Gen. Pharmac. Vol. 23, No. 3, pp. 309-316, 1992 Printed in Great Britain.All rights reserved

0306-3623/92$5.00+ 0.00 Copyright © 1992PergamonPress Ltd

MINIREVIEW GABAB-MEDIATED MODULATION OF THE VOLTAGE-GATED Ca 2÷ CHANNELS HIDEHARUTATEBAYASHIand NOBUKUNIOGATA* Department of Pharmacology, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan (Received 9 September 199 !)

The amino acid, ?-aminobutyric acid (GABA), activates two different receptor types (Bowery et al., 1980; reviewed by Ogata, 1990a). 2. GABAA receptors are bicuculline-sensitiveand are coupled to CI- channels, while activation of bicuculline-insensitiveGABAB receptors has been implicated in the modulation of Ca2+ (Dunlap and Fischbach, 1981) and K + (Gahwiler and Brown, 1985; Inoue et al., 1985a,b; reviewed by Ogata, 1990b) channels. 3. Baclofen is a specific agonist for GABABreceptors (Bowery et al., 1980). In rat sensory neurones, baclofen suppresses the membrane Ca2+ current (/ca) by a mechanism involvinga partussis toxin-sensitive G protein (Holz et al., 1986; Scott and Dolphin, 1986). 4. It has been shown that the inhibitory effect of baclofen is more potent on the early portion of Ic~ than on the later portion and consequently the rate of/ca activation is slowed (Deisz and Lux, 1985; Dolphin and Scott, 1986). 5. The mechanisms underlying these GABAB-mediatedmodulation of/ca is not fully understood. This article reviews the inhibitory action of baclofen on /Ca in sensory neurones. Abstract--l.

lcl OF RAT DRG NEURONES

Multiple types of Ca 2+ channels exist in excitable membranes. It is widely accepted that /ca of DRG neurones can be classified into two major categories, i.e. a low voltage-activated (LVA) /ca and a high voltage-activated (HVA) lc~ (Carbone and Lux, 1984; Nowycky et al., 1985; reviewed by Bean, 1989a). The LVA-Ica is elicited by a step depolarisation to - 6 0 mV from a holding potential (Vh) of --100 mV and is inactivated completely within 50-100msec [Fig. I(A)]. This type of/Ca is observed in about 30% of D R G neurones (Fedulova et al., 1985; Regan et al., 1991; Tatebayashi and Ogata, 1992). The HVA-Ic, is induced by a step depolarisation to potentials more positive than - 3 0 m V from Vh of - 8 0 m V [Fig. I(B-1)]. The HVA-Ica shows a relatively slow inactivation during a 300 msec step depolarisation. When evoked from Vh of - 4 0 mV, the HVA-Ica shows no or an extremely slow inactivation during the 300msec step depolarisation [Fig. l(B-2)]. EFFECTS OF BACLOFEN ON

1991), dopamine or noradrenaline (irreversible inhibition, chick DRG neurones: Marchetti et al., 1986), or l-oleoyl-2-acetyl-sn-glycerol, a protein kinase C activator (GH3 cells: Marchetti and Brown, 1988). On the contrary, adenosine (rat hippocampal CA3 neurones: Madison et aL, 1987), ~ opioid receptor agonist (mouse DRG neurones: Gross and MacDonald, 1987), 6 opioid receptor agonist (NG108-15 cells: Tsunoo et al., 1986) or noradrenaline (rabbit vesical parasympathetic ganglia: Akasu et al., 1990) have been shown to have no effect on LVA-Ica. As to the effect of baclofen on the LVA-Ica, several investigators have reported that a high concentration of baclofen (50-100 #M) partially reduces the LVA-Ica (by 22%, Deisz and Lux, 1985; by 31%, Dolphin et al., 1990). However, our recent reinvestigation showed that a high concentration (50 #M) baclofen had no detectable effect on the LVA-Ica in any of the six cells examined [Fig. I(A)]. The physiological significance of the GABA B mediated modulation of LVA-Ica remains to be elucidated. EFFECTS OF BACLOFEN ON

LVA-~,

A variety of neurotransmitters or neuromodulators modulate the LVA'Ica. Complete inhibition of LVA/ca by noradrenaline (frog D R G neurones: Bean, 1989b) or intracellular GTP-7-S (rat DRG neurones: Dolphin et al., 1989b) have been reported. In addition, partial inhibition occurs with p opioid receptor agonist (rat D R G neurones: Schroeder et al., *To whom all correspondence should be addressed.

HVA-Ic.

