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A pertussis toxin resistant alpha2-adrenoceptor pathway in fish melanophores
Life Sciences, Vol. 46. Printed in the U.S.A.
pp.
Pergamon Press
1099-1101
A PERTUSSIS TOXIN RESISTANT ALPHA2-ADRENOCEPTOR PATHWAY IN FISH MELANOPHORES N. Grundstrgm, J.O.G. Karlsson, S.P. Svensson and R.G.G. Andersson Department of Pharmacology, University of Linkoping S-581 85 Linkoping, Sweden (Received in finalformFebruary
7, 1990)
Summary pigment The effect of pertussis toxin (PT) on the aggregation of granules in melanophores from cuckoo wrasse (Labrus ossifagus L.) was studied. The results indicate the presence of a PT resistant a2-adrenoceptor signal transduction mechanism.
Melanophores are innervated, highly branched cells filled with translocatable, black pigment granules (c.f. I). The pigment aggregation are mediated by a pharmacologically classified a2-adrenoceptor subtype (2). Melanophores have post-junctional ozproved to be a useful model system when studying adrenoceptors. Methods Scales from cuckoo wrasse (Labrus ossifagus L.) were removed and transferred to a buffer solution as previously described (3). The state of pigment aggregation was evaluted in a microscope according to the melanophore index (3,4) The melanophores were stimulated to aggregate their pigment granules, either by the addition of noradrenaline 3x10-' M or by electrical field stimulation of intrinsic nerves, as previously described (3). A Grass S88 stimulator equipped with an isolation unit (Grass SIU 5) was used to deliver pulse trains of 15 s at a frequency of 20 Hz (1 ms bipolar square wave pulses, 60 V nominally out from the stimulator).
Results and Discussion A discrepancy in the effect of PT was found between pigment granule aggregation elicited exogenous noradrenaline or electrical field by stimulation (fig 1.). PT-treatment effectively attenuated the effect of exogenuos noradrenaline; within 5 min of incubation a near maximal inhibition of the pigment granule aggregation was achieved. In contrast, effect of electrical field stimulation was totally the resistant to PT-treatment, however 10v5 M yohimbine (a2-selective antagonist) almost completely inhibited the effect. Possible explanations for this could be: that the nerves may, in addition to noradrenaline, release a (i) cotransmitter, or (ii) the noradrenaline released from the nerves may activate another subpopulation or subtype of adrenoceptors that are resistant to PT, and not activated by exogenous noradrenaline (The latter receptor population could of course be distinguished from the other receptors by just being inaccessible for PT) or (iii) the receptors could utilize multiple transduction mechanisms. 0024-3205190 $3.00 +.oo Copyright (c) 1990 Pergamon Press plc
1100
Pertussis
Toxin
Resistant
a2-adrenoceptors
5.0
5
10 Frsqumcy
15
20
Vol.
46, No. 15. 1990
t
25
CHzj
FIG
1.
Effect of yohimbine and pertussis toxin (PT) on electrically induced pigment aggregation.(A Frequency-res onse curves in the absence (0) or in the presence of 10-4 M (v) or 10 -8 M (m) yohimbine. The scales were preincubated in saline buffer for 1 h. When yohimbine was used it was included during the last 10 min. of preincubation. (“) Frequency-response curves in the absence (0) or presence (0) of PT (10 The scales were preincubated in saline buffer with or without PT g/ml 3. (log/ml) for 1 h. The control curve is identical to the control curve in (A). Vertical bars indicate S.E.M. (n-5)
Recent oceptors relevant for these suggestions concern compartm:~:P:YKtY~n T:Tadrensubtype heterogeneity (6) and multiple transduction mechanisms We have previously observed indications of receptor (8). heterogeneity among the melanophore a2-adrenoceptors (2,7) but also indications of multiple transduction pathways (Svensson et al. unpublished observations). In summary, the a2-adrenoceptor signal transduction mechanism that is activated by noradrenaline released from sympathetic nerves, may not be the same mechanism utilized by exogenously applied noradrenaline, since PT are able to uncouple the effect of exogenous noradrenaline but not the effect of electrical field stimulation. Whether there are multiple signal transduction mechanisms is unknown at present, but work is proceeding at our laboratory to resolve this issue. Acknowledgements Purified pertussis toxin was a kind gift from Dr. Bacteriological Laboratory, Stockholm, Sweden. grants from TRION AB, The Swedish National Board and Centrala Fbrsbksdjursntimnden (89-43).
