µ-Opioid receptors (MORs) exhibit rapid desensitisation and internalisation on exposure to agonists such as Met-enkephalin (ME) and DAMGO. Morphine, however, induces neither rapid desensitisation nor internalisation, an effect not attributable to its relatively low efficacy (Bailey et al., 2003). Prolonged exposure of cells to morphine does however induce cellular tolerance (Williams et al., 2001). In the present study, we have examined whether protein kinase C (PKC) activation can enhance the desensitisation of MORs by morphine and other MOR agonists.

Whole cell patch clamp recordings (Vh -60mV) were made from visually identified locus coeruleus LC neurones in 250 mm thick pontine brain slices prepared from male Wistar rats (120-150g) as described previously (Bailey et al., 2003). All data are presented as mean ± S.E.M. and statistical significance assessed by the Student's t test.

Receptor saturating concentrations of ME (30µM) and morphine (30µM) evoked outward potassium currents of 232 ±- 18 pA and 140 ± 12 pA respectively (both n=13). The ME-induced current rapidly desensitised by 46 ± 2% after 7 min exposure, whereas the morphine-induced current showed little desensitisation as the evoked current only decayed by 8 ± 3% over 7 min (Table 1).

ME-induced desensitisation was unaffected by inhibition of PKC by chelerythrine (5 µM: response declined by 47 ± 6% over 7 mins). However, activating PKC with the phorbol-12-myristate-13-acetate (PMA; 1µM) significantly increased the level of desensitisation, an effect not mimicked by the inactive analogue, phorbol-12,13-didecanoate (PDD; 1µM) (Table 1). This facilitatory effect of PMA was completely blocked by the PKC inhibitor chelerythrine (5µM).

PKC activation converted morphine into a MOR desensitising agonist. In the presence of PMA (1µM) the morphine-evoked current decayed rapidly (Table 1). The effect of PMA was due to PKC activation since it was blocked by chelerythrine (5µM), and PDD (1µM), the inactive phorbol, had no effect.

	control	+PMA	+PDD	PMA+chelerythrine
morphine	8 ± 3%	32 ± 3%*	9 ± 3%	14 ±5%
ME	46 ± 2%	60 ± 4%*	43 ±10%	47 ± 3%

Table 1. Percentage desensitisation following 7 min incubation of morphine or Met-Enkephalin. * = p<0.05 vs. control. = p<0.05 vs. PMA, n = 3-13.

The data presented here suggest that, when levels of PKC are elevated, morphine can indeed cause rapid desensitisation of MORs. Thus, cellular tolerance induced by morphine may occur through MOR desensitisation in tissues with elevated PKC levels, perhaps through ongoing Gq-coupled receptor activation.

Bailey, C.P. et al. (2003). J. Neurosci., in press.
Williams, J.T. et al. (2001). Physiol. Rev., 81, 299-343.