UP-REGULATION OF THE LEVELS OF EXPRESSION AND FUNCTION OF A CONSTITUTIVELY ACTIVE MUTANT OF THE HAMSTER ALPHA(1B)-ADRENOCEPTOR BY LIGANDS THAT ACT AS INVERSE AGONISTS

The alpha(1)-adrenergic agonist phenylephrine stimulated phospholipase D (PLD) activity in Rat 1 fibroblasts transfected to express either t he wild-type hamster alpha(1B)-adrenoceptor or a constitutively active mutant (CAM) form of this receptor. The EC50 for agonist stimulation of PLD activity was substantially lower at the CAM receptor than at th e wild-type receptor as previously noted for phenylephrine stimulation of phosphoinositidase C activity. Sustained treatment of cells expres sing the CAM alpha(1B)-adrenoceptor with phentolamine resulted in a ma rked upregulation in levels of this receptor with half-maximal effects produced within 24 h and with an EC50 of approx. 40 nM, Such an up-re gulation could be produced with a range of other ligands generally vie wed as alpha(1)-adrenoceptor antagonists but equivalent treatment of c ells expressing the wild-type alpha(1B)-adrenoceptor was unable to mim ic these effects. After sustained treatment of the CAM alpha(1B)-adren oceptor expressing cells with phentolamine, basal PLD activity was inc reased and phenylephrine was now able to stimulate PLD activity to gre ater levels than in vehicle-treated CAM alpha(1B)-adrenoceptor-express ing cells. The EC50 for phenylephrine stimulation of PLD activity was not altered, however, by phentolamine pretreatment and the associated up-regulation of the receptor. After phentolamine-induced up-regulatio n of basal PLD activity, a range of alpha(1)-antagonists were shown to possess the characteristics of inverse agonists of the CAM alpha(1B)- adrenoceptor as they were able to substantially decrease the elevated basal PLD activity.