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Title: Natural and synthetic factors which influence the calcium sensitivity of chemically-skinned rat cardiac muscle
Author: Steele, Derek S.
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1990
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The work which comprises this thesis is concerned with natural and synthetic substances which influence the Ca2+ -sensitivity of the intracellular systems in chemically-skinned cardiac muscle. Preparations skinned with Triton-X100 retain only traces of membrane structure while the contractile proteins remain functional. This form of treatment is ideal for investigating factors which influence the Ca2+-sensitivity of the contractile proteins. Selectively-(saponin-) skinned trabeculae retain the intracellular membranes associated with the s. r. and mitochondria, while the surface membrane is rendered permeable to the bathing solution. This type of preparation has been used to investigate interventions which influence the functioning of the s. r. In chapter 3 the experimental protocol employed to study the functioning of the s. r. is described. This involved producing trains of caffeine contractures in selectively-treated preparations. Between exposures to caffeine, the muscle was 'Ca2+-loaded' in a solution with a [Ca2+] subthreshold for tension production. The contracture amplitude increased as the duration of Ca2+-loading or the [Ca2+] of the Ca2+-loading solution was increased. Such trains of contractures proved reproducible over prolonged periods of time. Using this method, it was possible to study the effects of various substances which influence the functioning of the s. r. under strictly controlled Ca2+-loading conditions. However, adequate interpretation of these results requires an understanding caffeine's action on the s. r. The reported effects of caffeine on a variety of preparations are discussed during the introduction of this chapter. Following this, a method is described which allows a direct assessment of the ability of the s. r. to effect relaxation in saponin-skinned preparations. The action of caffeine on net s. r. Ca2+-accumulation was studied using this method. The results suggest that millimolar concentrations of caffeine render the s. r. incapable of net Ca2+-accumulation under these experimental conditions. This led to the suggestion that the s. r. is unlikely to contribute significantly to the relaxation of the caffeine contracture in selectively-skinned trabeculae. The possible involvement of the mitochondria in the caffeine-induced response was also studied. However, a mitochondrial contribution was excluded as the caffeine contractures were unaffected by addition of azide or changing the [Na] of the bathing solutions. Chapter 4 addresses the possibility that enzymes which influence the intracellular levels of cAMP may persist following saponin treatment. This could potentially influence the results presented in the following chapters as many of the substances studied are known to affect phosphodiesterase (PDE) activity and other cellular systems which alter the levels of cyclic nucleotides in intact cells. The Effects of PDE inhibition, and stimulation of adenylate cyclase were studied. In addition, an attempt was made to characterise the response of the s. r. and contractile proteins to exogenous cAMP. Caffeine-induced contractures were potentiated in the presence of cAMP. This finding is consistent with previous studies in a variety of preparations. However, the contractile proteins were unaffected by exogenous cAMP unless the duration of skinning was reduced to an unacceptable level. Addition of forskolin toxin potentiated the caffeine contracture. This introduced the possibility that (i) cAMP production may continue following saponin-skinning and (ii) interventions which influence the adenylate cyclase activity may potentiate the caffeine response via a cAMP mediated mechanism. The results presented in the following chapters were interpreted with reference to these findings. Chapter 5 is a comparative study of the effects of caffeine and sulmazole on the contractile proteins and s. r. Both substances induce s. r. Ca2+-release and increase myofilament Ca2+-sensitivity. The contribution of increased Ca2+-sensitivity to transient sulmazole and caffeine contractures was assessed. The opposing effects of sulmazole and caffeine on Cmax were also studied. In chapter 6, the intracellular actions of ORG30029 (N-hydroxy-5,6-dimethoxy-benzo[b]thiophene -2-carboximidamide) were investigated. Chapter 7 considers the effects of various natural and synthetic modulators of Ca2+-sensitivity on the development of rigor tension. Chapter 8 investigates the effect of taurine on s. r. Ca2+-accumulation and myofilament Ca2+-sensitivity. Taurine was found to produce a small increase in Ca2+-sensitivity but a marked increase in the ability of the s. r to accumulate Ca2+. An attempt was made to correlate the intracellular actions of taurine described in this chapter with the reported effects of the amino acid on intact cardiac preparations. The possible physiological role of taurine in the heart is discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available