Inhibitory effects of botulinum toxin on 5-HT1C receptor-induced Cl− current in Xenopus oocytes

European Journal of Pharmacology - Molecular Pharmacology Section, 266 (1994) 19-24

19

© 1994 Elsevier Science Publishers B.V. All rights reserved 0922-4106/94/$07.00

EJPMOL 90538

Inhibitory effects of botulinum toxin on 5-HTlc receptor-induced C1current in Xenopus oocytes Michihisa T o h d a

a

T o s h i y u k i T a k a s u a Junji N a k a m u r a a, N a r i t o M o r i i b, S h u h N a r u m i y a b a n d Y a s u y u k i N o m u r a a,c,,

a Department of Pharmacology, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060, Japan, b Department of Pharmacology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan, and c Department of Neuroscience, Research institute for Oriental Medicine (Wakan-yaku), Toyama Medical and Pharmaceutical University, Toyama 930-01, Japan Received 19 May 1993; revised MS received 27 July 1993; accepted 13 August 1993

Several low molecular weight G proteins have been identified, but their functional roles remain unclear. To clarify the involvement of low molecular weight G protein in receptor-stimulated turnover of polyphosphoinositide (PI) turnover, influences of botulinum toxins on serotonin (5-HT)-stimulated C1- current mediated by PI turnover were investigated using Xenopus oocytes injected with rat brain mRNA. Treatment with botulinum toxin C, D or purified ADP-ribosyltransferase of botulinum toxin (botulinum toxin C3 enzyme) inhibited the 5-HT-induced CI- current in oocytes, and ADP-ribosylated 23 kDa proteins. Both botulinum toxin C3 enzyme-induced inhibition of the current and ADP-ribosylation were suppressed by pretreatment with antibotulinum toxin C3 enzyme antibody. Botulinum toxin D treatment of oocytes was ineffective in the response of C1- current induced by injection of 50 pmol inositol 1,4,5-trisphosphate and 50 pmol Ca 2+. It is suggested that low molecular weight G proteins ADP-ribosylated by botulinum toxin C3 enzyme are involved in phospholipase C activation in Xenopus oocytes. G protein (low molecular weight); Polyphosphoinositide turnover; Botulinum toxin; ADP-ribosylation, 5-HT (5-hydroxytryptamine, serotonin); Xenopus oocyte

I. Introduction

G T P binding proteins (G proteins) are mainly divided into two types: heterotrimeric and low molecular weight G proteins. Heterotrimeric G proteins transduce receptor signalling to effectors such as adenylate cyclase, Ca 2÷ channels (Yatani et al., 1988), K ÷ channels (Cerbai et al., 1988; VanDongen et al., 1988), phospholipase A 2 (Murayama et al., 1990) and cGMP phosphodiesterase. It has been reported that phospholipase C activity is regulated by heterotrimeric G proteins, but the characterization of the G proteins, particularly in terms of pertussis toxin sensitivity, is distinct in different cells (Moriarty et al., 1989), receptors (Socorro et al., 1990) and even by different in authors (Ueda et al., 1989: Moriarty et al., 1990). Low molecular weight G proteins (molecular mass 20-25 kDa) are divided into several superfamily, such as ras, rab, arf and rho (Hall, 1990). Although the functions including neuronal differentiation (Noda et

* Corresponding author. Tel.: 011-716-2111, ext. 3246; Fax: 011-7075632.

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al., 1985: Hancock et al., 1989: Nishiki et al., 1990), actin polymerization (Chardin et al., 1989), vesicular transport (Balch, 1990), and smooth muscle contraction (Hirata et al., 1992) have been investigated, the detail has not been clarified. Some low molecular weight G proteins, belonging to the rho family are ADP-ribosylated by botulinum toxins (Kikuchi et al., 1988; Narumiya et al., 1988; Braun et al., 1989). It is of interest to examine the effects of botulinum toxin on neuronal functions, in particular, cellular signalling mechanism. In this study, we found that low molecular weight G proteins ADP-ribosylated by botulinum toxin were involved in 5-HTlc receptor-induced polyphosphoinositide turnover in Xenopus oocytes injected with rat brain mRNA.

