In this multidisciplinary study on the role of the sarcoplasmic reticulum (SR) in malignant hyperthermia (MH), we will: (1) elucidate the molecular mechanisms of action of halothane and dantrolene on the SR; (2) develop in vitro screening methods for MH-inducing drugs as well as MH-inhibiting drugs; and (3) develop a non-invasive method to screen for MH using the subject's red blood cells. We hypothesized that: (i) Halothane does not influence the Ca2+ uptake of the SR at physiologically realistic concentrations; (ii) Halothane increases both Ca2+ permeability and Ca2+ affinity of the Ca2+ release channel of the SR membrane; (iii) The Ca2+ channel of the MH-SR has a higher Ca2+ permeability and a greater Ca2+ affinity than that of normal SR; (iv) These abnormalities of the Ca2+-channel in MH-SR are secondary to a genetic defect in the phospholipid structure of the MH-SR membrane; (v) These abnormalities of MH-SR causes abnormal (increased) Ca2+ release from the SR upon exposure to halothane in vivo. The released Ca2+ further stimulates Ca2+ release via the Ca2+-induced Ca2+ release mechanism and results in a massive Ca2+ release (this is the cause of the muscle contracture and heat production seen in MH); (vi) The membrane of red blood cells of MH-susceptible animals is also abnormal in the sense that it is more easily perturbed by halothane than that of normal animals. We will first use isolated SR from genetically MH pigs, and take the following four approaches: (I) kinetic measurements of Ca2+ efflux from isolated SR and reconstituted liposome vesicles; (II) EPR spin-probe experiments to measure the fluidity of the SR and liposome membranes; (III) isolation and purification of Ca2+ release channel proteins from the SR using high pressure liquid chromatography; and (IV) chemical analysis of phospholipids prepared from MH-SR and normal SR. We will also use red blood cells from both MH and control pigs to test whether or not the halothane effect on the membrane fluidity of the red cell membrane can be utilized to screen for MH in pigs. To date, the mechanism of MH has not been well-elucidated. Therefore, there can be no way to further develop either the therapeutic means to alleviate MH symptoms to develop non-invasive screening methods. Now that we have developed methods to study the mechanisms, the results of this project will help not only in the understanding of the pathogenesis of MH, but also in the development of simple new methods to screen drugs as well as a non-invasive method to screen for MH.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Surgery, Anesthesiology and Trauma Study Section (SAT)
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University City Science Center
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