Liver cell plasma membrane lipids and the origin of biliary phospholipid.

The liver cell plasma membranes of fed male Wistar rats were separated into a fraction rich in bile canaliculi and the remainder of the plasma membrane. Electron-microscopically, the bile canalicular fraction consisted almost exclusively of intact bile canaliculi with thier contiguous membranes. The remaining plasma membrane fraction consisted primarily of vesicles and sheets of membranes essentially free from the bile canaliculi. The bile canalicular membrane fraction contained relatively more total lipid, cholesterol, and phospholipid, and relatively less protein. Although the phospholipid composition of the two fractions was the same, the specific activity of the bile canalicular membrane phosholipids, up to 12 h following in vivo administration of [2-3H]glycerol, was always significantly greater than that of the remaining plasma membranes, and showed a biphasic response not found in the latter. The specific activity of the phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine of the bile canalicular membranes rose to a peak within 40 min after administration of the label, fell sharply and then rose to a second peak after 120 min. The specific activity of the sphingomyelin and phosphatidylserine plus phosphatidylinositol of the bile canalicular membranes and of all the phospholipids of the remaining plasma membranes diphasic pattern but increased steadily to reach a maximum at 120 min. The specific activity of biliary phosphatidylcholine followed a pattern identical to that of the phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine of the bile canalicular membrane fraction. These results show that the average rate of turnover of phospholipid in the bile canalicular membranes is considerably greater than that in the remaining plasma membrane and other cell membrane fractions; they indicate that the phospholipid of the bile canalicular membranes exists in two or more pools, turning over a different rates; and they support the concept that biliary phospholipid is derived from the bile canalicular membrane. The results also suggest that bile canalicular phospholipid may be derived from two different sources, in contrast to the remainong plasma membrane.     

The ultrastructural localization of antigen B10 of the hepatocyte plasma membrane in mouse hepatomas

The ultrastructure of the murine hepatocyte plasma membrane antigen (Ag B10) was studied by immunoelectron microscopy in 5 spontaneous and 3 chemical-induced hepatomas. Ag B10 was associated with plasmalemma of bile canaliculi and membrane of microvilli as in normal liver. Sometimes it was connected with plasmalemma of lateral domain of tumor cells. The availability of Ag B10 in the matrix of bile canaliculi and within microvilli was shown.     

Plasma membranes of the rat liver. Isolation and enzymatic characterization of a fraction rich in bile canaliculi

A method is described for the rapid isolation of a plasma membrane fraction containing a high concentration of intact bile canaliculi from the rat liver. Isolated bile canaliculi retain most of the ultrastructural features exhibited in the intact liver cell. The final fraction contains 5''-nucleotidase activity at approximately the same concentration as that in previous preparations of plasma membranes. In the presence of 0.01 M Mg⁺⁺, 5''-nucleotidase exhibits a double pH optimum at pH values of 7.5 and 9.5. The activities of glucose-6-phosphatase and alkaline phosphatase are present in low amounts. Cytochrome P-450 is not detectable. Na⁺-K⁺-activation of ATPase is observed to the extent of 20–36% in about half of the assays. The availability of a method for preparation of intact bile canaliculi should prove useful for studying the biochemical events associated with the transport of bile constituents into canaliculi.     

A morphometric study on growth of bile canaliculi after partial hepatectomy

19-33 h after partial hepatectomy there is a time-dependent increase of the bile canalicular luminal volume, of the total length of bile canaliculi, and of their non-microvillous, smooth surface, when measured per unit volume of lobular parenchyma. On average the luminal volume fraction is increased by a factor of 2.8, the length per volume by a factor of 1.4, and the density of smooth surface by a factor of 2.0, when compared with sham-operated controls. On the other hand, the volume and surface density of bile canalicular microvilli are only slightly increased after partial hepatectomy. These findings are interpreted as indicating disproportional growth of bile canaliculi which is due predominantly to the formation of new, at first non-microvillous, membrane.     

