Charles H. C. Twort

Direct action of BRL 38227 and glibenclamide on intracellular calcium stores in cultured airway smooth muscle of rabbit.

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 (10 μm) reduced loading of the inositol 1,4,5‐trisphosphate (InsP3)‐sensitive intracellular store by 26.5% ± 1.0; this effect was antagonized by 1 μm glibenclamide. BRL 38227 itself did not release calcium and had no effect on guanosine 5′‐0‐(3‐thiotriphosphate)‐induced calcium release. Glibenclamide (≥5 μm) 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.

Localisation and expression of β-adrenoceptor subtype mRNAs in human lung

Abstract The cellular localisation and distribution of mRNAs encoding β-adrenoceptor subtypes in human lung were studied by in situ hybridisation and Northern blot analysis. The 851-bp SmaI/PvuII fragment of human β 1 -adrenoceptor cDNA, the 439-bp SmaI fragment of human β 2 -adrenoceptor cDNA and the 975-bp SmaI fragment of human β 3 -adrenoceptor cDNA bound to single mRNA species of approximately 3.2 kb, 2.2 kb and 2.3 kb in size, respectively. Human lung and heart and rabbit lung expressed both β 1 - and β 2 -adrenoceptor mRNAs with no detectable level of β 3 -adrenoceptor mRNA, while rabbit perirenal adipose tissue expressed β 1 -, β 2 - and β 3 -adrenoceptor mRNAs. Cultured human airway epithelial cells and airway smooth muscle cells expressed only β 2 -adrenoceptor mRNA. In situ hybridisation in human lung, using 35 S-labelled antisense RNA probes revealed a high level of expression of β 1 - and β 2 -adrenoceptor mRNAs in the pulmonary blood vessels, high level of expression of β 2 -adrenoceptor mRNA in the alveolar walls with less expression of β 1 -adrenoceptor mRNA. There was a moderate expression of β 2 -adrenoceptor but not β 1 -adrenoceptor mRNA in airway epithelium and smooth muscle of peripheral airways and no detectable β 3 -adrenoceptor mRNA in any lung structures.

Inositol 1,4,5-trisphosphate- and guanosine 5'-O-(3-thiotriphosphate)-induced Ca2+ release in cultured airway smooth muscle.

1 The interaction between inositol 1,4,5‐trisphosphate (InsP3) and guanosine 5′‐O‐(3‐thio triphosphate) (GTPγS) releasable calcium (Ca2+) pools was examined using 45Ca effluxes in permeabilized cultured airway smooth muscle cells from rabbit trachea. 2 Addition of InsP3 or GTPγS caused a concentration‐dependent release of intracellular Ca2+. The release of Ca2+ by InsP3 was much greater than with GTPγS. Pretreatment with maximally effective InsP3 (10 μm) abolished the GTPγS‐induced Ca2+ release, whereas pretreatment with 100 μm GTPγS reduced the InsP3‐induced Ca2+ release by 25%. 3 Ryanodine (100 μm), also gave a large release of intracellular Ca2+. After pretreatment with 100 μm ryanodine, GTPγS did not induce Ca2+ release, and InsP3‐induced Ca2+ release was reduced by 76%. 4 Caffeine (50 mm), produced a slow release of intracellular Ca2+. Pre‐exposure to 50 mm caffeine had no effect on the GTPγS‐induced Ca2+ release but reduced the InsP3 releasable Ca2+ by 58%. 5 Pretreatment 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 GTPγS, and the third by InsP3 alone. Ca2+ may be able to move from the GTPγS‐sensitive pool into the InsP3‐ and ryanodine‐sensitive pool when this becomes depleted. In contrast, the opposite movement of Ca2+ cannot occur.

Differences in sensitivity to the specific protein kinase C inhibitor Ro31-8220 between small and large bronchioles of the rat.

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 Ro31‐8220 and Ro31‐7549 and the non‐specific inhibitor H7 on carbachol‐, 5‐hydroxytryptamine (5‐HT)‐ and 4β‐phorbol dibutyrate (4β‐PDBu)‐induced contractions in large and small bronchioles. 2 The study was performed on isolated bronchioles of the rat with internal diameters of 574 μm ± 11 (small, n= 128), and 1475 μm ± 32 (large, n = 93), using a Mulvaney‐Halpen small vessel myograph. 3 In these preparations 4β‐PDBu had no effect if added on its own. However, after precontracting with 30 mm K+, 0.5 μm 4β‐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. Ro31‐8220, Ro31‐7549 and H7 all showed concentration‐dependent inhibition of this response. 4 In small bronchioles 10 μm Ro31‐8220 shifted both the carbachol and 5‐HT concentration‐ response curves to the right, and reduced the maximum response. In contrast, 10 μm Ro31‐8220 had no significant effect on the EC50 to carbachol of larger bronchioles, although the maximum response was reduced, and had no significant effect on the 5‐HT concentration‐ response curve. 200 μm 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 IOIIM Ro31‐8220 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 μm 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.

Regulation of Airway Smooth Muscle

Publisher Summary This chapter focuses on the regulation of airway smooth muscle (ASM) function. In the past, it was assumed that an abnormal contractility of ASM was the underlying defect in asthma. Smooth muscle from asthmatic patients, however, does not demonstrate increased contractile responses to agonists such as histamine (H) in vitro suggesting that it is not the muscle itself but the control of ASM function and of airway calibre in vivo which is fundamentally abnormal. Although contraction is considered to be the most significant function of the ASM cell, the less well recognized processes of relaxation and proliferation should not be ignored because they also play important roles in the pathogenesis of asthma. The smooth muscle of the airways is morphologically similar to that of the vascular, alimentary, and urogenital systems. The ASM cells are spindle shaped, with a central nucleus and prominent nucleoli, packed with longitudinally arranged myofilaments, sarcoplasmic reticulum (sr), and mitochondria. Bundles of smooth muscle cells are arranged helically around the airway, but predominantly in a circular rather than a longitudinal orientation so that contraction leads to a narrowing rather than a shortening of the airway. ASM is rich in receptors for many of the inflammatory mediators. It relies heavily on pharmacomechanical coupling mechanisms for the transduction of extracellular signals.