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.

The effects of BRL 38227 and glibenclamide on intracellular calcium stores were investigated in permeabilized cultured airway smooth muscle cells of the rabbit using 45Ca effluxes. BRL 38227 (10p1M) reduced loading of the inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular store by 26.5% + 1.0; this effect was antagonized by 1 gM glibenclamide. BRL 38227 itself did not release calcium and had no effect on guanosine 5'-0-(3-thiotriphosphate)-induced calcium release. Glibenclamide ( > 5 M) also reduced calcium loading of the intracellular store, and enhanced calcium release. These results suggest that BRL 38227 has a direct effect on intracellular calcium handling.

The role of protein tyrosine kinases (PTK) in modulating contractility has not been investigated in airway smooth muscle (ASM). We have examined the effects of the PTK inhibitors ST638, genistein, and tyrphostin A47 on contractions induced by carbachol, serotonin, ionomycin, and 75 mM KCl in isolated bronchioles of the rat with internal diameters of 614 +/- 16 microm (small, n = 143), and 1,433 +/- 39 microm (large, n = 57). ST638 caused a dose-dependent decrease in the maximum response to carbachol, and shifted the carbachol concentration-response curve to the right. This effect was greater in small bronchioles. Tyrphostin A47 (100 microM) and genistein (74 microM) had a similar effect to ST638. ST638 caused a concentration-dependent relaxation (EC50 approximately 7.2 microM) in bronchioles precontracted with 0.5 microM carbachol, and was maximally effective at 50 microM when tone was reduced by 82.5 +/- 3.8% in small bronchioles, and 57.2 +/- 2.8% in large bronchioles. ST638 also reduced the maximal response to serotonin, and caused a large shift to the right of the serotonin concentration-response curve. Pretreatment with ST638 (50 microM) reduced the response to 75 mM KCl in both small and large bronchioles in the presence of atropine (small: by 88.9 +/- 5.6%, n = 11; large: by 90.1 +/- 4.4%, n = 11). Tyrphostin A47 (100 microM) had a similar effect (91%). ST638 (50 microM) and tyrphostin A47 (100 microM) substantially relaxed small bronchioles contracted with 1.5 microM ionomycin (ST638: by 86.7 +/- 1.8%, n = 6; tyrphostin: by 89.3 +/- 1.7%, n = 5). We have therefore demonstrated that PTK inhibitors can suppress contraction induced by a number of different mechanisms in ASM. These results suggest that PTK signaling pathways are not only important for proliferation of ASM, but also fon contractile function.

1 The interaction between inositol 1,4,5-trisphosphate (InsP3) and guanosine 5'-O-(3-thio triphosphate) (GTPyS) releasable calcium (Ca2") pools was examined using 45Ca effiuxes in permeabilized cultured airway smooth muscle cells from rabbit trachea. Caffeine (50 mM), produced a slow release of intracellular Ca2 . Pre-exposure to 50 mM caffeine had no effect on the GTPyS-induced Ca2 + release but reduced the InsP3 releasable Ca2 + by 58%. 5Pretreatment with ryanodine abolished the caffeine-induced Ca2+ release, and addition of caffeine before ryanodine reduced the ryanodine-induced Ca2+ release by 64.4%. 6 These results suggest that there are at least three pools of Ca2 + present within airway smooth muscle cells. The largest pool is released by InsP3 or ryanodine, another is released either by a high concentration of InsP3 or on application of GTPyS, and the third by InsP3 alone. Ca2+ may be able to move from the GTPyS-sensitive pool into the InsP3-and ryanodine-sensitive pool when this becomes depleted. In contrast, the opposite movement of Ca2 + cannot occur.

No abstract