| pA2 online © Copyright 2004 The British Pharmacological Society |
056P
GKT, University of London Winter Meeting December 2003 |
|
Involvement
of protein kinase C in µ-opioid receptor internalization in
HEK 293 cells |
|
Print abstractµ-Opioid receptors (MORs) can undergo rapid desensitization and subsequent internalization on exposure to some opioid agonists, whereas others such as morphine produce little of either (Bailey et al, 2003a). In this study we have investigated the role of protein kinase C (PKC) in MOR internalization in stably transfected HEK 293 cells.
HEK 293 cells were stably transfected with T7-epitope tagged receptors of the MOR1 subtype. Internalization was measured by ELISA using a colourimetric alkaline phosphatase assay adapted from Bailey et al (2003a). Data are expressed as means ± S.E.M and were compared using unpaired Student's t-tests.
A receptor saturating concentration of the MOR agonist DAMGO (30µM) caused a high degree of receptor internalization after 30 min (48.5 ± 1.5%, n=5) whereas a receptor saturating concentration of morphine (30µM) produced only a low level of MOR internalization after 30 min (7.2 ± 2.7%, n=5). Phorbol 12-myristate 13-acetate (PMA, 1µM), an activator of PKC caused MOR internalization (24.8 ± 4.4%, n=5) in the absence of any agonist.
The internalization produced by PMA was blocked by the PKC inhibitor GF 109203X (-0.83 ± 4.3%, n=6, p<0.05) but was unaffected by the PKC inhibitor chelerythrine (29.5 ± 2.7%, n=5, p>0.05). This may suggest that the PMA-induced internalization of the MOR is produced by one of the novel isoforms of PKC as these are inhibited by GF109203X but not by chelerythrine.
Internalization produced by morphine and DAMGO was shown to be PKC-independent as neither GF 109203X nor chelerythrine inhibited the responses (Table 1).
|
%
internalization
|
n
|
|
| morphine |
16.0
± 3.2
|
13
|
| morphine & GF 109203X |
17.6
± 5.1
|
6
|
| morphine & chelerythrine |
12.2 ± 3.1
|
5
|
| DAMGO |
50.3 ± 2.7
|
9
|
| DAMGO & GF 109203X |
45.0
± 4.7
|
4
|
| DAMGO & chelerythrine |
51.1 ± 1.5
|
5
|
These data suggest that MOR internalization can occur through stimulation of PKC as well as through agonist activation. A possible pathway for the PKC mediated internalization is through the activation of phospholipase D and ADP ribosylation factor proteins, both of which have been implicated in the regulation of MOR trafficking (Koch et al, 2003).
The effect of PMA seen here in HEK 293 cells is markedly different from that seen in mature CNS neurones, where PMA alone is unable to promote MOR desensitization, but can enhance the desensitization produced in response to morphine exposure (Bailey et al, 2003b).
Bailey, C.P. et al.,
(2003a) J. Neurosci. in press.
Bailey, C.P. et al., (2003b) This meeting.
Koch, T. et al., (2003) J. Biol. Chem. 278, 9979-85.