Microscope laser light-scattering spectroscopy of vesicles within canaliculi of rat hepatocyte couplets

Abstract

Employing microscope laser light-scattering spectroscopy, we investigated "primary" bile secretion into canalicular spaces of rat hepatocyte couplets in monolayer culture. Time-dependent scattered light intensities were fitted by bi-exponential decays. The "slow" decay was attributed to an undulating canalicular membrane motion, whereas the "fast" decay was consistent with rapidly diffusing intracanalicular vesicles with mean hydrodynamic radii (+/- SD) of 479 +/- 53 A. After addition of micromolar concentrations of common bile salts, increases in the amplitude of the fast component facilitated a quantitative estimate of vesicle secretion rates. A dose-response relationship with 0.1-200 microM sodium taurocholate was characterized by an initial concentration-dependent increase and then a decrease in the amplitude of the fast canalicular component. Since these taurocholate concentrations are nontoxic to cultured hepatocytes, the maximum in vesicle-sized particles at 10 microM taurocholate suggested that its critical micellar concentration of approximately 5 mM was attained within the canalicular spaces. Sodium taurolithocholate resulted in time- and dose-dependent diminution in vesicle secretion rates, which after 2 h was followed by spontaneous canalicular recovery. This suggested that acute bicellular "cholestasis" was followed by oxidative metabolism and detoxification of the monohydroxy bile salt. Microscope laser light-scattering spectroscopy should facilitate further physical-chemical and pathophysiological studies of bile secretion at the cellular level.