Isolation of rat hepatocyte plasma membranes. I. Presence of the three major domains

A rat liver plasma membrane preparation was isolated and characterized both biochemically and morphologically. The isolation procedure was rapid, simple and effective in producing a membrane fraction with the following biochemical characteristics: approximately 40-fold enrichment in three plasma membrane markers, 5'-nucleotidase, alkaline phosphodiesterase I (both putative bile canalicular membrane enzymes), and the asialo-glycoprotein (ASGP) receptor (a membrane glycoprotein present along the sinusoidal front of hepatocytes); a yield of each of these plasma membrane markers that averaged approximately 16%; and minimal contamination by lysosomes, nuclei, and mitochondria, but persistent contamination by elements of the endoplasmic reticulum. Morphological analysis of the preparation revealed that all three major domains of the hepatocyte plasma membrane (sinusoidal, lateral, and bile canalicular) were present in substantial amounts. The identification of sinusoidal membrane was further confirmed when ASGP binding sites were localized predominantly to this membrane in the isolated PM using electron microscope autoradiography. By morphometry, the sinusoidal front membrane accounted for 47% of the total membrane in the preparation, whereas the lateral surface and bile canalicular membrane accounted for 6.8% and 23% respectively. This is the first report of such a large fraction of sinusoidal membrane in a liver plasma membrane preparation.     

Electron microscopic cytochemical characterization of bile canaliculi and bile ducts in vitro.

Electron microscopic cytochemical localization of Mg++-activated adenosine triphosphatase (Mg++-ATPase) and 5-nucleotidase (AMPase) was investigated in bile canaliculus-rich and bile duct-containing fractions isolated from rat liver. Comparative cyochemical studies between prefixed and non-prefixed fractions revealed that the activity of both enzymes could be detected in the fractions under appropriate experimental conditions. However, the cytochemical activity of AMPase was much more sensitive to glutaraldehyde than that of Mg++-ATPase. Mg++-ATPase and AMPase reaction products were localized primarily on bile canalicular microvilli, that is, along the outer (luminal) surface of canalicular plasma membranes, but they were never observed on bile ductal microvilli. AMPase was also detectable on lateral hepatic plasma membranes. Mg++-ATPase demonstrated by the cytochemical technique described is a reliable enzyme marker for isolated bile canalicular membranes. At high magnification, Mg++-ATPase reaction product was also observed on the microfilaments surrounding isolated bile canaliculi. The possibility that the reaction product on the pericanalicular microfilaments may result from the hydrolysis of ATP byan actomyosin ATPase-like enzyme associated with these filaments is briefly discussed.     

Analysis of hepatocyte plasma membrane domains during rat development using monoclonal antibodies

The plasma membrane of adult rat hepatocyte consists of three domains, which have been identified by the monoclonal antibodies A39 and A59 as markers of the sinusoidal domain, B1 of the lateral, and B10 of the canalicular domains (Eur J Cell Biol 39:122, 1985). These monoclonal antibodies were used to study, by indirect immunocytochemistry, formation of the hepatocyte plasma membrane domains during development, from day 15 of gestation to day 35 post partum. The antigens defined by A39, B1, and B10 were detected, from day 15, over the major part of the hepatocyte plasma membrane except for the membranes of newly formed bile canaliculi, which were not labeled by B1 and only poorly labeled, if at all, by A39 and B10. As soon as fetuses were 16 days old, B1 labeled predominantly the lateral domain, as in the adult. Labeling with B10 progressively intensified on the membranes of bile canaliculi, but localization was not exclusively canalicular until day 21 post partum. A39 intensely labeled the canalicular membranes at 19-21 days of gestation, while at 35 days post partum it exhibited the predominantly sinusoidal labeling observed in adult hepatocytes. The antigen defined by A59 was not detected before birth and was found exclusively on the sinusoidal domain, as in the adult. These results show that the patterns of antigen distribution on different plasma membrane domains establish themselves at different rates. The marked differences observed between fetal or neonatal and adult hepatocytes might be responsible for immaturity of liver functions in the neonate.     

