Having demonstrated chloride-coupled Na transport in purified rabbit ileal brush border membrane vesicles, the studies in the coming year will focus on demonstrating stimulatory or inhibitory effects of various clinically important compounds on that transport mechanism. Cathartics such as phenolphthalein, bisacodyl, and anthroquinone will be tested for inhibitory effects on coupled NaCl influx across the rabbit ileal brush border membrane vesicle. In addition, anti-diarrheal compounds such as acetylsalicylic acid and both endogenous and exogenous opiates such as codeine and enkephalins will be tested for possible stimulatory effects on this influx mechanism. In addition, we will attempt to further purify rabbit ileal basolateral membranes. Our present techniques of differential centrifugation and sucrose density gradient ultracentrifugation yield highly purified (20-fold enrichment) brush border membranes, but our basolateral membranes are contaminated with endoplasmic reticulum. Various techniques, as reported in literature, will be utilized to attempt to further purify these plasma cell membranes. If successful, we will seek to demonstrate coupled NaCl influx mechanisms in these membranes as we have done with brush borders. Basolateral membranes from both villus and crypt cells will be studied. Anion-stimulated Na uptake will be studied by measuring 22Na influx into either mannitol or KCl-valinomycin treated vesicles. These studies should increase our understanding of electrolyte transport by the enterocyte and how normal transport mechanisms are perturbed by clinically important drugs.
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