Role of L-type Ca²⁺ channels in pertussis toxin induced antagonism of U50,488H analgesia and hypothermia

Abstract

Previous studies have shown that the κ-opioid effects are sensitive to pertussis toxin (PTX) and affected by Ca²⁺ fluxes. However, the possible involvement of Ca²⁺ channels in PTX-induced inhibition of κ-opioid effects has not been reported. The effect of intracerebroventricular (i.c.v.) treatment of pertussis toxin (1 μg/rat, PTX) or saline on the κ-opioid agonist, U-50,488H (U5H) induced tail-flick analgesia and hypothermia in rats was determined. The effect of nimodipine (NIM), a dihydropyridine (DHP)-sensitive Ca²⁺ channel blocker (CCB), on PTX-induced modulation of U5H effects was examined. The DHP ligand, [³H]PN200–110 binding was also determined in both PTX and saline treated rats to study the possible involvement of L-type Ca²⁺ channels in PTX modulation of κ-opioid agonist effects. The analgesia and change in colonic temperature were determined using tail-flick analgesiometer and telethermometer, respectively. U5H (40 mg/kg, i.p.) produced significant analgesic and hypothermic responses. PTX treatment significantly (P<0.01) antagonized the analgesic and hypothermic effects of U5H. Acute pretreatment of NIM (1 mg/kg, i.p.) 15 min prior significantly (P<0.01) reversed the PTX-induced antagonism of U5H effects. In the binding study, PTX treatment (72 h before) resulted in a significant (P<0.005) upregulation (+45% vs. saline control) of DHP binding (B_max) with no change in affinity (K_d). The results showed significant upregulation of DHP binding in accordance with PTX-induced antagonism of U5H effects and this blockade was reversed by NIM. Thus, present results suggest that U5H-induced analgesia and hypothermia may be mediated through PTX-sensitive transducer G-proteins (G_i/o) coupled to L-type Ca²⁺ channels.

Introduction

Opioid receptors belong to the family of G-protein coupled receptors and mediate the inhibition of adenylate cyclase, opening of voltage gated calcium (Ca²⁺) channels and promote the opening of potassium (K⁺) channels [6] by coupling mainly to G_i/o proteins [23]. These proteins (G_i/G_o) are heterotrimeric in nature with α, β and γ subunit composition and shown to be sensitive to pertussis toxin (PTX) produced by Bordetella pertussis[6], [20], [39]. At the molecular level, PTX has been shown to ADP-ribosylate the α-subunit of guanine nucleotide regulatory proteins (G_i/G_o) and hence interfere selectively with opioid receptor mediated inhibitory signal transduction [20], [39]. The PTX-sensitive G_i and G_o proteins are known to mediate the inhibitory effects of opioids such as analgesia, hypothermia, etc. [6]. The κ-opioid receptor agonists are potent analgesics with minimal respiratory depression and physical dependence [27]. However, in addition to analgesia, κ-opioid agonists also produce hypothermia [3], [5], [14], [16], [30], [36].

Several studies have used PTX pretreatment as a tool to assess the role of G-protein mediated events on the pharmacodynamics of morphine. The pretreatment of PTX has been shown to reduce the analgesic effect of μ-opioid agonists [18], [26], [33], [34], [37], [40], [41], [46]. Moreover, PTX pretreatment converts the hypothermia produced by a high dose of morphine into consistent hyperthermia [2].

However, only few in vivo studies have assessed the effect of PTX pretreatment on the pharmacodynamics of κ-opioid agonists. In mice, PTX pretreatment blocked the U5H tail-flick analgesia [12]. The PTX pretreatment has been shown to antagonize the hypothermia and behavioral effects of κ-opioid agonists [4], [42]. In addition, PTX has been shown to regulate the Ca²⁺ channel currents through G-proteins (mainly G_o) in neuroblastoma×glioma hybrid cells [17] and rat spinal cord–dorsal root ganglion co-cultures [1] providing a clear evidence for the role of PTX-sensitive G-protein coupling to Ca²⁺ channels. These authors reported that opiates inhibit the voltage-dependent Ca²⁺ channel currents in a PTX-sensitive manner. Moreover, there is ample evidence for direct coupling of κ-opioid receptors to L-type Ca²⁺ channels [1], [10], [11]. In addition, upregulation of L-type Ca²⁺ channels has been shown as a possible mechanism of hyperalgesia to morphine in mice following PTX pretreatment [32]. Furthermore, CCBs have been shown to potentiate κ-opioid agonist-induced analgesia [8], [28], [38] and hypothermic responses [14], [36]. In a recent study, we showed CCBs to potentiate κ-opioid agonist-induced analgesia and inhibit tolerance [15]. Despite the biochemical evidence for a direct coupling of κ-opioid receptors to voltage-sensitive Ca²⁺ channels via PTX-sensitive G-proteins [1], there are no in vivo studies assessing the role of Ca²⁺ channels in the effect of PTX pretreatment on the pharmacodynamics of κ-opioid agonists. So, the aim of the present study is to determine the (1) effect of PTX pretreatment on κ-opioid agonist, U5H-induced analgesic and hypothermic effects, (2) effect of NIM on PTX modulation of U5H-induced analgesia and hypothermia and (3) status of L-type Ca²⁺ channels in both saline and pertussis toxin (i.c.v.) pretreated rat brain membranes using [³H]PN200–110, a highly selective DHP radioligand.