Identification of rat hepatocyte plasma membrane proteins using monoclonal antibodies

We have localized and identified five rat hepatocyte plasma membrane proteins using hybridoma technology in combination with morphological and biochemical methods. Three different membrane preparations were used as immunogens: isolated hepatocytes, a preparation of plasma membrane sheets that contained all three recognizable surface domains of the intact hepatocyte (sinusoidal, lateral, and bile canalicular), and a glycoprotein subfraction of that plasma membrane preparation. We selected monoclonal IgGs that were hepatocyte specific and localized them using both immunofluorescence on 0.5-micron sections of frozen liver and immunoperoxidase at the ultrastructural level. One antigen (HA 4) was localized predominantly to the bile canalicular surface, whereas three (CE 9, HA 21, and HA 116) were localized predominantly to the lateral and sinusoidal surfaces. One antigen (HA 16) was present in all three domains. Only one antigen (HA 116) could be detected in intracellular structures both in the periphery of the cell and in the Golgi region. The antigens were all integral membrane proteins as judged by their stability to alkaline extraction and solubility in detergents. The apparent molecular weights of the antigens were established by immunoprecipitation and/or immunoblotting. In a related study (Bartles, J.R., L.T. Braiterman, and A.L. Hubbard, 1985, J. Cell. Biol., 100:1126-1138), we present biochemical confirmation of the domain-specific localizations for two of the antigens, HA 4 and CE 9, and demonstrate their suitability as endogenous domain markers for monitoring the separation of bile canalicular and sinusoidal lateral membrane on sucrose density gradients.     

Comparison of specific binding of human epidermal growth factor (EGF) to sinusoidal and bile canalicular membranes isolated from rat liver

The binding characteristics of human epidermal growth factor (EGF) were compared between highly purified canalicular (CMV) and sinusoidal (basolateral) rat liver plasma membrane (SMV) preparations. The dissociation constants (2-3 nM) for these membranes were comparable, while the binding capacity for CMV was approximately half that for SMV. The binding capacity for CMV was too high to be accounted for only by the contamination with sinusoidal membranes, since the measurements of specific activities of various enzymes (Na+,K+-ATPase, alkaline phosphatase, and leucine aminopeptidase) indicated that the extents of the cross contamination with other membrane fractions were at most 10%. Although the physiological function of specific binding of EGF to bile canalicular membrane domain remains to be determined, it may have a role in biliary excretion of EGF. The specific binding of EGF to bile canalicular membranes from rat liver was identified for the first time.     

Monoacylglycerol acyltransferase activity in the rat liver plasmalemma fractions

Bile canalicular membranes and plasma membranes free of bile canalicular membranes were prepared from rat livers and their lipolytic activities were measured. Both preparations catalyzed hydrolysis and transacylation when monoacylglycerol and phosphatidylethanolamine were used as substrates. The specific enzymatic activity in the plasmalemma free of bile canalicular membranes was slightly higher than that in bile canalicular membranes. Neither preparation attacked the triacylglycerol of chylomicra, which indicates the lack of a lipoprotein lipase. Heparin and CaCl₂ stimulated the activities in both preparations. On the basis of these data, we suggest that monoacylglycerol acyltransferase can serve two distinct roles in the liver cell, depending upon the mumbrane fraction of association.     

Dynamics of rat liver ecto-ATPase during development suggests its involvement in bile acid efflux

We studied cytochemical localization of ectoadenosine triphosphatase in the rat liver during development from 15-day-old fetus to 4-week-old and adult animal. First signs of the enzyme activity were found in some of the primitive bile canaliculi of 15-day-old fetuses. The majority of canaliculi, however, did not reveal any reaction product. Although intensity of the cytochemical reaction increased at 20 days of gestation, it still remained relatively low. Intensity of the reaction increased significantly and its product became readily detectable in the liver of newborn rats. Liver of 1-, 2-, and 4-week-old animals showed strong reaction for ecto-ATPase at the luminal surface of the plasma membrane of the bile canaliculi. Liver of adult rats contained a prominent reaction product similar to that seen in newborns, 1-, 2-, and 4-week-old animals. At all stages of fetal development, as well as in postnatal and adult rats, reaction was found only within the hepatic bile canalicular system and exclusively at the luminal surface of the canalicular plasma membrane. Using diethyl pyrocarbonate (DEPC), a specific inhibitor of ecto-ATPase activity, cytochemical reaction was blocked in all examined samples. Results of the present study, taken together with established biochemical and immunological data, provide conclusive morphological evidence in support of the view that canalicular ecto-ATPase is involved in bile acid efflux.     

