The mechanisms of bile acid uptake have been studied with primary monolayer cultures of rat hepatocytes. Hepatocytes were incubated with taurocholic acid (TC), glycocholic acid (GC), cholic acid (CA), glycochenodeoxycholic acid (GCDC), chenodeoxycholic acid (CDCA), deoxycholic acid (DOCA), lithocholic acid (LCA), or cholylglycylhistamine (CCH), a neutral bile acid derivative for 10 s to 60 min in medium containing sodium chloride, sodium chloride with 1 mM ouabain, or choline chloride. Cells were washed free of radioactive tracer, cell-associated radioactivity was quantitated, and bile acid uptake rates, kinetic parameters of uptake, and steady-state bile acid content were calculated. Two mechanisms for bile acid uptake were identified. Uptake of TC, GC, CA, and GCDC occurred predominantly via a sodium-dependent, ouabain-suppressible saturable mechanism, presumably sodium-coupled transport. Estimates of apparent Km and Vmax for these bile acids were TC, 33 micro M and 0.36 nmol . min-1 . mg prot-1; GC, 18 micro M and 0.22 nmol . min-1 . mg prot-1; CA, 13 micro M and 0.10 nmol . min-1 . mg prot; and GCDC, 6 micro M and 0.21 nmol . min-1 . mg prot, respectively. Uptake via this sodium-coupled mechanism exhibited considerable substrate selectivity. It was enhanced by increased ring hydroxylation and amino acid conjugation and decreased by further conjugation with a neutral histamine group (CGH). In contrast, uptake of CDCA, DOCA, LCA, and CGH occurred primarily via a nonsaturable sodium-independent mechanism, possibly simple diffusion. This mechanism accounted for only a small portion of uptake of TC, GC, CA, and GCDC at low bile acid concentrations. Nonsaturable bile acid uptake rates appeared to correlate with decane-buffer partition coefficients and to be related to bile acid structure.

Transport of sodium, chloride, and taurocholate was studied in primary cultures-ofadult rat hepatocytes incubated in a balanced electrolyte solution containing 150 mM NaCI, various concentrations oftaurocholate, and 'Na, 31C1, [3H] Bile acids are the predominant organic solute in bile and their importance in bile formation is attested to by the observation that the rate of canalicular bile formation is linearly related to bile acid output in all species thus far examined (1). Previous investigations (2-8) have suggested that bile acid uptake is saturable, concentrative, and partially sodium dependent. While these observations are consistent with a sodium-facilitated or sodium-cotransport mechanism, no simultaneous measurement of bile acid and sodium fluxes has been reported, and information regarding the nature of the dependence of concentrative bile acid uptake on extracellular sodium and the sodium gradient is not available. Moreover, little is known regarding the transport by hepatocytes ofeither sodium or chloride, which together account for more than half oftotal bile osmolality.The experiments described in this paper concern three principal areas. First, we report a technique that permits measurement of uptake of sodium and chloride as well as of other inorganic and organic solutes by cultured rat hepatocytes. Second, uptake kinetics and apparent steady-state intracellular concentration of sodium and chloride in cultured rat hepatocytes have been determined. Third, the relationship of uptake and intracellular accumulation of taurocholate to extracellular sodium concentration, sodium uptake, and the transmembrane sodium gradient have been defined. The (14); or ouabain (1 mM), taurocholate, or both were added.Technique. Coverslips with adherent hepatocytes were preincubated 10 min in medium identical to that used in the uptake study but without radioisotope or added tauroctholate. A 1-hr preincubation in ouabain-containing medium was utilized in studies ofthe effect ofouabain on taurocholate transport. The coverslips were then transferred to 26 x 33 mm incubation wells (Flow Laboratories) containing 3 ml of appropriate medium and 1-2 puCi of radioisotope (1 Ci -= 3.7 x 10"°becque-rels). At time intervals varying between 10 sec and 2 hr, the cellcoated coverslips were washed by dipping them for 30 sec in each of eight 30-ml beakers containing 25 ml of ice-cold incubation medium without isotope. The cells were then scraped into Lowry's solution (0.1 M NaOH/0. 189 M Na2CO3) with a rubber spatula. Radioactivity and protein in the Lowry's solution and radioactivity in the incubation medium were measured 986The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