Baclofen inhibits the HVA-Ica in neurones of DRG [Deisz and Lux, 1985; Dolphin and Scott, 1986; Green and Cottrell, 1988; Grassi and Lux, 1989; Tatebayashi and Ogata, 1992; see Fig. I(B)]. A concentration-response curve for baclofen measured at the peak or at the end of the 300msec step depolarisation is shown in Fig. I(C). In the presence of 50 #M baclofen, the amplitude of the HVA-Ica was reduced by about 60 or 30% when measured at the peak or at the end of the step depolarisation,

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Baclofen concentration (gM) Fig. h Effects of (-)-baclofen on the Ca 2+ current (Ic~) in cultured neurones of the newborn rat dorsal root ganglia (DRG). (A) The low voltage-activated Ca 2+ currents (/ca) were evoked by 300 msec voltage steps to - 6 0 mV from a holding potential (Vh) of - 100 mV. The inward current in the control solution was not affected by 100 pM baclofen, whereas it was totally blocked by 50 #M Cd 2+. To exclude a possible contamination of sodium currents, total amounts of NaCI were substituted with an equimolar amounts of choline-Cl and 100 nM TTX was added. (B) High voltage-activated (HVA)/ca were evoked by a step depolarisation to - 1 0 mV from Vh of - 8 0 mV (B-I) or - 4 0 mV (B-2), during and after application of baclofen. (C) Concentration-response relationships for the inhibition of HVA-Ic~ by baclofen. Individual currents were evoked by a step depolarisation to - 10mV from Vh of - 8 0 mV during repeated applications of various concentrations of baclofen. The numerals attached to current traces indicate the concentration of baclofen. The current amplitudes were measured at 7 msec after the onset of the 300msec step depolarisation (open circles) or at the end of the depolarisation (solid circles). In this and subsequent figures the Ca 2÷ currents were subtracted from the current remaining after application of 50 pM Cd 2+ [except for Fig. I(A)], the "control" response was recorded after washout of the drug solution and downward deflections represent an inward currents. External Ca 2+ concentration was 1.8 mM throughout the experiments. respectively. The half-maximal concentration for the inhibition o f the peak current was 1 . 1 7 p M . This value is much lower than the value previously reported in the same preparation (15 p M, Dolphin and Scott, 1987), but is similar to values reported in the

chick D R G (Dunlap and Fischbach, 1978; Canfield and Dunlap, 1984; Holz et al., 1989). An intriguing feature o f the baclofen-induced inhibition o f HVA-Ic~ is the slowing o f the activation phase o f the current [Deisz and Lux, 1985; D o l p h i n

GABAB and Ca2+ channels and Scott, 1986; Green and Cottrell, 1988; Dolphin et al., 1989a; Sah, 1990; Tatebayashi and Ogata,

199 i; see Fig. 1(B- 1)]. Similar slowing of the HVA-Ica has also been observed after applications of a variety of neurotransmitters (adenosine on hippocampal CA3 neurones, Madison et al., 1987; muscarinic receptor agonist on rat sympathetic neurones, Wanke et al., 1987; dynorphin A on mouse DRG neurones, Gross and MacDonald, 1987; ~-adrenergic receptor agonist on frog sympathetic neurones, Lipscombe and Tsien, 1987; Lipscombe et al., 1989; G proteins in PC12 or rat sympathetic neurones, Plummer et al., 1989; noradrenaline on rabbit vesical parasympathetic neurones, Akasu et al., 1990; N M D A receptor agonists, Chernevskaya et al., 1991; /~ opioid receptor agonists, Schroeder et al., 1991; for reviews see Tsien et al., 1988; Hess, 1990). Therefore, the slowing of the activation phase of the HVA-Ica appear to be a common feature of the transmittermediated inhibition of the HVA-Ica. M E C H A N I S M UNDERLYING THE SLOWED ACTIVATION OF T H E HVA-I¢,

