Agonist induced desensitization of the human beta3-adrenoceptor

Wim Vrydag, Astrid E. Alewijnse, Martin C. Michel. Academic Medical Center, Amsterdam, The Netherlands.

Beta3-Adrenoceptors mediate the relaxation of smooth muscle in e.g. the urinary bladder, the gastrointestinal tract and several blood vessels. Beta3-Adrenoceptor agonists are currently in clinical trials for the treatment of voiding disorders and depression, both requiring chronic treatment. Early studies on beta3-adrenoceptors expressed in Chinese hamster ovary (CHO) cells indicated that the beta3-adrenoceptor is rather resistant towards agonist-induced desensitization, possibly due to a lack of phosphorylation sites; on the other hand, desensitization may occur in human embryo kidney (HEK293) cells (Chaudhry et al. 1994). The underlying mechanisms are largely unknown. Therefore, we have compared desensitization in CHO and HEK293 cells transfected with human beta3-adrenoceptors and investigated underlying mechanisms in the latter.

CHO and HEK293 cells were stably transfected with the human beta3-adrenoceptor at a density of approximately 200 and 100 fmol/mg protein, respectively. Unless otherwise indicated cells were treated for 24 hours with 10 μM isoprenaline or vehicle. After washing 3 times with buffer, agonist was added for 30 minutes in the presence of 100 μM each of the phosphodiesterase inhibitors isobutylmethyl-xanthine and Ro20-1724 (4-[(3-butoxy-4-methoxyphenyl)-methyl]-2-imidazolidinone) using a LANCE® kit (Perkin Elmer) as described (Jongsma et al., 2006). Data are means ± SEM of 3-6 experiments, and a p<0.05 (t-test) was considered significant.

Isoprenaline pre-treatment had little effect on the subsequent on cAMP responses to isoprenaline in CHO cells, but reduced the maximal response in HEK293 cells from 712 ± 33 to 260 ± 16 (p<0.05) without major chances of agonist potency, and all further experiments were done in HEK293 cells. The desensiti-zation of the cAMP response was time and concentration dependent. Treatment with pertussis toxin (100 ng ml-1 for 24 h) did not alter the desensitization. Moreover isoprenaline pre-treatment also reduced the forskolin induced cAMP accumulation to an at least similar extent. A 24 h treatment with 10 μM forsko-lin for 24 hours also desensitized the subsequent response to isoprenaline. The common beta3-adreno-ceptor polymorphism Trp64Arg did not affect its susceptibility towards agonist-induced desensitization.

We conclude that the agonist induced desensitization of the human beta3-adrenoceptor is cell-type specific. It does functionally not involve Gi protein, but may involve reduced adenylyl cyclase function, possibly explaining why it can happen despite a lack of phosphorylation sites. The desensitization can be mimicked by adenylyl cyclase activation and is not affected by the Trp64Arg polymorphism.

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Jongsma et al, BML-241 fails to display selective antagonism at the sphingosine-1-phosphate receptor, S1P(3), Br. J. Pharmacol., 2006, 149, 277-82