Philip M. Best, Ph.D. - US grants

The amount of force produced by a muscle cell is a function of the intracellular calcium ion concentration. An intracellular organelle called the sarcoplasmic reticulum sequesters calcium from the cytoplasm to cause muscle relaxation and releases it to initiate contraction. Although it is known that a change in the electrical voltage across the cell surface membrane can stimulate calcium release from the SR, the cellular mechanisms that control and regulate the release process are poorly understood. This project is designed to gain a greater understanding of these mechanisms by studying the way in which a variety of drugs and ions modulate calcium release from the sarcoplasmic reticulum. Single amphibian muscle cells will be used. The surface membranes of these cells are removed by microdissection so that the conditions surrounding the SR can be rigorously controlled. This technique causes only minor disruption of normal cellular structure. A quantitative measure of the rate of calcium release from the sarcoplasmic reticulum in these isolated cells will be obtained using an optical technique employing the calcium sensitive dye Arsenazo III. In other experiments the permeability of the SR membrane to potassium ion will be monitored using radioactive tracer techniques. Specific experiments are designed to determine the mechanism of action of calcium channel blocking agents in decoupling the spread of activation from the transverse tubular membranes to the sarcoplasmic reticulum. In addition, the importance of counterion movement in modulating the magnitude of calcium efflux will be investigated by using potassium channel blocking agents. The results of these experiments will add to our knowledge of the cellular processes involved in the activation of contraction in skeletal muscle. A better understanding of the mechanisms underlying this process in healthy cells will improve our understanding of the changes which occur in the activation of cells under a variety of pathological states.

The lone-term goal of the work described in this proposal is to understand how growth factors and hormones effect the expression and function of ion channels. The proposal will test the specific hypothesis that growth hormone (GH) and/or insulin like growth factor 1 (IGF-1) are important regulators of the T-type calcium current in atrial myocytes. Both electrophysiological and molecular biological techniques will be used to study the expression of T-current in myocytes. AIM 1: The direct effects of GH and IGF-1 on T-current expression will be studied in cultured myocytes. Use of an in vivo system allows precise control of the composition of the cell environment thus simplifying to some extent experimental design. The role of second messenger pathways and of intracellular processes such as protein synthesis, RNA translation, and DNA transcription will be determined. AIM 2: Postnatal changes in the density and biophysical properties of T-type calcium current will be determined in myocytes acutely isolated from a growth hormone deficient (GHD) rat. A naturally occurring "knockout", this mutant provides a unique whole animal model to assess the effects of GH and IGF-1 on T- current expression. Reintroduction of GH and IGF-1 by osmotic minipumps will allow separation of the effects of these agents in the complex environment represented by the intact animal. AIM 3: Molecular biological approaches will be used to clone the T-channel alpha-subunit. Probes will be constructed and the relationship between current density and mRNA levels investigated to provide additional information about the regulation of T-current expression. AIM 4: The correlation between the expression of different beta-isoform subunits and T-current density will be determined to investigate the possible involvement of this subunit in regulating T-current density. The results will provide new information about the long-term regulation of electrical excitability in cardiac tissue both during normal postnatal development and in response to altered physiological states. While the proposal is focused on T-current in cardiac myocytes, this calcium current is widely distributed in neuronal and neural secretory tissues. The information gained should therefore have broad applicability to other excitable cells.