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Functional and biochemical evidence for the presence of protein kinase C in bovine ciliary muscle
Pharmacological Research, Vol. 22, Supplement 2,1990
271
FUNCTIONAL AND BIOCHEMICAL EVIDENCE FOR THE PRESENCE OF PROTEIN KINASE C IN BOVINE CILIARY MUSCLE. M.D. Log~, E. Daniele, M.Trabucchi* and S.Govoni. Pharmacobiol. Dept. Univ. Bari, * Dept. Exp. Med. Biochem. Sci. Univ. Rome, Italy. The presence of protein kinase C (PKC) has been described in rabbit ciliary body. Experiments using active phorbol esters have suggested a role of PKC in modulating the adenyl ate cyclase system and aqueous humor production. Phorbol esters are utilized in binding and functional approaches as specific markers of PKC. The aim of the present study was to investigate the presence and the role of PKC in bovine ciliary muscle. The specific binding of 3H-Phorbol-l2,13-dibutyrate (3HPdBu) to membranes prepared from bovine ciliary muscle was used as probe to measure the presence of PKC. Binding experiments were performed using membranes prepared from freshly dissected bovine ciliary muscles. The binding data show the existence of a high affinity saturable specific binding. Specific binding was 80% of total binding and was linear with the amount of protein added in the range of 10-100 pg/ml. The labelled PdBu was displaced by cold phorbol myristate acetate and by phorbol l2-l3-dibutyrate but not by 4-alpha-phorbol, a phorbol derivative which does not activate PKC. The involvement of PKC in modulating the contractility of the bovine ciliary muscle was investigated in functional studies using activators and inhibitors of PKC. A pretreatment with PdBu (1 pM for 15 min) abolished the contractions induced by carbachol and other agonists (PGF2alpha and Substance P). On the contrary H7 (30 pM) and staurosporine (3 pM), inhibitors of PKC, enhanced these responses; interestingly the repeated addition of carbachol was followed by desensitization which was counteracted by H7. The results are in support of the existence of PKC in bovine ciliary muscle and indicate the involvement of this transduction system in the modulation of carbachol response.
1043--6618/90/22110271-0I/$03.00/0
© 1990The Italian Pharmacological Society
271
FUNCTIONAL AND BIOCHEMICAL EVIDENCE FOR THE PRESENCE OF PROTEIN KINASE C IN BOVINE CILIARY MUSCLE. M.D. Log~, E. Daniele, M.Trabucchi* and S.Govoni. Pharmacobiol. Dept. Univ. Bari, * Dept. Exp. Med. Biochem. Sci. Univ. Rome, Italy. The presence of protein kinase C (PKC) has been described in rabbit ciliary body. Experiments using active phorbol esters have suggested a role of PKC in modulating the adenyl ate cyclase system and aqueous humor production. Phorbol esters are utilized in binding and functional approaches as specific markers of PKC. The aim of the present study was to investigate the presence and the role of PKC in bovine ciliary muscle. The specific binding of 3H-Phorbol-l2,13-dibutyrate (3HPdBu) to membranes prepared from bovine ciliary muscle was used as probe to measure the presence of PKC. Binding experiments were performed using membranes prepared from freshly dissected bovine ciliary muscles. The binding data show the existence of a high affinity saturable specific binding. Specific binding was 80% of total binding and was linear with the amount of protein added in the range of 10-100 pg/ml. The labelled PdBu was displaced by cold phorbol myristate acetate and by phorbol l2-l3-dibutyrate but not by 4-alpha-phorbol, a phorbol derivative which does not activate PKC. The involvement of PKC in modulating the contractility of the bovine ciliary muscle was investigated in functional studies using activators and inhibitors of PKC. A pretreatment with PdBu (1 pM for 15 min) abolished the contractions induced by carbachol and other agonists (PGF2alpha and Substance P). On the contrary H7 (30 pM) and staurosporine (3 pM), inhibitors of PKC, enhanced these responses; interestingly the repeated addition of carbachol was followed by desensitization which was counteracted by H7. The results are in support of the existence of PKC in bovine ciliary muscle and indicate the involvement of this transduction system in the modulation of carbachol response.
1043--6618/90/22110271-0I/$03.00/0
© 1990The Italian Pharmacological Society