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Studies on Receptor Activation in Intact and Permeabilized Smooth Muscle

The aim of this thesis was to study agonist-induced contractions in smooth muscle. The majority of the work was undertaken on rat anococcygeus, although some has been performed on guinea pig portal vein longitudinal muscle. The agonists which were examined (noradrenaline, phenylephrine and acetylcholine) all cause contraction in rat anococcygeus. They are thought to have their effect mediated by a G-protein which in turn stimulates the phosphatidylinositol cycle and promotes contraction. Work was carried out therefore, to analyse the effect of receptor activation at two levels: (a) the G-protein; and (b) the phosphatidylinositol cycle. To examine the intracellular effects of receptor activation the muscle was permeabilized. Part of this thesis comprises an investigation into the effectiveness of the three putative permeabilizing techniques. ANALYSIS OF THREE DIFFERENT PERMEABILIZATION TECHNIQUES The three permeabilization techniques examined were: (a) saponin-treatment; (b) EGTA-treatment; and (c) alpha-toxin-treatment. To analyse the effectiveness of each treatment several tests were carried out and the behaviour of the permeabilized smooth muscle after the three different treatments was compared with that of an intact muscle. The muscles were examined to see: (i) if they contracted readily to calcium; (ii) if they produced rigor crossbridges when ATP and CrP were removed from the bathing medium; (iii) how they responded when two factors known to have a direct effect on the contractile proteins, cyclic AMP and inorganic phosphate, were added to the bathing medium; and (iv) how they responded to the application of noradrenaline at a low calcium concentration. It was found that calcium-activated force decayed with time in saponin-and alpha-toxin-treated muscle. Possible mechanisms for the decay were examined. These included: (a) loss of calmodulin; (b) reduced or increased ionic strength; and (c) reduced or increased pH. These were all examined in saponin-treated muscle. The effect of lowering ionic strength was examined in toxin-treated muscle. RECEPTOR ACTIVATION AND CONTRACTION Once a suitable permeabilization technique had been established, further experimentation was undertaken to examine the effect of receptor activation on contraction in intact and permeabilized muscle. The permeabilization technique adopted was alpha-toxin treatment. Originally it had been hoped that this method would allow the analysis of receptor activation by noradrenaline and acetylcholine. However, it was found that alpha-toxin-treated rat anococcygeus muscle was unable to contract in response to acetylcholine. Possible reasons for this is examined in Chapter 4. (a) G-protein Kitazawa et al (1989) had already reported that GTP had to be included in the bathing medium before phenylephrine could produce a contraction in alpha-toxin permeabilized guinea pig portal vein. This led these workers to conclude that a G-protein was involved in receptor activation. The rest of Chapter 4 aims to assess the involvement of G-proteins in receptor activation in alpha-toxin-treated rat anococcygeus and guinea pig portal vein. Receptor activation was examined by looking at its effect on calcium release and calcium-activated force. The involvement of G-proteins was examined by using GTP, GTP-gamma-S (a non-hydrolysable analogue of GTP) and GDP-B-S (a non-hydrolysable analogue of GDP). A striking feature of the noradrenaline-activated contractions in both tissues was that their amplitude decayed upon repeated exposure. Several factors were examined to ascertain the cause of this decay. These included examining the effect of GTP on the decay, the accessibility of the calcium store and the sensitivity of the calcium store to Ins(1,4,5)P3 before and after the decay in noradrenaline-activated force. (b) phosphatidylinositol cycle The fifth chapter attempts to assess the role of the phosphatidylinositol cycle in receptor activation. This was examined by chronically treating rats with lithium chloride which is known to block the phosphatases which normally are responsible for the breakdown of Ins(1,4,5)P3. This blockade eventually causes the rundown of inositol within the cell and, therefore, eventually of PtdIns(4,5)P2, the precursor of Ins(1,4,5)P3 and of diacylglycerol (DG). The effects of chronic lithium treatment of rats on (i) the concentration response curves to different agonists; and (ii) the intracellular component produced by different agonists were examined in intact anococcygeus muscle. In the second set of experiments, chronic lithium treatment was undertaken at the same time as the animals were being given myo-inositol in their drinking water. (Abstract shortened by ProQuest.).