In isolated hamster hepatocytes, ursodeoxycholic acid (UDCA) mobilized intracellular free calcium ([Ca2+]i) and activated phosphorylase a with a half-maximally effective concentration of 188 and 9 microM, respectively. Addition of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) did not affect the maximum [Ca2+]i mobilized by UDCA; however, [Ca2+]i returned to basal levels in 4-5 min compared with > 10 min in the absence of EGTA. Both UDCA and vasopressin activated phosphorylase a to the same extent in the presence and absence of extracellular Ca2+, and the effect of both agents was abolished when the cells were depleted in Ca2+. Vasopressin (100 nM) did not further mobilize [Ca2+]i or activate phosphorylase a when combined with 500 microM UDCA. However, unlike vasopressin, UDCA did not stimulate inositol 1,4,5-trisphosphate (IP3) formation. In contrast to taurine-conjugated UDCA (TUDCA), concentration < or = 500 microM of glycine-conjugated UDCA (GUDCA) did not affect either [Ca2+]i or phosphorylase a. Lithocholic acid and taurolithocholic acid (TLCA) displayed the highest affinity for Ca2+. In addition, TLCA, chenodeoxycholic acid, and NaF stimulated Ca2+ efflux at concentrations as low as 100 microM, 200 microM, and 5 mM, respectively. Conversely, UDCA, TUDCA, and GUDCA presented the lowest affinity for Ca2+ and had no effect on Ca2+ efflux. The 28% increase in Ca2+ release induced by TLCA alone was further augmented to approximately 60% when TLCA was combined with UDCA, TUDCA, or GUDCA. However, Ca2+ efflux induced by NaF was not further increased by UDCA and its conjugates.(ABSTRACT TRUNCATED AT 250 WORDS)

The uptake of ursodeoxycholic acid (UDCA) was studied in isolated hamster hepatocytes. The uptake was rapid and linear up to 60 seconds for each concentration studied. When the uptake rate was plotted against UDCA concentration, the curve was nonlinear, indicating both saturable and nonsaturable uptake mechanisms. The nonsaturable process had a diffusion constant of 0.01 nmol.s-1.g of cell.mumol/L-1. The saturable component was characterized by a maximum rate of uptake (Vmax) of 5.68 nmol.s-1.g of cell-1 and a Michaelis constant (Km) of 224 mumol/L. In the presence of monensin, ouabain, and amiloride, the uptake of UDCA was significantly decreased by 35% to 55%, whereas the sodium-independent uptake of UDCA was not affected by either monensin or amiloride, thereby confirming sodium dependence of UDCA uptake. The sodium-dependent uptake of UDCA was characterized by a Vmax and a Km of 1.57 nmol.s-1.g of cell-1 and 46 mumol/L, respectively. The rate of uptake of UDCA was maximal at extracellular sodium concentrations > or = 20 mmol/L. Furthermore, the uptake of UDCA was competitively inhibited by both taurocholic acid and cholic acid with an inhibitory constant (Ki) of 60 mumol/L and 48 mumol/L, respectively. Finally, 1 mmol/L of 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS) inhibited solely the sodium-dependent uptake of cholic acid and UDCA. These findings confirm that the hepatocellular uptake of UDCA involves, at least in part, a sodium-dependent, ouabain, amiloride, and DIDS-sensitive transporter.

In isolated hamster hepatocytes, the Ca2+ ionophore A-23187 immediately decreased the uptake rate of taurocholic acid (TCA) by 60-70%, whereas it slowly inhibited that of ursodeoxycholic acid (UDCA) by a maximum of 35-45%, with an inhibition constant (Ki) of 0.36 and 1.93 microM, respectively. In contrast to ionomycin, which mimicked the effect of A-23187, vasopressin inhibited the bile acid uptake rate by 40 and 45%, respectively, only after a 5- to 10-min preincubation. The Na(+)-dependent bile acid transport was exclusively inhibited by these agents, and this inhibition was independent of extracellular Ca2+. However, intracellular Ca2+ depletion with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid or chelation with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid resulted in 40-50% inhibition of the uptake rate of both bile acids. The exogenous protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), but not the nonactive 4 alpha-phorbol, significantly inhibited TCA uptake rate. Although both A-23187 and ionomycin immediately increased and decreased the cellular Na+ and K+ concentration, respectively, neither vasopressin nor PMA had a significant effect on the cellular concentration of these cations, even after a 10-min incubation. Furthermore, the effect of A-23187 and ionomycin on TCA uptake and Na+ flux, respectively, disappeared after a 40-min preincubation, and additional ionophore remained without effect. However, after a 40-min incubation with A-23187, PMA was still able to inhibit TCA uptake. Therefore, A-23187 and ionomycin transiently inhibited Na(+)-dependent uptake of both TCA and UDCA, in part because of transient alteration of the cellular Na+ and K+ concentration. Vasopressin and PMA inhibited Na(+)-dependent bile acid uptake, at least in part, through protein kinase C activation.