/point) using an on-line data acquisition system (pClamp, Axon Instruments). Drug solutions were applied to single neurons by pressure ejection (15 psi) from seven-barrel pipettes [1,2]. All drugs were obtained from Sigma, with the exception of 5-HT hydrochloride, MDL 72222, tropisetron, and m-chlorophenylbiguanide (Research Biochemicals). Stock solutions of steroids were prepared in dimethyl sulfoxide (®nal concentration, 0.5%, v/v). To obviate the possible effect of dimethyl sulfoxide on the 5-HT-induced currents, all other drug solutions, including 5-HT and external buffer (in the pressure pipette), also contained 0.5% dimethyl sulfoxide. In all experiments, neurons received a 10-s prepulse of either external buffer or steroid solution, followed by a 10-s application of 5-HT or 5-HT plus steroid, and further by a 20-s pulse of external buffer solution. A period of 3±4 min was allowed between successive applications of 5-HT. At a holding potential of 270 mV, 5-HT produced inward currents, which reversed at or near 0 mV in standard recording solutions, as expected for Na 1 and K 1-mediated currents (data not shown). The current induced by 3 mM 5-HT was completely blocked by 100 nM MDL 72222 (n 3) or 100 nM tropisetron (n 3), consistent with the activation of 5-HT3 receptors. In agreement with the previous work [6], the current induced by 3 mM 5-HT quickly desensitized to a steady level. The selective 5-HT3 agonist m-chlorophenylbiguanide (5 mM) induced a current with similar kinetics (data not shown). The effect of progesterone on the current induced by 3 mM 5-HT in male rat neurons is illustrated in Fig. 1. Pressure application of 100 mM progesterone rapidly and reversibly inhibited the 5-HT response (Fig. 1A). Progesterone alone produced little or no direct response. The magnitude of the 5-HT response reduction (57 ^ 2.9%, n 10) by progesterone in female rat neurons was similar to that (64 ^ 3.6%, n 5) observed in male rat neurons, indicating that the effect of progesterone is sex-independent. The current induced by 5 mM mchlorophenylbiguanide was also reduced (84 ^ 6.4%, n 4) by 100 mM progesterone, suggesting that progesterone acts on 5-HT3 receptors or related areas. Not all steroids inhibit the 5-HT response. The progesterone reduced metabolite 5a-pregnan-3a-ol-20-one at 50 mM (which represents its maximal solubility in the external buffer) did not exert any signi®cant effect on the 3 mM 5-HT-induced current (2 ^ 3.0% potentiation, n 5). To quantitatively evaluate the potency and ef®cacy of progesterone for 5-HT3 receptors, pooled data were used to construct the dose-response curve for inhibition of the 3 mM 5-HT response by progesterone. Progesterone inhibited the 5-HT-induced current in a dose-dependent manner, with an EC50 of 31 mM, a maximal inhibition of 75%, and a Hill coef®cient of 1.27 (Fig. 1B). The threshold concentration for an effect of progesterone on the 5-HT-induced current was 10 mM. The maximal effect on the 5-HT response was achieved at approximately 200 mM. To determine whether progesterone acts intracellularly to
Fig. 1. Progesterone inhibits the 5-HT response. (A) 100 mM progesterone (P) inhibits rapidly and reversibly the current induced by 3 mM 5-HT. Horizontal bar above each trace, period of drug application. (B) Dose-response curve for inhibition of the 5-HT (3 mM) response by progesterone. Data points, percentage change in peak current in the presence of progesterone (mean of four to eight experiments). Error bars, standard errors. Progesterone dose-response curve is ®tted with the logistic equation [3]. (% inhibition)/(% inhibitionmax P nH = P nH 1 EC50 nH ) where [P] is the concentration of progesterone, and nH is the Hill coef®cient.
inhibit the 5-HT response, we tested the effect of a saturating concentration (200 mM) of intracellular progesterone on inhibition of the 5-HT response by extracellularly applied progesterone (100 mM). Inclusion of progesterone in the electrode buffer had no signi®cant effect on the 5-HTinduced current at 18 min after the cell was ruptured and did not block the effect of extracellular progesterone (Fig. 2A). In ®ve cells, average currents induced by 3 mM 5-HT at 6, 12, and 18 min after cell rupture were, respectively 2462 ^ 514.8, 2322 ^ 468.9, and 2287 ^ 475.3 pA, which did not differ signi®cantly from that (2505 ^ 541.5 pA) immediately (0 min) after cell rupture (P . 0:05, paired t-test). In addition, the average inhibition produced by extracellular progesterone with progesterone inside was 59 ^ 4.1% (n 5), which was not signi®cantly different from that (64 ^ 3.6%, n 5) measured without intracellular progesterone (P . 0:05, unpaired t-test) (Fig. 2B). This result indicates that progesterone acts at the extracellular surface of the membrane. To investigate whether inhibition of the 5-HT response by progesterone is competitive, pooled data were used to construct dose-response curves for 5-HT in the presence and absence of 100 mM progesterone. All responses were normalized to the peak current induced by 3 mM 5-HT to
F.-S. Wu et al. / Neuroscience Letters 278 (2000) 37±40
Fig. 2. Progesterone acts extracellularly to inhibit the 5-HT response. (A) Dialyzing the inside of the cell with a saturating concentration (200 mM) of progesterone in¯uences neither the 5HT (3 mM)-induced current nor inhibition by extracellularly applied progesterone (100 mM) of the 5-HT-induced current. Time after cell rupture is shown above each tracing. (B) Averaged data for the effect of extracellularly applied progesterone (100 mM) on the 5-HT-induced current in the absence (control) and presence (P inside) of intracellular progesterone (200 mM). Number of cells is indicated in parentheses.
