Analysis of inhibition by H89 of UCP1 gene expression and thermogenesis indicates protein kinase A mediation of beta(3)-adrenergic signalling rather than beta(3)-adrenoceptor antagonism by H89

Although it has generally been assumed that protein kinase A (PKA) is essen tial for brown adipose tissue function, this has not as vet been clearly de monstrated. H89. an inhibitor of PKA, was used hen to inhibit PKA activity, In cell extracts, it was confirmed that norepinephrine stimulated PKA acti vity, which was abolished by H89 treatment. In isolated brown adipocytes, H 89 inhibited adrenergically induced thermogenesis (with an IC50 of approx. 40 mu mM), and in cultured cells, adrenergically stimulated expression of t he uncoupling protein-1 (UCP1) gene was abolished by H89 (full inhibition w ith 50 muM). However, HS? has been reported to be an adrenergic antagonist on beta (1)/beta (2)-adrenoceptors (AR). Although adrenergic stimulation of thermogenesis and UCP1 gene expression are mediated via beta (3)-AKs, Ir w as deemed necessary to investigate whether H89 also had antagonistic potenc y on beta (3)-ARs. It mas found that EC50 values for beta (3)-AR-selective stimulation of cAMP production (with BRL-37344) in brown adipose tissue mem brane fractions and in intact cells were not affected by H89. Similarly, th e EC50 of adrenergically stimulated oxygen consumption was not affected by H89, As H89. also abolished forskolin-induced UCP1 gene expression? and pot entiated selective beta (3)-AR-induced cAMP production, H89 must be active downstream of cAMP. Thus, no antagonism of H89 on beta (3)-ARs could be det ected. We conclude that H89 can be used as a pharmacological tool for eluci dation of the involvement of PKA in cellular signalling processes regulated via beta (3)-ARs: and that the results are concordant with adrenergic stim ulation of thermogenesis and UCP1 gene expression in brown adipocytes being mediated via a PKA-dependent pathway. (C) 2001 Elsevier Science B.V. AII r ights reserved.