On the basis of recent findings that neuronal membranes contain multiple types of voltage-gated Ca 2+ channels (Nowycky et al., 1985; Tsien et al., 1988), Green and Cottrell (1988) suggested that baclofen inhibits predominantly an inactivating type of lc~. On the contrary, a different mechanism of the inhibitory action of baclofen has been postulated in chick D R G neurones (Grassi and Lux, 1989) and in rat DRG neurones (Scott and Dolphin, 1990; Dolphin et al., 1990; Swandulla et al., 1991). These reports suggested that the slowed activation of the HVA-Ica in the presence of baclofen is due to a gradual voltage-dependent recovery from the baclofen-induced block. Such a voltage-dependent unblocking of the HVA-Ica has also been reported for dopamine (chick DRG or sympathetic neurones, Marchetti et al., 1986), leucine-enkephalin (NG10815 cells, Tsunoo et al., 1986), and noradrenaline (bullfrog DRG neurones, Bean, 1989c). Recently, Bean (1989c) suggested that the slowed activation of the HVA-Ic~ by noradrenaline is due to a voltage-dependent gradual conversion of channels in the closed "reluctant" state to the closed, "willing" state which can open during moderate depolarisation. This postulation was based on the observation that in bullfrog DRG neurones the inhibition of Ic, by noradrenaline was nullified when a large depolarising test pulse to potentials more positive than + 50 mV was used. According to this postulation, there is little or no change in number of functional channels when activated by very large depolarisations. However, this postulation appears to be untenable for the effect of baclofen on vertebrate DRG neurones, because Dolphin and Scott (1990) showed that the baclofeninduced inhibition of the HVA-Ic, was persistent even when extremely positive (+120mV) step depolarisations were used. SELECTIVE INHIBITION OF THE INACTIVATING HVA-lc, BY BACLOFEN

Since baclofen reduced not only the HVA-Ica activated from negative ( - 8 0 m V ) Vh but also /ca

311

activated from positive ( - 30 mV) Vh when measured at the end of 100 msec depolarising test pulse in rat DRG neurones, it was suggested that baclofen reduces both inactivating and sustained component of the HVA-Ica (Dolphin and Scott, 1986; Dolphin et al., 1990). However, Schroeder et al. (1991) observed preferential inhibition of the transient component of HVA-/ca by p opioids when they examined using a longer (1 sec) duration pulse. The inactivating HVA-Ica firstly described in chick DRG neurones (N-type current) shows relatively rapid inactivation (z < 100 msec) and inactivates with Vh of - 4 0 mV (Nowycky et al., 1985). However, the slowly inactivating HVA-Ica has been reported in NG108-15 cells (z about 800 msec, Docherty, 1988), in rat sympathetic neurones (z > 500 msec, Hirning et al., 1988), and in mouse DRG neurones (z about 175 msec, Gross and MacDonald, 1989). In addition, Shroeder et al. (1991) have shown in rat DRG neurones that Vh of - 4 0 m V only partially inactivates the inactivating HVA-Ica. Thus, a commonly employed protocol for isolation of the steady-state (non-inactivating) component with a relatively short test pulse (>400 msec) from Vh of - 4 0 mV appears to be insufficient to completely remove the inactivating component. The diversity of N-type Ica has also been inferred from single channel recordings. Kongsamut et al., (1989) have shown in frog sympathetic neurones that the time-course of N-channel inactivation is quite variable from patch to patch. In addition, Plummer and Hess (1991) have reported in rat sympathetic neurones that individual N-channels can exhibit both short (an average duration of 40msec) and long (lasting for over 1 sec) bursting patterns. Furthermore, a possible involvement of the two components in the N-type Ic~ within one cell has been pointed out in PC12 cells (Plummer et al., 1989) and in SH-SY5Y cells (Seward and Henderson, 1990). A pharmacological separation of components of HVA-Ic~ has been attempted, co-conotoxin has been suggested to be a selective blocker for N-type HVAIc~ (Aosaki and Kasai, 1989; Plummer et al., 1989). Dihydropyridine calcium antagonists selectively block the sustained component of the HVA-Ica in chick DRG (Fox et al., 1987). However, the action of these drugs on neuronal Ca 2÷ channels are generally inconsistent (Bean, 1989b; Mogul and Fox, 1991; Regan et al., 1991; Tatebayashi and Ogata, unpublished data). Thus a protocol using a prolonged depolarising test pulse appears to be best suitable for separation of components of HVA-Ic~ (Akasu et al., 1990; Schroeder et al., 1991; Tatebayashi and Ogata, 1992). Using prolonged (2-4 sec) depolarising test pulses, we have recently obtained data in support of the hypothesis that the slowed activation of the HVA-Ica in the presence of baclofen may be due to a selective inhibition of the inactivating component of the HVA/Ca (Tatebayashi and Ogata, 1992). Figure 2(A) shows HVA-Ic~ evoked by 2 sec step depolarisations to - 1 0 m V from a Vh of - 8 0 m V in the presence or absence of 50/~M baclofen. Baclofen significantly reduced the initial component of the HVA-Ica, whereas it has little effect on the HVA-Ica at the end of 2 sec step depolarisation. The transient component