2. Materials and methods

2.1. Purification of poly(A) + mRNA, dissection of oocytes and microinjection of mRNA The maintenance and dissection of frogs, handling of oocytes, and detection of drug-evoked current in

20 electrophysiological recordings were carried out as previously reported (Tohda et al., 1989). Total R N A was extracted from whole brains of adult male Wistar rats by the cesium chloride method (Sambrook et al., 1989) and stored at - 8 0 ° C in sterile water until use (5 mg/ml). Oocytes defolliculated by collagenase treatment (1 m g / m l , in Ca2+-free MBS (see below) at 22°C for 30 rain) were injected with 50 ng of total R N A and cultivated at 22°C in sterile modified Barth's solution (MBS: 88 mM NaC1, 1 mM KCI, 0.4 mM CaC12, 0.33 mM Ca(NO3)2, 0.82 mM MgSO4, 2.4 mM NaHCO3, 7.5 mM Tris-HC1, pH 7.6) for 1 to 3 days.

2.2. Electrophysiological arrangements Following removal of the deteriorated oocytes, vigorous oocytes possessing negative membrane potential exceeding - 2 0 mV were used for electrophysiological recording. A single oocyte was placed in a small bath continuously perfused at room temperature (18-23°C), impaled with two microelectrodes and the membrane potential was maintained at - 6 0 mV. 5-HT was diluted with MBS and applied to oocytes in the bath at a rate of 1 ml/min. A period of 10-15 s was required to change all the solution in the bath. Inositol 1,4,5-trisphosphate (IP 3) or Ca 2÷ solution of 50 nl was injected into the oocyte by N 2 pressure.

bated at 70°C for 5 min. Then each sample (20/zg) was applied to 15% SDS-polyacrylamide gel and electrophorised. The gel was dried and autoradiographed at - 8 0 ° C using Kodak X-Omat film. Anti-C3-antibody was preincubated with botulinum toxin C3 enzyme enzyme at 30°C for 60 min, and then the mixture of botulinum toxin C3 enzyme and anti-botulinum toxin C3 enzyme antibody was added to oocytes.

2.4. Materials Botulinum toxin A, C, D were purchased from Wako (Japan). Botulinum toxin C3 enzyme was purified from the culture medium of Clostridium botulinum type C strain 003-9 and Anti-C3-antibody was prepared by the previously reported method (Morii et al., 1990).

3. Results

3.1. Effects of botulinum toxin on 5-HT-induced current and ADP-ribosylation Treatment with 2 /zg/ml and 4 / z g / m l botulinum toxin D and 2 /zg/ml botulinum toxin C significantly inhibited the 5-HT-induced current in Xenopus oocytes injected with rat brain mRNA, although botulinum toxin A was ineffective (Fig. 1). No oocytes died by the

2.3. Autoradiographic analysis of proteins [3ep]ADPribosylated by botulinum toxin Oocytes were homogenized in homogenizing buffer (50 mM Tris-HCl (pH 7,4), 10 mM MgC12, 1 mM EDTA, 100 U / m l aprotinin), centrifuged at 105,000 × g for 60 rain twice, and was the cytosol fraction then obtained as supernatant. The resultant pellet was resuspended in homogenizing buffer, centrifuged at 1000 × g for 10 min, and the supernatant was further centrifuged at 10,000 × g for 20 min twice to obtain the membrane fraction as the pellet. The pellet was suspended in stock buffer (50 mM Tris-HC1 (pH 7.4). 1 mM MgC12, 0.1 mM EDTA, 250 mM sucrose) and stored at -80°C. The membrane a n d / o r cytosol fraction (3 m g / m l of both or 0.3 m g / m l of membrane alone) were ADP-ribosylated with 5 izM, 185 k B q / t u b e [32p]NAD and 1 0 0 / z g / m l botulinum toxin A, C, D or 250 n g / m l botulinum toxin C3 enzyme in ADP-ribosylation buffer (total volume 100 tzl of the 50 mM Tris-HCl (pH 8.0). 2.5 mM MgCIz, 2.5 mM EDTA, 1 mM ATP, 10 mM dithiothreitol, 10 mM thymidine, 3 mM phosphocreatine, 100 U phosphocreatine kinase, 0.2% deoxycholate, 0.04 mg cardiolipin) at 30°C for 60 min. The reaction was terminated by the addition of 200 /zl lysing buffer (130 mM Tris-HC1 (pH 7.8), 4% sodium dodecyl sulfate, 20% glycerol, 0.01% bromophenol blue, 10% 2-mercaptoethanol) and incu-

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Fig. 1. Effects of several types of botulinum toxin on 5-HT-induced current in Xenopus oocytes injected with rat brain mRNA. The oocytes were treated with A, C, D of botulinum toxin for 17 h and the transmembrane currents were measured by voltage clamp methods, held at -60 mV. The number of the experiments is shown in parentheses. Each value shows the mean+S.E.. Significance: * P < 0.05, ** P < 0.01.