Per Askelgf at the National This work was supported by for Technichal Development
Vol. 46, No. 15, 1990
Pertussis Toxin Resistant ag-adrenoceptor
References 1. R. FUJII, In: Fish Physiology. Eds. W.S. Hoar and D.J. Randall, pp. 307-353 Academic Press,N.Y. (1969). 2. J.O.G. KARLSSON, R.G.G. ANDERSSON and N. GRUNDSTRbM, Br. J. Pharmacol. 91, 222-228 (1989). 3. R.G.G. ANDERSSON, J.O. KARLSSON and N. GRUNDSTRdM, Acta Physiol.Scand., 121, 173-180 (1984). 4. L.T. HOGBEN and D. SLOME, Proc. Roy. Sot. B., 108, lo53.(1931). 5. R.M. McKERNAN, M.J. HOWARD, H.J. MOTULSKY. and P.A. INSEL, Mol. Pharmacol. 32, 258-265 (1987). 6. D.B. BYLUND, Trends Pharmacol. Sci. 2, 356-361 (1988). 7. J.O.G. KARLSSON, H. ELWING, N. GRUNDSTRflM and R.G.G. ANDERSSON, J. Pharm. Exp. Ther. 246, 345-351 (1988). 8. S. COTECCHIA, B.K. KOBILKA, K. W. DANIEL, R.D. NOLAN, E.Y. LAPETINA, M.G. CARON, R.J. LEFKOWITZ and J.W. REGAN J. Biol. Chem. (in press).
1101
pp.
Pergamon Press
1099-1101
A PERTUSSIS TOXIN RESISTANT ALPHA2-ADRENOCEPTOR PATHWAY IN FISH MELANOPHORES N. Grundstrgm, J.O.G. Karlsson, S.P. Svensson and R.G.G. Andersson Department of Pharmacology, University of Linkoping S-581 85 Linkoping, Sweden (Received in finalformFebruary
7, 1990)
Summary pigment The effect of pertussis toxin (PT) on the aggregation of granules in melanophores from cuckoo wrasse (Labrus ossifagus L.) was studied. The results indicate the presence of a PT resistant a2-adrenoceptor signal transduction mechanism.
Melanophores are innervated, highly branched cells filled with translocatable, black pigment granules (c.f. I). The pigment aggregation are mediated by a pharmacologically classified a2-adrenoceptor subtype (2). Melanophores have post-junctional ozproved to be a useful model system when studying adrenoceptors. Methods Scales from cuckoo wrasse (Labrus ossifagus L.) were removed and transferred to a buffer solution as previously described (3). The state of pigment aggregation was evaluted in a microscope according to the melanophore index (3,4) The melanophores were stimulated to aggregate their pigment granules, either by the addition of noradrenaline 3x10-' M or by electrical field stimulation of intrinsic nerves, as previously described (3). A Grass S88 stimulator equipped with an isolation unit (Grass SIU 5) was used to deliver pulse trains of 15 s at a frequency of 20 Hz (1 ms bipolar square wave pulses, 60 V nominally out from the stimulator).