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Fig. 2. A selectiveblock of the fast component of the HVA-lc~by baclofen. (A) The HVA-Ica was evoked by 2 sec step depolarisation to - I0 mV from Vh of - 8 0 mV in the presence or absence of 50 p baclofen. (B) The HVA-Icas were evoked by a step depolarisation to -10mV from Vh of -80mV (B-l) and immediately after an interpulse interval of 5 sec held at - 30 mV (B-2). (C) The HVA-Icas were evoked by 4 sec step depolarisations during and after application of 50 pM baclofen. The decay phase of the HVA-Ic, during the 4 sec step depolarisation was fitted by a sum of two exponentials in the control solution (C-l). On the contrary, the decay phase of the current in the presence of 50/t M baclofen was fitted by a single exponential (C-2). of HVA-Ica [Fig. 2(B-I)] was markedly inactivated by a depolarising Vh of - 3 0 m V for 5sec [Fig. 2(B-2)]. The decay of the HVA-Ica evoked by a 4 sec step depolarisation was well fitted by the sum of two exponentials in the control solution [Fig. 2(C-1)]. The fast component (z = 420 msec) of HVA-Ica was completely abolished by 50pM baclofen, whereas a considerable portion of the slow inactivating component (z = 1220 msec) remained unaffected in the presence of baclofen [Fig. 2(C-2)]. The baclofensensitive component was sensitive to a change in Vh whereas the amplitude of the baclofen-insensitive component at Vh of - 110 mV was much the same as that at Vh of --30 mV (Tatebayashi and Ogata, 1992). When external Ca 2+ was completely replaced with Ba 2÷, the time constant for the fast component remained unaffected, whereas that for the slow component was prolonged (Tatebayashi and Ogata, 1992). These observations suggest that the fast baclofen-sensitive inactivation is due to voltagedependent inactivation, whereas the slower baclofeninsensitive decay mainly reflects the inactivation due to Ca 2+ entry into a cell (Brehm and Eckert, 1978; Tillotson, 1979; Schroeder et al., 1990).

VOLTAGE-DEPENDENT UNBLOCKING OF THE BACLOFEN-INDUCED INHIBITION OF THE HVA-Ic=

Grassi and Lux (1989) have suggested in chick DRG that the slowed activation of the HVA-Ica by GABA is reversed by a large depolarising prepulse. They demonstrated that, in the presence of GABA or in cells loaded with GTP-7-S, a prepulse to +40 mV delivered prior to the activation of the HVA-Ica prominently accelerated the rising phase of the HVAIca and increased its amplitude. A similar observation was also reported by Scott and Dolphin (1990) and Dolphin et al. (1990) in rat DRG neurones. Such a prepulse-induced potentiation of HVA-Ica in the presence of baclofen appears to be an important finding for the voltage-dependent unblocking of the baclofen-induced inhibition of the HVA-Ica. However, our recent observations suggest that the component which has been facilitated by the depolarising prepulse is the baclofen-insensitive component of HVA-Ic~ (Tatebayashi and Ogata, 1992). Figure 3(A) shows a facilitation of HVA-Ica by a prepulse of + 5 0 m V in the control medium [Fig. 3(A-l)] and in the presence of 50/~M baclofen [Fig. 3(A-2)]. Contrary to previous studies in chick

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Fig. 3. Facilitation of the HVA-Icaby a large depolarising prepulse. (A) Two identical step depolarisations to - 10 mV for 100 msec were applied 5 sec before (Vcontro0and AT msec after (Vt~t) the large conditioning depolarising prepulse to + 50 mV. The current during the prepulse was not shown in the current trace due to a large sampling rate at this period. The traces were recorded with AT of 4 msec in the control solution (A-l) and in the solution containing 50/~M baclofen (A-2). Icomroland Item, were shown superimposed in each trace. (B) Time-dependence of the prepulse-induced facilitation of the HVA-Ica. (B-l) AL the increment of the current amplitude produced by the prepulse (Item,-l¢o,t~ol), was plotted against AT. Only the period up to 256 msec was shown to facilitate the clarity of the plot. (B-2) AI at each time point was normalised with the maximal AI (AI at AT = 2 msec) and plotted against AT. D R G neurones (Gassi and Lux, 1989), significant enlargement of the HVA-Ica by a large depolarising prepulse was observed also in the control solution in rat DRG neurones. The facilitation of the HVA-Ica in the control solution has also been reported by Scott and Dolphin (1990). They interpreted this phenomenon as a voltage-dependent recovery from a partial tonic inhibition of HVA-Ic, by G proteins. In our study, the size of the current newly produced by a depolarising prepulse (AI) in the control solution was much the same as that in the presence of baclofen when an interval between prepulse and test pulse (AT) is longer than 64msec [Fig. 3(B-l)]. When interpulse interval was shorter than 64 msec, AI in the control solution was smaller than that in the presence of baclofen, because an inactivating component of the HVA-Ica in the control solution was largely inactivated by a large depolarising prepulse at this period. The time-course of the inactivation of the facilitated HVA-Ica in the presence of baclofen was almost identical to that in the absence of baclofen [Fig. 3(B-2)]. These observations are in support of the notion that the component which has been facilitated by the depolarising prepulse may be the baclofeninsensitive HVA-Ica. The facilitation of the sustained types of Ic~ by a prior depolarising pulse has been described in bovine chromaffin cells (Fenwick et al., 1982; Hoshi et al., 1984; Artalejo et al., 1990, 1991), in cardiac cells (guinea pig, Lee, 1987; rat, Pietrobon and Hess, 1990), and in sympathetic neurones (bullfrog, Jones