Studies on the preparation of rat liver plasma membrane fractions and on their polypeptide patterns

Plasma membrane and bile canalicular membrane fractions were prepared from rat liver using NaHCO3, NaHCO3--CaCl2, and K2HPO4-KH2PO4 buffers (all at pH 7.4). The amount (expressed as milligrams protein per gram liver) of plasma membrane fraction exceeded the amount of bile canalicular membrane fraction using each of these three media; the use of NaHCO3-CaCl2 afforded a substantially higher yield of both types of membranes. The two membrane fractions exhibited complex patterns of polypeptides (greater than 30) on sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. Several reproducible differences in polypeptide patterns were observable between the two membrane fractions; in particular, components possibly corresponding to the heavy chain of myosin and to action were prominent in the bile canalicular membrane fraction. The effects of incubation in the above three buffers and in Tris--HCl (pH 7.4) on the polypeptide patterns of both types of membrane were studied. Many polypeptides were released from each type of membrane in all of these media. Differential effects on the polypeptide patterns of either type of membrane fraction were observed among the various buffers. In terms of minimizing loss of polypeptides, in general, NaHCO3--CacCl2 appeared to be the best buffer and Tris--HCl the worst buffer. The significance of these results for the preparation and storage of liver cell plasma membrane fractions is briefly discussed.     

Fine structure of the liver in the larval lamprey, Petromyzon marinus L.; hepatocytes and sinusoids

The ultrastructure of hepatocytes, bile canaliculi, and hepatic sinusoids of the larval lamprey, Petromyzon marinus, was examined using thin-sectioned and freeze-fractured tissues. The liver is a "tubular gland" with hepatocytes arranged in a tubular fashion around large bile canaliculi. Hepatocytes are roughly conical in shape, with their tapered apices facing a bile canalicular lumen. They possess extensive rough and smooth endoplasmic reticulum, a well-developed Golgi complex, abundant mitochondria, and varying numbers of large secondary lysosomes. Both secondary lysosomes and the Golgi complex are concentrated in the apical or peribiliary cytoplasm, indicating a possible role in bile secretion. The apical surfaces of the hepatocytes bear numerous elongate microvilli and occasional cilia, which project into the bile canaliculi. The hepatocytes are joined, apically, by junctional complexes composed of zonulae occludentes and adhaerentes. In freeze-fracture, the zonulae occludentes are of variable apicobasal depth and consist of honeycomb-like meshworks of fibrils. Spaces of variable width frequently appear in the P-face grooves, indicating that the zonulae occludentes are "leaky." Numerous communicating (gap) junctions join the hepatocytes laterally. Varying numbers of lateral microvilli project into the intercellular spaces and, basally, the plasma membrane is deeply infolded, resulting in the formation of apparently interdigitating basal processes resting upon a thin basal lamina. Sinusoids are composed of both a heavily-fenestrated, continuous endothelium, and phagocytic reticulo-endothelial (Kupffer) cells. Depsite the difference in arrangement of their hepatocytes, the mammalian and lamprey livers show similar ultrastructural features.     

Newly synthesized canalicular ABC transporters are directly targeted from the Golgi to the hepatocyte apical domain in rat liver

Newly synthesized canalicular ectoenzymes and a cell adhesion molecule (cCAM105) have been shown to traffic from the Golgi to the basolateral plasma membrane, from where they transcytose to the apical bile canalicular domain. It has been proposed that all canalicular proteins are targeted via this indirect route in hepatocytes. We studied the membrane targeting of rat canalicular proteins by in vivo [(35)S]methionine metabolic labeling followed by preparation of highly purified Golgi membranes and canalicular (CMVs) and sinusoidal/basolateral (SMVs) membrane vesicles and subsequent immunoprecipitation. In particular, we compared membrane targeting of newly synthesized canalicular ABC (ATP-binding cassette) transporters MDR1, MDR2, and SPGP (sister of P-glycoprotein) with that of cCAM105. Significant differences were observed in metabolic pulse-chase labeling experiments with regard to membrane targeting of these apical proteins. After a chase time of 15 min, cCAM105 appeared exclusively in SMVs, peaked at 1 h, and progressively declined thereafter. In CMVs, cCAM105 was first detected after 1 h and subsequently increased for 3 h. This findings confirm the transcytotic targeting of cCAM105 reported in earlier studies. In contrast, at no time point investigated were MDR1, MDR2, and SPGP detected in SMVs. In CMVs, MDR1 and MDR2 appeared after 30 min, whereas SPGP appeared after 2 h of labeling. In Golgi membranes, each of the ABC transporters peaked at 30 min and was virtually absent thereafter. These data suggest rapid, direct targeting of newly synthesized MDR1 and MDR2 from the Golgi to the bile canaliculus and transient sequestering of SPGP in an intracellular pool en route from the Golgi to the apical plasma membrane. This study provides biochemical evidence for direct targeting of newly synthesized apical ABC transporters from the Golgi to the bile canaliculus in vivo.     