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A C D Fig. 2. ADP-ribosylation of Xenopus oocytes proteins by each type of botulinum toxin. The oocyte membrane and cytosol fractions were obtained by centrifugation. The 3 mg/ml proteins containing both membrane and cytosolic fractions were incubated with 100 tz/ml botulinum toxin A, C, or D and 5 p,M, 185 kBq/tube [32p]NAD at 30°C for 60 rain. Then the proteins (20 /xg) were applied to 15% SDS-polyacrylamide gel and electrophoresis was carried out. The gel was dried and autoradiographed.

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Fig. 4. ADP-ribosylation of Xenopus oocyte proteins by botulinum toxin C3 enzyme. The oocyte membrane (0.3 mg/ml) was ADPribosylated by 250 ng/ml botulinum toxin C3 enzyme and 2 /zg of protein was applied to electrophoresis and the electrophotogram was autoradiographed. Anti-C3-antibody (5 /xg/ml) was preincubated with botulinum toxin C3 enzyme at 30°C for 60 min, and then the mixture was added to incubate. (1) vehicle; (2) botulinum toxin C3 enzyme alone; (3) anti-C3-antibody alone; and (4) botulinum toxin C3 enzyme preincubated with anti-C3-antiboby.

treatment with botulinum toxin A, C, and D even at 10 /xg/ml (data not shown). Both botulinum toxin D and botulinum toxin C ADP-ribosylated 23 kDa proteins in 100

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Fig. 3. Effects of botulinum toxin C3 enzyme on 5-HT-induced current in Xenopus oocytes injected with rat brain mRNA. The oocytes were treated with botulinum toxin C3 enzyme for 17 h and the transmembrane currents were measured by voltage clamp methods, held at - 6 0 mV. The number of the experiments is shown in parentheses. Each value shows the mean + S.E..

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(pg/ml) Fig. 5. Effects of anti-C3-antibody on botulinum toxin C3 enzyme-induced inhibition of 5-HT-current in oocytes. The oocytes were treated with botulinum toxin C3 for 17 h and the transmembrane currents were measured by voltage clamp methods, held at - 6 0 mV. AntiC3-antibody was preincubated with botulinum toxin C3 enzyme for 60 min, and then the mixture was added. The number of the experiments is shown in parentheses. Each value shows the mean + S.E..

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membrane and cytosol fractions but botulinum toxin A did not (Fig. 2).

3.2. Effects of botulinum toxin C3 enzyme on 5-HT-induced current and ADP-ribosylation

Since it has been indicated that ADP-ribosylation of proteins by botulinum toxin is due to the ADP-ribosyltransferase activity of botulinum toxin C3 enzyme (Morii et al., 1990), influences of the purified botulinum toxin C3 enzyme on 5-HTtc receptor-induced current were also investigated. Botulinum toxin C3 enzyme inhibited the current in a concentration-dependent manner (Fig. 3). The ADP-ribosylation induced by 250 ng/ml botulinum toxin C3 enzyme was inhibited by pretreatment of the enzyme with 5 /zg/ml anti-botulinum toxin C3 enzyme antibody (Fig. 4). The antibody treatment (2/~g/ml) also significantly blocked the 0.1 tzg/ml botulinum toxin C3 enzyme-induced inhibition of 5-HT current (Fig. 5).

3.3. Effects of botulinum toxin D on IP3- and Ca 2 +-induced current

The pretreatment of oocyte for 17 h with 4 ~ g / m l botulinum toxin D was ineffective in the CI- current response induced by intracellular injection of 50 pmol I P3 and 50 pmol Ca 2+-induced current (Fig. 6).

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Fig. 6. Effects of botulinum toxin D on IP 3- or Ca2+-induced current in oocytes. The oocytes were treated with 4 ~ g / m l botulinum toxin D for 17 h and the transmembrane currents were measured by voltage clamp methods, held at - 6 0 inV. The 50 nl of I P 3 or Ca 2÷ was injected into the oocyte. The number of the experiments is shown in parentheses. Each value shows the mean + S.E..

