Microscope laser light scattering spectroscopy of vesicles within
canaliculi of rat hepatocyte couplets.
M[diaeresis]ockel, Gotthold-Mathias, Sridhar Gorti, Rakesh K. Tandon,
Toyoichi Tanaka, and Martin C. Carey.
Department of Medicine, Harvard Medical School, Brigham and Women's
Hospital and Harvard Digestive Diseases Center, Boston, MA 02115, and
Department of Physics, Center for Materials Science and Engineering,
and George R. Harrison Spectroscopy laboratory, Massachusetts
Institute of Technology, Cambridge, MA 02139
APStracts 2:0034G, 1995.
Employing the technique of microscope laser light scattering
spectroscopy we investigated $QUOTprimary$QUOT bile secretion into
canalicular spaces of rat hepatocyte couplets. Once short-term
monolayer culture was established, time-dependent light scattering
intensities were fitted by bi-exponential decays; the $QUOTslow$QUOT
decay was attributed to an undulating membrane motion possibly
canalicular in origin; whereas the $QUOTfast$QUOT decay was
consistent with rapidly diffusing intra-canalicular particles (mean
hydrodynamic radii +/-SD; 479 +/- 53 [angstrom]a) typical of
unilamellar vesicles. Following addition of [mu]M concentrations of
the common bile salts to the culture medium, an increase in the
amplitude of the fast (vesicular) component, facilitated a
quantitative estimation of lipid secretion rates per bile
canaliculus. Prolonged observations of up to 90 minutes on the same
canaliculus was aided by addition of cytochalasin D, which by
inhibiting F-actin, impeded spontaneous contractile closure of the
canalicular spaces. Primary bile secretion rates with added sodium
taurocholate, a choleretic bile salt, were characterized by a
concentration-dependent increase followed by a decrease in the
amplitude of the fast canalicular component. Since the taurocholate
concentrations (0.1-200[mu]M) employed are physiological and well
below levels considered toxic to cultured hepatocytes, the maximum
density of vesicle-sized particles which occured at an added bile
salt concentration of 10[mu]M is consistent with the attainment of
the critical micellar concentration ( nearly equal to 5mM) of
taurocholate and thenceforth vesicle dissolution in the canalicular
spaces. Contrarywise, addition of the cholestatic bile salt, sodium
taurolithocholate, resulted in time- and dose-dependent diminution in
the amplitude of the fast component consistent with decreased vesicle
secretion rates into the canalicular spaces. $QUOTCholestasis$QUOT,
as evidenced by the amplitude of the fast component was followed by
spontaneous recovery within 2 hrs. This result implied that rapid
detoxification of the monohydroxy bile salt occured by oxidative
metabolism and further verified the viability of hepatocyte couplet
systems during prolonged incubation. This microscope laser light
scattering techique should prove useful for further studies of
physical-chemical and pathophysiological events involved in bile
secretion at the cellular level.
Received 29 April 1994; accepted in final form 14 February 1995.
APS Manuscript Number G158-4.
Article publication pending Am. J. Physiol. (Gastrointest. Liver
Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 10 March 1995.