Plasma membrane specialization and intracellular polarity of freshly isolated rat hepatocytes

It was investigated whether rat hepatocytes maintain their plasma membrane specialization (sinusoidal, lateral and bile canalicular sites) and their intracellular polarity (peribiliary region, rich in lysosomes and poor in mitochondria) after isolation. The morphology of the hepatocytes and the cytochemical localization of marker enzymes for the bile canalicular membrane (alkaline phosphatase, adenosine triphosphatase and 5' nucleotidase), for the lysosomes (acid phosphatase) and for the mitochondria (beta-hydroxybutyrate dehydrogenase and succinate dehydrogenase) were studied in situ and directly after isolation using both light and electron microscopy. The morphology of the cells and the cytochemical activity of acid phosphatase, succinate dehydrogenase and beta-hydroxybutyrate dehydrogenase showed that in isolated cells, as in situ, the lysosomes were concentrated in bands, devoid of mitochondria. Unlike in situ the reaction product of alkaline phosphatase, adenosine triphosphatase and 5'nucleotidase was evenly distributed along the entire plasma membrane of the isolated cells. Morphologically, no tight or gap junctions or desmosomes could be detected in the isolated cells, while the plasma membrane appeared to be homogeneously covered with uniform microvilli. In conclusion it can be stated that during isolation the hepatocytes loose their distinct plasma membrane specialization, but maintain their peribiliary region rich in lysosomes and poor in mitochondria.     

Endogenous and exogenous domain markers of the rat hepatocyte plasma membrane

We have used a combined biochemical and morphological approach to establish the suitability of certain endogenous and exogenous domain markers for monitoring the separation of rat hepatocyte plasma membrane domains in sucrose density gradients. As endogenous domain markers, we employed two of the integral plasma membrane protein antigens, HA 4 and CE 9, localized to the bile canalicular and sinusoidal/lateral domains, respectively, of the hepatocyte plasma membrane in rat liver tissue (Hubbard, A. L., J. R. Bartles, and L. T. Braiterman, 1985, J. Cell Biol., 100:1115-1125). We used immunoelectron microscopy with a colloidal gold probe to demonstrate that HA 4 and CE 9 retained their domain-specific localizations on isolated hepatocyte plasma membrane sheets. When the plasma membrane sheets were vesiculated by sonication and the resulting vesicles were centrifuged to equilibrium in sucrose density gradients, quantitative immunoblotting revealed that the vesicles containing HA 4 and those containing CE 9 exhibited distinct density profiles. The density profile for the bile canalicular vesicles (marked by HA 4) was characterized by a single peak at a density of 1.10 g/cm3. The density profile for the sinusoidal/lateral vesicles (marked by CE 9) was bimodal, with a peak in the body of the gradient at a density of 1.14 g/cm3 and a smaller amount in the pellet (density greater than or equal to 1.17 g/cm3). We used this sucrose gradient fractionation as a diagnostic procedure to assign domain localizations for several other hepatocyte plasma membrane antigens and enzyme activities. In addition, we used the technique to demonstrate that 125I-wheat germ agglutinin, introduced during isolated liver perfusion at 4 degrees C, can serve as an exogenous domain marker for the sinusoidal domain of the rat hepatocyte plasma membrane.     

Localization of plasma membrane CD38 is domain specific in rat hepatocyte

CD38 is a 42- to 45-kDa type II transmembrane glycoprotein with the ability to synthesize cADPR, a metabolite with potent calcium mobilizing properties independent of IP(3). We report here the primary characterization and localization of CD38 in the plasma membrane fraction of rat hepatocyte. Western blot analysis of a partially purified plasma membrane fraction with a panel of polyclonal antibodies against CD38 detected a 42- to 45-kDa protein band which is characteristic of CD38. ADP-ribosyl cyclase activity was found to be present in the plasma membrane fraction, indicating the presence of functionally active CD38. Subfractionation of the plasma membrane to the sinusoidal and bile canalicular membrane fractions showed the presence of ADP-ribosyl cyclase activity in both fractions with the sinusoidal membrane fraction having a 10-fold higher specific activity than the bile canalicular membrane fraction. Immunohistochemical staining with the same panel of polyclonal antibodies showed exclusive differential spatial localization to both the nuclei and sinusoidal domain of the plasma membrane. It is possible that the different spatial distribution of CD38 in the rat hepatocyte might be responsible for its myriad of previously known functional roles.     