4. Discussion

Botulinum toxin D and botulinum toxin C inhibited the 5-HT-induced current, although botulinum toxin A was ineffective (Fig. 1). Botulinum toxin are divided into 7 types, from A type to G type, by immunoreactivity (Simpson, 1981). Although every type of toxins exert inhibitory effects on acetylcholine release (Murayama et al., 1987), only C and D type toxins possess ADPribosylation activity (Matsuoka et al., 1987), suggesting that ADP-ribosylation is not involved in the inhibition of ACh release. A recent paper reported that protease of the toxin blocked the transmitter release by cleavage of synaptobrevin, a protein that seems to play a key part in the release (Schiavo et al., 1992). It has been also reported that ADP-ribosylation activity in both botulinum toxin C and botulinum toxin D is carried by botulinum toxin C3 enzyme, molecular mass 25 kDa (Aktories et al., 1987; Ohashi and Narumiya, 1987). The botulinum toxin C3 enzyme inhibited the 5-HT-induced current in a concentration-dependent manner (Fig. 3) and ADP-ribosylated 25 kDa membrane proteins (Fig. 4). Botulinum toxin D, which contains the ADP-ribosyltransferase activity (Fig. 2), did not influence I P 3- and CaZ+-induced current (Fig. 6). These results suggest that low molecular weight G proteins ADP-ribosylated by botulinum toxin C3 enzymes are involved in PI turnover, probably in phospholipase C activities. However, there is no report to indicate that these G proteins are directly involved in PI turnover. It has been reported that several low molecular weight G proteins are ADP-ribosylated by botulinum toxin including rho A (22 kDa) (Narumiya et al., 1988; Ogorochi et al., 1989), rho B (22 kDa) (Kikuchi et al., 1988), rho C (22.5 kDa) (Chardin et al., 1989). Since ADP-ribosylation of rho induces actin depolymerization (Chardin et al., 1989), rho appears to be involved in some functions in cytoskeleton. Recently, interesting findings that the actin binding proteins are detected by antiphosphatidylinositol 4,5-bisphosphate (PIP 2) antiboby have been reported, suggesting that the actin binding proteins possess PIP 2 (Fukami et al., 1992). It is presumed that ADP-ribosylation of rho by botulinum toxin reduces the supply of PIP 2 which bind to actin binding proteins through actin depolymerization, resulting in the inhibition of 5-HTlc-stimulated PI turnover. Wang et al. (1989b) found a novel G protein with high affinity to phospholipase C in cytosolic fraction of bovine thymic lymphocytes. They purified this novel G protein with molecular mass of 21 kDa from membrane fractions (Wang et al., 1989a). It has not been clarified, however, whether the novel G proteins are ADP-ribosylated by botulinum toxin. Although I P 3 and diacylglycerol contents are increased in Ha-ras transfected fibroblasts (Huang et al., 1988), Ha-ras protein does not seem to be involved in 5-HT]c-in-

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duced PI turnover in the present experiment, since ADP-ribosylation site by botulinum toxin C3 enzyme is the aspartic acid of 41st amino acid from N-terminal (Sekine et al., 1989) and ras does not possess this site. It has been reported that low molecular weight G protein is involved in activation of phosphatidylinositol 4-phosphate 5-kinase, causing an increase in the content of PIP 2 in rat hepatocytes (Urumow and Wieland, 1990), although the botulinum toxin sensitivity of the G protein has never been reported. Some of heterotrimeric G proteins, including Gi (Ueda et al., 1989) and Go (Moriarty et al., 1990), are also relevant to PI turnover. Recently, a G protein which selectively stimulates /31-subtype of phospholipase C is purified from bovine liver membranes (Taylor et al., 1990). The G protein, named Gq, is pertussis toxin-insensitive, and the molecular mass is 42 kDa (Smrcka et al., 1991). It cannot be ruled out that low molecular weight G protein modulates these heterotrimeric G proteins activities. It has been suggested that many of the phospholipase C subtypes exist (Rhee et al., 1989). As found in the case of Gq/phospholipase C/31 described above, each phospholipase C may selectively couple or be specifically regulated by respective G proteins. The results presented here show that low molecular weight G proteins ADP-ribosylated by botulinum toxin C3 enzymes are involved in 5-HTlc receptor mediated PI turnover. It is of interest to identify the low molecular weight G proteins involved in the transmembrane signalling mechanism and to clarify whether the low molecular weight G proteins are generally concerned with other receptors/phospholipase C systems or are 5-HTlc specific. Moreover, it is important whether the low molecular weight G proteins directly transduce the receptor signal to phospholipase C or indirectly modulate the receptor/heterotrimer G protein/phospholipase C system through a mechanism such as actin polymerization.

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