Evidence for inverse agonism of SR141716A at human recombinant cannabinoid CB1 and CB2 receptors.

The cannabinoid receptor antagonist SR141716A has been suggested to be an inverse agonist at CB1 receptors in some isolated intact tissues. We found that the basal incorporation of [35S]-GTPgammaS in Chinese hamster ovary cells expressing human recombinant CB1 and CB2 receptors was inhibited by SR141716A (mean pEC50s 8.26 and 6.00, respectively), whereas cannabinol (10 microM) had no significant effect at hCB1 receptors but inhibited the binding at hCB2 receptors. As cannabinol had no effect on basal [35S]-GTPmicroS binding at hCB1 at a concentration 100 fold higher than its binding affinity (K = 0.1 microM), we conclude that endogenous cannabinoid receptor agonists are not a confounding factor and suggest the actions of SR141716A at the hCB1 receptor, and the actions of SR141716A and cannabinol at the hCB2 receptor, are due to inverse agonism.

Characterization of human recombinant alpha(2A)-adrenoceptors expressed in Chinese hamster lung cells using extracellular acidification rate changes.

1. Human alpha(2A)-adrenoceptors heterologously expressed in Chinese hamster lung (CHL) fibroblasts have been characterized pharmacologically using a cytosensor microphysiometer to measure ligand-induced extracellular acidification rate changes. 2. In untransfected CHL cells, noradrenaline had no effect at concentrations up to 100 microM. In alpha(2A)-adrenoceptor transfected cells the rank order of agonist potency was A-54741 (mean pEC(50)=8.96)>dexmedetomidine (8.88)>UK-14304 (8.42)>B-HT 920 (7.05)>noradrenaline (6.92). A-54741, UK-14304 and noradrenaline had the same maximum response while dexmedetomidine and B-HT 920 behaved as partial agonists. 3. The selective alpha(2)-adrenoceptor ligand rauwolscine antagonized acidification rate changes with an affinity independent of the agonist used; the affinity (mean pK(B)) against noradrenaline was 8.43. 4. The selective alpha(1)-adrenoceptor ligands prazosin and doxazosin (each 3 microM) had no effect on noradrenaline responses. 5. Acidification rate changes induced by each agonist were abolished by pre-treatment of cells with pertussis toxin. 6. These data suggest that agonist-induced acidification rate responses in CHL cells transfected with the human alpha(2A)-adrenoceptor are mediated exclusively by the recombinant protein, via pertussis toxin sensitive G(i/o) proteins.

Characterization of human recombinant alpha(2A)-adrenoceptors expressed in Chinese hamster lung cells using intracellular Ca(2+) changes: evidence for cross-talk between recombinant alpha(2A)- and native alpha(1)-adrenoceptors.

1. Human alpha(2A)-adrenoceptors expressed in Chinese hamster lung (CHL) fibroblasts have been pharmacologically characterized by measuring intracellular calcium (Ca(2+)(i)) changes using the Ca(2+)-sensitive dye Fluo3-AM, in conjunction with a fluorometric imaging plate reader (FLIPR). 2. Several alpha-adrenoceptor agonists were examined including the alpha(2)-adrenoceptor agonists UK-14304, B-HT 920, dexmedetomidine and A-54741, the selective alpha(1)-adrenoceptor agonist phenylephrine and the non-selective adrenergic agonist noradrenaline. Of these only noradrenaline (mean pEC(50)=6.49) and A-54741 (6.90) evoked changes in Ca(2+)(i); A-54741 was a partial agonist relative to noradrenaline, achieving only 33% of the noradrenaline maximum. 3. Ca(2+)(i) changes induced by noradrenaline and A-54741 were antagonized by the alpha(2)-selective antagonist rauwolscine (10 nM) and by the alpha(1)-selective antagonists prazosin (0.1 nM) and doxazosin (1.0 nM). 4. Phenylephrine (100 microM) and UK-14304 (10 microM) alone were ineffective in causing Ca(2+)(i) increase. In the presence of a fixed concentration of UK-14304 (3.0 microM), phenylephrine induced concentration-dependent increases in Ca(2+)(i) (mean pEC(50)=5.33). In the presence of phenylephrine (30.0 microM) UK-14304 induced Ca(2+)(i) release (pEC(50)=6.92). The effects of phenylephrine were abolished by prazosin (1.0 nM) or rauwolscine (100 nM). 5. In saturation radioligand binding experiments using membranes of parental (non-transfected) CHL cells there was a small, specific binding of [(3)H]-prazosin (B(max)=24 fmol mg protein(-1); pK(D)=10. 24). 6. Collectively, these data suggest that alpha-adrenoceptor agonist-induced Ca(2+)(i) release in CHL fibroblasts transfected with the human alpha(2A)-adrenoceptor is dependent upon co-activation of the recombinant receptor and a native alpha(1)-adrenoceptor.