Results and Discussion A discrepancy in the effect of PT was found between pigment granule aggregation elicited exogenous noradrenaline or electrical field by stimulation (fig 1.). PT-treatment effectively attenuated the effect of exogenuos noradrenaline; within 5 min of incubation a near maximal inhibition of the pigment granule aggregation was achieved. In contrast, effect of electrical field stimulation was totally the resistant to PT-treatment, however 10v5 M yohimbine (a2-selective antagonist) almost completely inhibited the effect. Possible explanations for this could be: that the nerves may, in addition to noradrenaline, release a (i) cotransmitter, or (ii) the noradrenaline released from the nerves may activate another subpopulation or subtype of adrenoceptors that are resistant to PT, and not activated by exogenous noradrenaline (The latter receptor population could of course be distinguished from the other receptors by just being inaccessible for PT) or (iii) the receptors could utilize multiple transduction mechanisms. 0024-3205190 $3.00 +.oo Copyright (c) 1990 Pergamon Press plc
1100
Pertussis
Toxin
Resistant
a2-adrenoceptors
5.0
5
10 Frsqumcy
15
20
Vol.
46, No. 15. 1990
t
25
CHzj
FIG
1.
Effect of yohimbine and pertussis toxin (PT) on electrically induced pigment aggregation.(A Frequency-res onse curves in the absence (0) or in the presence of 10-4 M (v) or 10 -8 M (m) yohimbine. The scales were preincubated in saline buffer for 1 h. When yohimbine was used it was included during the last 10 min. of preincubation. (“) Frequency-response curves in the absence (0) or presence (0) of PT (10 The scales were preincubated in saline buffer with or without PT g/ml 3. (log/ml) for 1 h. The control curve is identical to the control curve in (A). Vertical bars indicate S.E.M. (n-5)
Recent oceptors relevant for these suggestions concern compartm:~:P:YKtY~n T:Tadrensubtype heterogeneity (6) and multiple transduction mechanisms We have previously observed indications of receptor (8). heterogeneity among the melanophore a2-adrenoceptors (2,7) but also indications of multiple transduction pathways (Svensson et al. unpublished observations). In summary, the a2-adrenoceptor signal transduction mechanism that is activated by noradrenaline released from sympathetic nerves, may not be the same mechanism utilized by exogenously applied noradrenaline, since PT are able to uncouple the effect of exogenous noradrenaline but not the effect of electrical field stimulation. Whether there are multiple signal transduction mechanisms is unknown at present, but work is proceeding at our laboratory to resolve this issue. Acknowledgements Purified pertussis toxin was a kind gift from Dr. Bacteriological Laboratory, Stockholm, Sweden. grants from TRION AB, The Swedish National Board and Centrala Fbrsbksdjursntimnden (89-43).
Per Askelgf at the National This work was supported by for Technichal Development
Vol. 46, No. 15, 1990
Pertussis Toxin Resistant ag-adrenoceptor
References 1. R. FUJII, In: Fish Physiology. Eds. W.S. Hoar and D.J. Randall, pp. 307-353 Academic Press,N.Y. (1969). 2. J.O.G. KARLSSON, R.G.G. ANDERSSON and N. GRUNDSTRbM, Br. J. Pharmacol. 91, 222-228 (1989). 3. R.G.G. ANDERSSON, J.O. KARLSSON and N. GRUNDSTRdM, Acta Physiol.Scand., 121, 173-180 (1984). 4. L.T. HOGBEN and D. SLOME, Proc. Roy. Sot. B., 108, lo53.(1931). 5. R.M. McKERNAN, M.J. HOWARD, H.J. MOTULSKY. and P.A. INSEL, Mol. Pharmacol. 32, 258-265 (1987). 6. D.B. BYLUND, Trends Pharmacol. Sci. 2, 356-361 (1988). 7. J.O.G. KARLSSON, H. ELWING, N. GRUNDSTRflM and R.G.G. ANDERSSON, J. Pharm. Exp. Ther. 246, 345-351 (1988). 8. S. COTECCHIA, B.K. KOBILKA, K. W. DANIEL, R.D. NOLAN, E.Y. LAPETINA, M.G. CARON, R.J. LEFKOWITZ and J.W. REGAN J. Biol. Chem. (in press).
1101