The development of suberin lamellae in the hypodermis of Zea mays cv. LG 11 was observed by electron microscopy and the presence of suberin inferred from autoliuorescence and by Sudan black B staining in nodal (adventitious) and primary (seminal) root axes. Suberin lamellae were evident at a distance of 30-50 mm from the tip of roots growing at 20°C and became more prominent with distance from the tip. Both oxygen deficiency and growth at 13°C produced shorter roots in which the hypodermis was suberized closer to the root tip. There were no suberin lamellae in epidermal cells or cortical collenchyma adjacent to the hypodermis. Plasmodesmata were not occluded by the suberin lamellae: there were twice as many of them in the inner tangential hypodermal wall (1,14 μn ) as in the junction between the epidermis and hypodermis (0.54 μm ). Water uptake by seminal axes (measured by micropotometry) was greater at distances more than 100 mm from the root lip than in the apical zone where the hypodermis was unsuberized. In the more mature zones of roots grown at 13°C rates of water uptake were greater than in roots grown at 20°C even though hypodermal suberization was more marked. Sleeves of epidermal/hypodermal cells (plus some accessory collenchyma) were isolated from the basal 60 mm of nodal axes by enzymatic digestion (drisclase). The roots were either kept totally immersed in culture solution or had the basal 50 mm exposed to moist air above the solution surface. In both treatments the permeabilities to tritiated water and Rb were low (circa 10 mms ) in sleeves isolated from the extreme base. In roots grown totally immersed, however, the permeability of sleeves increased 10 to 50-fold over a distance of 40 mm. In roots exposed to moist air the permeability remained at a low level until the point where the root entered the culture solution and then increased rapidly (> 50-fold in a distance of 8 mm). Growth of roots in oxygen depleted (5% O ) solutions promoted the development of extensive cortical aerenchymas. These developments were not associated with any reduction in permeability of sleeves isolated from the basal 40 mm of the axis. It was concluded that the presence of suberin lamellae in hypodermal walls does not necessarily indicate low permeability of cells or tissues to water or solutes. The properties of the walls (lamellae?) can be greatly changed by exposure to moist air, perhaps due to increased oxygen availability.

A B S T R A C T To characterize the transport mechanisms responsible for formation of canalicular bile, we have examined the effects of ion substitution on bile acid-dependent and bile acid-independent bile formation by the isolated perfused rat liver. Complete replacement of perfusate sodium with choline and lithium abolished taurocholate-induced choleresis and reduced biliary taurocholate output by >70%. Partial replacement of perfusate sodium (25 of 128 mM) by choline reduced bile acid-independent bile formation by 30% and replacement of the remaining sodium (103 mM) by choline reduced bile acid-independent bile formation by an additional 64%. In contrast, replacement of the remaining sodium (103 mM) by lithium reduced bile acid-independent bile formation by only an additional 20%, while complete replacement of sodium (128 mM) by lithium reduced bile formation by only 17%, and lithium replaced sodium as the predominant biliary cation. Replacement of perfusate bicarbonate by Tricine, a zwitterionic amino acid buffer, decreased bile acid-independent bile formation by >50% and decreased biliary bicarbonate output by -60%, regardless of the accompanying cation. In separate experiments, replacement of sodium by lithium essentially abolished Na,K-ATPase activity measured either as ouabain-suppressible ATP hydrolysis in rat liver or kidney homogenates, or as ouabain-suppressible 86Rb uptake by cultured rat hepatocytes.These studies indicate that bile acid(taurocholate)-dependent bile formation by rat liver exhibits a specific requirement for sodium, a finding probably attribPortions of this work were presented at the 82nd Annual

Chloride is the predominant inorganic anion in bile, and it has been proposed that active chloride transport, possibly via a sodium-coupled mechanism, may contribute to that portion of canalicular bile formation not directly related to bile acid transport (bile acid-dependent bile formation or BAIBF). We have therefore examined the anion specificity of BAIBF using the isolated perfused rat liver and have studied sodium-chloride flux coupling and the sodium dependence of intracellular chloride content using 22Na and 36Cl transport by cultured rat hepatocytes. BAIBF by the isolated rat liver was unaltered by replacement of chloride with nitrate or benzenesulfonate but was significantly reduced by replacement of chloride with sulfate or thiocyanate. In cultured hepatocytes, sodium entry rate was reduced when chloride in the incubation medium was replaced by cyclamate, benzenesulfonate, or sulfate and mannitol but was unaffected when chloride was replaced by nitrate, gluconate, or thiocyanate. Conversely, chloride entry rate was decreased when sodium was replaced with choline but was unaffected when sodium was replaced by lithium or when ouabain was added to the medium. Thus no consistent evidence of sodium-chloride flux coupling was observed. Steady-state exchangeable intracellular chloride in the cultured hepatocytes was unaffected by ouabain or by replacement of sodium with choline and was increased when sodium was replaced by lithium. These findings indicate that basal BAIBF exhibits no specific chloride requirement. Although they do not exclude the possible existence in rat liver of sodium-coupled chloride transport, they provide no evidence that such a mechanism accounts for a major portion either of chloride transport by individual rat hepatocytes or of basal BAIBF by intact rat liver.