and Marks, 1989; Elmslie et al., 1990; rat, Ikeda, 1991). Scott and Dolphin (1990) have reported that the effect of the large depolarising prepulse on HVA-Ica was not affected by a dihydropyridine antagonist, (-)-202-791, in rat DRG neurones. However, the facilitated Ic~ in bovine chromaffin cells was sensitive to dihydropyridine agonists or antagonists (Artalejo et al., 1991). Therefore, facilitations of Ic~ in DRG neurones and bovine chromaffin cells may be induced by different mechanisms.

PHYSIOLOGICAL SIGNIFICANCEOF SLOWED ACTIVATION OF HVA-Icj A functional significance of the slowed activation of HVA-Ic~ by neurotransmitters has not been characterised despite a number of investigations on this topic. Although a detailed mechanism underlying the slowed activation of the HVA-Ica remains to be elucidated, two explanations have been proposed, one, voltage-dependent unblocking of the transmitter-induced inhibition, the other, preferential blocking of the inactivating HVA-Ica (see above). A physiological consequence of the slowed activation induced by the two mechanisms is expected to be quite distinct from each other. If we assume that the slowed activation is due to the voltage-dependent unblocking, then the inhibitory action of neurotransmitters on HVA-Ica could be kinetically modulated by the depolarising impingement to the channel. If

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the depolarising i m p i n g e m e n t is a prolonged depolarisation, the inhibition could be attenuated due to the unblocking o f /ca during the prolonged depolarisation. On the contrary, if the depolarising impingem e n t is a brief depolarisation, then the inhibition could be maximal, because the unblocking is negligible. Thus, the voltage-dependent unblocking can work as a " d u r a t i o n - s e n s i t i v e " filter against the transmitter-induced Ca 2÷ channel block. C o n t r a r y to the voltage-dependent unblocking o f the HVA-Ica, the preferential inhibition of the inactivating HVA/ca by n e u r o t r a n s m i t t e r s is d e p e n d e n t only on the c o n c e n t r a t i o n of transmitters. CONCLUSION Since the original proposal of D u n l a p and Fischbach (1978), a n u m b e r o f investigations confirmed t h a t a variety o f n e u r o t r a n s m i t t e r s including G A B A inhibit the n e u r o n a l H V A - I c , . These n e u r o t r a n s m i t t e r s slow the activation phase o f the n e u r o n a l HVA-Ica. One possible explanation for this slowing of the activation phase is the voltaged e p e n d e n t removal of the transmitter-induced block. A n alternate explanation is the preferential block of the inactivating HVA-Ica which forms a rapid rising phase of this current, thus slowing the activation phase of the total HVA-Ica. In view of i m p o r t a n t roles of Ca :+ ions in n e u r o t r a n s m i t t e r release, signal t r a n s d u c t i o n a n d a variety o f biochemical events, inhibition o f / c a by n e u r o t r a n s m i t t e r s is t h o u g h t to contribute to various kinds of n e u r o n a l functions. A n i m p o r t a n t question is whether this type of inhibition which has been observed in most cases in cell bodies is relevant to n e u r o t r a n s m i t t e r release or not. In this respect, in Aplysia neurones a good correlation between inhibition of/Ca in cell bodies and a decrease in neurot r a n s m i t t e r release from its nerve terminals has been reported (Kretz et al., 1986). It is n o w quite possible t h a t in nerve terminals G A B A or other n e u r o t r a n s mitters suppress the HVA-Ica a n d then the release of neurotransmitters. Acknowledgement--We thank Professor H. Kuriyama for his support and advice. REFERENCES

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