9505910 Allen Osmoregulation is the process by which all cells establish their water and salt balance keeping the cell's cytosol slightly hypertonic or isotonic to its external environment. In fresh water protozoa the organelle generally regarded to be responsible for osmoregulation is the contractile vacuole complex (CVC) which is thought to secrete a hypotonic solution. However, as cellular membranes are permeable to water, how these or any cells can accumulate, store and expel water or hypotonic solution uphill against a concentration gradient of water remains a paradox. This project studies the poorly understood CVC organelle system with electrophysiological, immunocytochemical and cell biological methods. The model organism selected is Paramecium, a ciliate which offers the advantages (1) of having recognizable and separate CVC compartments for fluid uptake, fluid transport and fluid storage/expulsion and (2) of allowing an easy assay for determining total cellular fluid output. The host laboratory has extensive experience with this cell and its CVC, having studied in detail its CVC ultrastructure. Monoclonal antibodies to different parts of the CVC of this ciliate are available from preliminary work. An understanding of how osmoregulation is achieved in "primitive" single-celled eukaryotes will establish the fundamental principles on which multicellular organisms may have fashioned their own osmoregulatory systems as they evolved. (1) Ion-selective microelectrodes are used to determine the ionic contents of the in vivo CV fluid. (2) The electrical potential, capacitance, and ionic conductance of the CV membrane is measured and compared with the values found for plasma membrane. Membrane ion channels and electrogenic pump activity are discriminated electrophysiologically and pharmacologically, usine specific ion channel and pump blockers. Patch-clamping is used on isolated CV membrane to determine the properties of individual channels or pum ps. (3) Confirmation that 15 nm "peg" structures represent the V-type H+ ATPase is made using monoclonal antibodies to specific polypeptides of the pump. The tubules containing the pump are isolated and characterized using the techniques of molecular biology. (4) A fluid segregation mechanism is proposed. The effects of osmotic stress on the CVC is studied to see if these effects are consistent with the mechanism. The outcome of these studies contribute to an understanding of the basic mechanism of osmoregulation in this single-celled fresh- water eukaryote as well as give a start on understanding CVC regulation and provides a step in the eventual understanding of the path of evolutionary development of osmoregulation in higher animals. %%% Cells must maintain their content of salts and water at a concentration which is favorable for life even though the cells live in a harsh environment. If these concentrations waver from the norm, the cell will not be able to carry on its metabolic functions and will die. This project is an exploration of how a single cell living in fresh water is capable of secreting water to maintain the proper balance of water and salts. Protozoa have a contractile vacuole system (CVC) which presumably does this job. However, what is not understood is how the water is accumulated within this CVC since all membranes including those surrounding the CVC are permeable to water. One way around the paradox would be for the cell to pump salts into the CVC to maintain a solution within the CVC equal in salt concentration to the cell's cytoplasm. In this way water would enter the CVC and there would be net backflow of water into the c cytoplasm. Both water and salts would then be secreted from the cell. However, what salts the cell is capable of continually excreting remains unknown. Thus the investigation is undertaken to determine what the contents of the CVC are in the fresh water protozoan Paramecium, thereby determining what the ce ll is excreting through its CVC. (1) Ion- selective microelectrodes are inserted into a living CVC to measure the concentration of different ions. (2) Microelectrodes are also used to determine the electrical potential, resistance, and capacitance of the CVC membrane. Clues about the types of ion channels and ion pumps driven by the electrical potential across the membrane will be followed-up with experiments using drugs which block specific channels or pumps. The biochemistry of individual ion carriers can be determined using by assaying the electrical properties in an isolated patch of membrane (patch clamping). (3) Using monoclonal antibodies against pump components, the identity of the pump can be verified visually. (4) A probable mechanism of how the CVC works in this protozoa is suggested and tested. This study gives us clues about how osmoregulation (the balance of salt and water in cells) is made possible in nature and how this process may have evolved from "simpler" forms of life to its present state in higher organisms.