μ-Opioid receptor activation inhibits N- and P-type Ca2+ channel currents in magnocellular neurones of the rat supraoptic nucleus

The whole-cell voltage-clamp technique was used to examine opioid regulation of Ba2+ currents (IBa) through voltage-sensitive Ca2+ channels in isolated magnocellular supraoptic neurones (MNCs). The effects of local application of μ-, δ- or κ-opioid receptor selective agonists were examined on specific components of high voltage-activated (HVA) IBa, pharmacologically isolated by use of Ca2+ channel-subtype selective antagonists. The μ-opioid receptor selective agonist, DAMGO, suppressed HVA IBa (in 64/71 neurones) in a naloxone-reversible and concentration-dependent manner (EC50 = 170 nm, Emax = 19.5 %). The DAMGO-induced inhibition was rapid in onset, associated with kinetic slowing and voltage dependent, being reversed by strong depolarizing prepulses. Low-voltage activated (LVA) IBa was not modulated by DAMGO. Administration of κ- (U69 593) or δ-selective (DPDPE) opioid receptor agonists did not affect IBa. However, immunostaining of permeabilized MNCs with an antibody specific for κ1-opioid receptors revealed the presence of this opioid receptor subtype in a large number of isolated somata. μ-Opioid-induced inhibition in IBa was largely abolished after blockade of N-type and P-type channel currents by ω-conotoxin GVIA (1 μm) and ω-agatoxin IVA (100 nm), respectively. Quantitation of antagonist effects on DAMGO-induced reductions in IBa revealed that N- and P-type channels contributed roughly equally to the μ-opioid sensitive portion of total IBa. These results indicate that μ-opioid receptors are negatively coupled to N- and P-type Ca2+ channels in the somatodendritic regions of MNCs, possibly via a membrane-delimited G-protein-dependent pathway. They also support a scheme in which opioids may act in part to modulate cellular activity and regulate neurosecretory function by their direct action on the neuroendocrine neurones of the hypothalamic supraoptic neucleus. Opiates and endogenous opioid peptides (here collectively termed opioids) are known to inhibit the secretion of the neurohormones Arg-vasopressin (AVP) and oxytocin (OT) from the posterior pituitary (see Falke, 1991 for review). Early studies have shown that opioids can modulate this release by acting at the level of the supraoptic nucleus (SON), inhibiting the activity of magnocellular SON neurones (Wakerley et al. 1983; Wuarin & Dudek, 1990; Pumford et al. 1993; Inenaga et al. 1994) either directly or via presynaptic inhibition of their afferent input. Opioids also attenuate release of these neurohormones at the level of the neural lobe through activation of presynaptic opioid receptors located on the nerve endings of the magnocellular SON neurones (Zhao et al. 1988; Rusin et al. 1997). In addition, recent results obtained from recordings of SON neurones in vitro and from microdialysis experiments in vivo have strengthened the case for somatodendritic release of AVP and OT from these magnocellular neurones and provided evidence for its inhibitory regulation by opioids (Douglas et al. 1995; Ingram et al. 1996). Hence, there is ample reason to postulate that opioids act to modulate activity-dependent release of AVP and OT by engaging multiple cellular mechanisms widely distributed within the hypothalamo-neurohypophysial axis. There is general agreement that opioids can influence neuronal function via three distinct subtypes of opioid receptors, designated as μ-, δ- and κ-opioid receptors. Each of these opioid receptor subtypes shows a unique and preferential pattern of distribution within the central nervous system, with κ-opioid receptors being the predominant species expressed throughout the rodent hypothalamus and posterior pituitary (Herkenham et al. 1986; Mansour et al. 1995). Autoradiographic (Sumner et al. 1990) and in situ hybridization studies (George et al. 1994; Minami et al. 1994) have further revealed only moderate levels of μ-opioid receptor binding and mRNA within the magnocellular hypothalamic nuclei, while failing to demonstrate the presence of δ-opioid receptors within the hypothalamus or posterior pituitary. Consistent with these anatomical data are the findings from studies in vivo that μ-and κ-opioid receptor agonists act at sites in the SON to inhibit the firing rate of identified SON neurones (Ludwig et al. 1997). Similarly, only κ-opioid receptor selective agonists and non-selective agonists such as etorphine have been reported to inhibit depolarization-evoked release of AVP and OT from isolated neural lobes and neurosecretosomes prepared from rat neurohypophysis (Dayanithi et al. 1992; Kato et al. 1992). Magnocellular SON neurones have recently been shown to express several types of voltage-sensitive calcium channels (VSCCs) that can be distinguished based on their distinct biophysical and pharmacological profiles (Fisher & Bourque, 1995a; Foehring & Armstrong, 1996). These include a low voltage-activated (LVA) T-type Ca2+ channel and a number of high-voltage activated (HVA) Ca2+ channels, including L-, N-, P-, Q- and R-types, similar to those described in many other peripheral and central neurones (see Scott et al. 1991 for review). A novel LVA L-type channel has also been described in these neurones (Fisher & Bourque, 1995a). Interestingly, Lemos and colleagues (1989, 1994) have shown that the nerve terminals of magnocellular SON neurones express only HVA Ca2+ channels. Furthermore, while the latter include a conventional L-type channel, the rapidly inactivating N-type (Nt) (Lemos & Nowycky, 1989; Wang et al. 1992) and P-type (Fisher & Bourque, 1995b) channel currents expressed in these terminals have biophysical and pharmacological characteristics that differ from those of the corresponding N- and P-type somatic currents. It is tempting to speculate that such differences may reflect distinct Ca2+-dependent functions required for cellular bursting activity and neurosecretion subserved by the somatic and the nerve terminal regions of magnocellular hypothalamo-neurohypophysial neurones, respectively. The differential expression of VSCC subtypes in somatic and terminal regions may also support different capabilities for modulation of these functions by opioid receptors. It is currently unknown whether modulation of VSCCs represents an important mechanism whereby opioids exert regulatory effects on SON neural excitability and secretory function. Intracellular recordings from magnocellular SON neurones in slices of rat and guinea-pig hypothalamus have revealed both δ- and μ-opioid receptor-mediated reductions in action potential firing (Wakerley et al. 1983; Wuarin & Dudek, 1990), although these effects were not accompanied by changes in postsynaptic membrane properties. In contrast, Inenaga (1994) reported that superfusion of rat hypothalamic slices with κ-opioid receptor agonists not only decreased spontaneous firing rates, but also shortened the duration of Ca2+ action potentials recorded under conditions where potassium conductances were blocked. There are also initial reports that κ-opioid receptor selective agonists can suppress Ca2+ currents in cultured neonatal (Mason et al. 1988) and acutely dissociated adult rat SON neurones (Bourque et al. 1993). However, mediation of the latter effects by κ-opioid receptors was not established by use of antagonists selective for this opioid receptor subtype. This is of some concern since the κ-opioid agonists used in these studies (U50 488H and dynorphin A, respectively), when administered at the concentrations employed, can cross react with μ-, and to a lesser extent δ-opioid binding sites (Goldstein, 1987). The present experiments were designed to determine whether Ca2+ channel currents in magnocellular SON neurones are regulated by activation of a particular subtype(s) of opioid receptor and, if so, to identify the specific Ca2+ channel subtype(s) that are subject to opioid modulation. A preliminary report of this has been presented in abstract form (Soldo & Moises, 1996).