A three-dimensional study of organelle interrelationships in regenerating rat liver. 3. Organelles related to bile.

A number of cell structures are described which show a morphological relationship to the bile canaliculi. Two types of peribiliary vesicles are identified: osmication positive ones occurring between the bile canaliculi and the osmicated immature Golgi cisternae and probably deriving from the latter, and osmication negative ones related to MVB, on which they appear as buds. Small coated vesicles are seen attached to this second type. Large lacunae may originate from MVB, as suggested by the MVB-like internal vesicles they may contain. Some stay in luminal continuity with the bile canaliculi. Canalicular coated vesicles are seen as parts of the canalicular plasma membrane and free in the cytoplasm.     

Redistribution and fate of colchicine-induced alkaline phosphatase in rat hepatocytes: possible formation of autophagosomes whose membrane is derived from excess plasma membrane

The redistribution and fate of colchicine-induced alkaline phosphatase (ALPase) in rat hepatocytes were investigated by electron microscopic enzyme cytochemistry and biochemistry. ALPase activity markedly increased in rat hepatocytes after colchicine treatment (2.0 mg/kg body weight, intraperitoneal injection). At 20–24 h after colchicine treatment, the liver showed the highest activity of ALPase. Thereafter, ALPase activity decreased and returned to normal levels at 48 h. In normal hepatocytes from control rats, ALPase activity was seen only on the bile canalicular membrane. However, at 20–24 h after colchicine treatment, colchicine-induced ALPase was redistributed in the sinusoidal and lateral (basolateral) membranes as well as in the bile canalicular membrane. At 30–36 h after colchicine treatment, ALPase activity on the basolateral membrane gradually decreased. In contrast, ALPase in the bile canalicular membrane increased along with the enlargement of bile canaliculi, suggesting that ALPase in the basolateral membrane had been transported to the bile canalicular membrane. Furthermore, ALPase-positive vesicles, cisternae and autophagosome-like structures were frequently seen in the cytoplasm. ALPase was also positive in some lysosomal membranes. ALPase in hepatocytes at 48 h after colchicine treatment returned to almost the same location as in control hepatocytes. Altogether, it is suggested that excessively induced ALPase is at least partially retrieved by invagination of the bile canalicular membrane and then transported to lysosomes for degradation. In addition, this study indicates that excess plasma membrane might be a possible origin of autophagosomal membrane.     

Vasopressin-induced morphological changes in polarized rat hepatocyte multiplets: dual calcium-dependent effects

Calcium-mobilizing hormones and neurotransmitters are known to affect cell morphology and function including cell differentiation or division. In this study, we examined vasopressin (AVP)-induced morphological changes in a polarized system of rat hepatocytes. Light and electron microscope observations showed that AVP induced microvilli formation and a remodeling of the isolated hepatocyte F-actin submembrane cytoskeleton, these two events being correlated. We showed that these effects were rapid, reversible, observed at nanomolar AVP concentration and mediated by the V(1a) receptor. On polarized multicellular systems of hepatocytes, we observed a rapid reduction of the bile canaliculi lumen at the apical pole and micovilli formation at the basolateral domain with an enlarged F-actin cytoskeleton. Neither activation of protein kinase C nor A via phorbol ester or dibutyryl cAMP induced such rapid morphological changes, at variance with ionomycin, suggesting that AVP-induced intracellular calcium rise plays a crucial role in those effects. By using spectrofluorimetry and cytochemistry, we showed that calcium release from intracellular stores was involved in bile canaliculus contraction, while calcium entry from the extracellular space controlled microvilli formation. Taken together, AVP and calcium-mobilizing agonists differentially regulate physiological hepatocyte plasma membrane events at the basal and the apical domains via topographically specialized calcium-dependent mechanisms.