The Isoquinoline Sulfonamide Inhibitors of Protein Phosphorylation, H-7, H-8, and HA-1004, Also Inhibit RNA Synthesis: Studies on Responses of Adipose Tissue to Growth Hormone

To evaluate the possibility that some of the metabolic effects of GH in rat adipose tissue depend upon phosphorylation-dephosphorylation reactions, we examined the effects of the isoquinoline sulfonamide family (H-7, H-8, and HA-1004) of protein kinase inhibitors on the actions of GH. In the course of these studies it became clear that these compounds may also block RNA synthesis. In the concentration range of 50–200 μM, H-7, H-8, and HA-1004 completely blocked lipolysis in response to the combination of 100 ng/ml dexamethasone and 30 ng/ml human GH in segments of epididymal fat from normal rats, but were less effective in blocking lipolysis in response to either 1 μM (Bu)₂cAMP or 1 ng/ml isoproterenol, which are known to depend upon activation of protein kinase-A. Activation of protein kinase-C with phorbol myristate nearly doubled the rate of glucose oxidation in segments of normal adipose tissue, and this insulin-like response was completely inhibited with 200 nM H-7. At concentrations as high as 500 μM, H-7, H-8, and HA-1004 failed to inhibit the insulin-like response to GH in tissue segments of either normal or hypophysectomized rats. However, when 200 μM H-7 or H-8, but not HA-1004, was present during the first 3 h of treatment with GH, it prolonged the duration of the insulin-like response (acceleration of glucose oxidation) from its normal termination within 2-3 h to more than 4 h. Identical results were obtained with 5 μg/ml actinomycin- D. The effect of H-7 or H-8 was reversible and required the continuous presence of these agents, whereas actinomycin- D was required only during the first 60 min after GH. Termination of the insulin-like response normally is followed by a period of several hours in which the tissues are refractory to further insulin-like stimulation by GH. When actinomycin-D, H-7, H-8, or HA-1004 was added to tissues of hypophysectomized rats 60 min after GH, the insulin-like response terminated at its normal time, but the tissues were not refractory to insulinlike stimulation upon reexposure to GH. These agents also prevented GH from sustaining refractoriness in normal adipose tissue. At the same concentration at which GH effects are blocked (200 μM), H-7 and H-8 inhibited the incorporation of [³H]uridine into RNA by about 80%; HA-1004 was somewhat less potent. Since only those effects of GH that are also sensitive to actinomycin-D were inhibited by H-7, H-8, and HA-1004, it is likely that the observed inhibitory effects of these agents on the actions of GH are due to their previously unrecognized inhibitory effects on RNA synthesis rather than their inhibitory effects on protein kinase. With regard to GH action, these experiments suggest that temporally separate periods of RNA synthesis may be required for termination of the insulin-like response to GH and for the induction of refractoriness. (Endocrinology126: 441–450, 1990)