obviate cell-to-cell variability with respect to the maximal current induced by 5-HT. Progesterone decreased the 5-HT maximal response with little effect on the 5-HT EC50 (Fig. 3), suggesting that the blocking action of progesterone on the 5-HT response was non-competitive. To determine whether block of the 5-HT response by progesterone is voltage-dependent, we tested the effect of 100 mM progesterone on the current induced by 3 mM 5-HT at two different holding potentials. In ®ve cells, the average inhibition produced by progesterone at 150 mV (57 ^ 1.9%) was not signi®cantly different from that at 270 mV (59 ^ 1.7%) (P . 0:05, paired t-test), indicating that the blockade by progesterone of the 5-HT response was not voltage-dependent. The blockade by progesterone was immediate and affected peak and plateau currents equally, and the recovery of the 5-HT response after washout of progesterone was rapid, suggesting that progesterone inhibition of the 5-HT response was not agonist-dependent and did not require opening of the 5-HT-gated channels. In the present study, the neurosteroid progesterone inhibited 5-HT3 receptor-mediated currents. The high lipophilicity of the steroids raised the possibility that the effect of progesterone was due to a non-speci®c mechanism of action, such as perturbation of the membrane lipids surrounding the 5-HT3 receptor protein. However, 5a-pregnan-3a-ol-20-one, a reduced metabolite of progesterone,
39
produced no signi®cant effect on the 5-HT response. This ®nding argued that inhibition of the 5-HT-induced current by progesterone was a speci®c effect. The ineffectiveness of 5a-pregnan-3a-ol-20-one on the 5-HT response indicated that reduction converted an active steroid to an inactive steroid inhibitor, suggesting that 5a-reductase and 3ahydroxysteroid oxidoreductase could play an important role in regulating 5-HT3 receptor activity in the mammalian nervous system. Recent studies on human embryonic kidney cells (HEK 293 cells) stably expressing 5-HT3 receptors from mouse neuroblastoma cells have shown that both progesterone and 5a-pregnan-3a-ol-20-one inhibit the 5-HT-induced current [13]. This is contrary to our observation that progesterone, but not 5a-pregnan-3a-ol-20-one, antagonizes the 5HT response in acutely dissociated rat nodose ganglion neurons. It may be that rat nodose ganglion neurons are less sensitive than HEK 293 cells to the modulatory effect of 5a-pregnan-3a-ol-20-one at the 5-HT3 receptor due to possible differences in subunit composition of the 5-HT3 receptors expressed in these systems. Intracellular progesterone affects neither the 5-HT response nor inhibition by extracellularly applied progesterone of the 5-HT response (Fig. 2), indicating that the progesterone modulatory site is located externally. There are a number of potential sites at which progesterone could exert its blocking action including: (1) competitive inhibition at the 5-HT binding site; (2) blockade of the 5-HT3 receptor channel; or (3) non-competitive inhibition or allosteric modulation at a distinct site. First, progesterone inhi-
Fig. 3. Antagonism of the 5-HT response by progesterone is noncompetitive. Dose-response curves for 5-HT in the presence and absence of 100 mM progesterone. All responses are normalized to the peak current (*) induced by 3 mM 5-HT. Data points, normalized peak currents (mean of four to eleven experiments). Error bars, standard errors. Error bars are not indicated when smaller than the size of the circle. Each set of data points is ®tted with the nH logistic equation [3]. I=I max 5±HT nH = 5±HT nH 1 EC50 ) where Imax is the maximal normalized current, [5±HT] is the concentration of 5-HT, and nH is the Hill coef®cient. In the absence of progesterone, EC50 5:0 mM, I max 2:68, and nH 1:03. In the presence of progesterone, EC50 4:0 mM, I max 1:19, and nH 1:66.
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F.-S. Wu et al. / Neuroscience Letters 278 (2000) 37±40
bits the 5-HT maximal response with little effect on the 5HT EC50 (Fig. 3), demonstrating that the action of progesterone is non-competitive. This contrasts with the competitive mechanism of action of tetraethylammonium [10], cocaine [6], and morphine [5] on the 5-HT3 receptor and suggests that progesterone and these three drugs act through different sites to inhibit the 5-HT response. Second, inhibition of the 5-HT-induced current by progesterone is neither voltage- nor agonist-dependent, arguing against the possibility that progesterone acts as an open-channel blocker. The highest reported concentration of progesterone in rat plasma is about 20 mM in late pregnancy [9], which is close to our EC50 value for progesterone effect on the 5-HT response (Fig. 1). Due to the highly lipophilic nature of progesterone and its possible localized accumulation in the peripheral and central nervous system, it is likely that progesterone under normal physiological conditions may modulate the 5-HT3 receptor in the mammalian nervous system. The study was supported by the National Science Council (NSC 88±2314-B006±135) of Taiwan. [1] Chan, C.Y. and Farb, D.H., Modulation of neurotransmitter action: control of the g-aminobutyric acid response through the benzodiazepine receptor. J. Neurosci., 5 (1985) 2365± 2373. [2] Choi, D.W. and Fischbach, G.D., GABA conductance of chick spinal cord and dorsal root ganglion neurons in cell culture. J. Neurophysiol., 45 (1981) 605±620. [3] De Lean, A.P., Munson, P.J. and Rodbard, D., Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay, and physiological doseresponse curves. Am. J. Physiol., 235 (1978) E97±E102. [4] Derkach, V., Surprenant, A. and North, R.A., 5-HT3 receptors are membrane ion channels. Nature, 339 (1989) 706±709. [5] Fan, P., Nonopioid mechanism of morphine modulation of
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