1 The involvement of protein kinase C (PKC) in constriction of small bronchioles has never been investigated. In this study we have examined the effects of the specific PKC inhibitors Ro3l-8220 and Ro31-7549 and the non-specific inhibitor H7 on carbachol-, 5-hydroxytryptamine (5-HT)-and 4p-phorbol dibutyrate (4p-PDBu)-induced contractions in large and small bronchioles.2 The study was performed on isolated bronchioles of the rat with internal diameters of 574 Am ± 11 (small, n = 128), and 1475 jam ± 32 (large, n = 93), using a Mulvaney-Halpen small vessel myograph.3 In these preparations 4P-PDBu had no effect if added on its own. However, after precontracting with 30 mM K+, 0.5 AM 4P-PDBu caused a contractile response of 110.4 ± 7.0% TK (TK = maximum response to 75 mM K+ in small and 69.3 ± 6.5% TK in large bronchioles. Ro3l-8220, Ro3l-7549 and H7 all showed concentration-dependent inhibition of this response. 4 In small bronchioles 10 JM Ro3l-8220 shifted both the carbachol and 5-HT concentration-response curves to the right, and reduced the maximum response. In contrast, 10 JIM Ro3l-8220 had no significant effect on the ECm to carbachol of larger bronchioles, although the maximum response was reduced, and had no significant effect on the 5-HT concentration-response curve. 200 JM H7 shifted the carbachol concentration-response curve to the right as well as reducing the maximal response in both small and large bronchioles. 5 Large bronchioles exhibited a greater rate of decay of carbachol-induced contraction than did small bronchioles. Pretreatment with Ro31-8220 accelerated the rate of decay. 6 Pretreatment with 10 JM Ro3l-8220 caused a small reduction in the response to 75 mM K+ in both small and large bronchioles (small: to 87.8 ± 3.0% TK; large: to 94.1 ± 0.8% TK). H7 at 200 JM caused a much larger reduction in both preparations (small: to 75.1 ± 3.0% TK); large: to 82.7 ± 0.6% TK). 7 Small bronchioles were more sensitive than larger bronchioles to agonists and phorbol ester. The protein kinase inhibitor Ro31-8220 could reduce agonist-induced constriction in small and large bronchioles, as well as reducing or abolishing phorbol ester-induced contractions. Small bronchioles were more sensitive than large bronchioles to Ro31-8220. These results suggest that there is a significant PKC involvement in constriction of bronchioles to carbachol and 5-HT, and that the proportion of the contractile response that can be attributed to PKC is greater in smaller than larger bronchioles.

A new and convenient method for the preparation of the four stereoisomers of dihexadecanoyl phosphatidylinositol has been developed. An enantiomeric pair of acid-labile, pentaprotected myo-inositol building blocks was synthesized in high yield and coupled with chiral phenyl dihexadecanoylglyceryl phosphates to give the fully protected phosphatidylinositols. These were subsequently deprotected by hydrogenolysis and self-hydrolysis in aqueous ethanol to give the desired pure products. Comparison of these compounds as potential substrates for a partially purified phosphatidylinositol 4-kinase (EC 2.7.1.67) derived from human erythrocyte membranes revealed that the chirality of the inositol ring is crucial for efficient phosphorylation, whereas the chirality of the glycerol moiety is relatively unimportant. Moreover, the similarity in phosphorylation rates of the naturally occurring mammalian phospholipid, I, and its synthetic stereochemical counterpart, compound 10a, suggests that the enzyme is relatively tolerant to changes in fatty acid composition.

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