Mohamad Y. El-Mir

Relationship between Bile Acid Transplacental Gradients and Transport across the Fetal-Facing Plasma Membrane of the Human Trophoblast

ABSTRACT: Bile acids and bilirubin are synthesized by the fetal liver very early on during intrauterine life. The main fate of these compounds is to be transferred to the mother. This excretory role of the placenta is primarily determined by the ability of the trophoblast to transport them, which is believed to occur mainly by carrier-mediated processes. The aim of this study was to investigate the role of the cholephilic organic anion exchanger located in the fetal-facing plasma membrane of the human trophoblast in placental “biliary-like‘’ function. No relationship between the magnitude of transplacental gradients for total bile acids and bilirubin was found. However, transport studies, which were carried out by using perified plasma membrane vesicles derived from the fetal-facing pole of the human trophoblast, revealed that [14C]taurocholate transport was affected by both another bile acid (taurochenodeoxycholic acid) and a non-bile acid cholephilic organic anion (bromosulfophthalein). On plotting the ability of different major bile acid species to inhibit radiolabeled taurocholate uptake by these vesicles versus their concentrations in fetal serum or the magnitude of their transplacental gradients, inverse relationships were found. Lower fetal serum concentrations and transplacental gradients were found for bile acid species with higher abilities to affect this transport and presumably to interact with the carrier. By contrast, the magnitude of the transplacental gradient for bile acid species was not correlated with their hydrophobic/hydrophilic balance, as would be expected if diffusion across the lipidic structures of the placental barrier would be the major pathway for the flux of bile acid across this organ. In summary, these results indicate that carriers located in the basal plasma membrane may play an important role in the control of the qualitative and quantitative fetal-maternal bile acid exchange. Moreover, they suggest that although both bile acids and bilirubin may share this pathway for access to the trophoblast, other additional mechanisms are probably responsible in part for the control of the magnitude of their transplacental gradients.

Bile acid secretion during synchronized rat liver regeneration

One major difficulty in interpreting the changes occurring during liver regeneration is the co-existence of non-activated cells and proliferating hepatocytes at all stages of differentiation. The aim of this study was to investigate bile acid (BA) secretion into bile during normal (NLR) and synchronized (SLR) liver regeneration in rats. Regeneration was synchronized by reversible inhibition of ribonucleotide reductase by 10 h treatment with hydroxyurea (HU) shortly after two-third partial hepatectomy. Total BA output as measured by GC-MS increased immediately after partial hepatectomy. This was followed by a further transient enhancement during the next day in the NLR. HU treatment did not significantly modify total BA output, but after releasing synchronized regeneration a marked reduction was observed. This was followed by a recovery to reach values close to those of NLR on day 7 of the regenerative process in SLR. Amidated BA output as measured by HPLC analysis revealed an early enhancement in the proportion of non-conjugated BAs in bile in NLR. However, the profile of conjugated BAs, which was not affected by HU treatment, matched that of total BAs during the first stage of SLR. By contrast, the increase in BA output observed on day 3 of the regenerative process in this group was accounted for by an enhancement in non-conjugated BA secretion. On day 7 of the regenerative process, the proportion of conjugated BA in bile was restored to approximately 100% in this group. Most BA molecules were conjugated with taurine rather than with glycine in all experimental groups, during both NLR and SLR. GC-MS determinations indicated that the magnitude of the cholic acid predominance in all bile samples was significantly modified during liver regeneration. This was increased early after partial hepatectomy and declined toward control values after few (2-3) days. Enhancement in the cholic acid predominance was due to a reduction in the proportion of all other major BAs, above all ursocholic acid and omega-muricholic acid. By contrast, minor BAs in normal control rat bile such as allo-cholic acid were increased during both NLR and SLR, and remained at detectable levels up to day 7. Changes in the proportion of secreted BA species were similar in SLR and NLR except that the early reduction in the proportion of BAs other than cholic acid was more pronounced in SLR and the quantitative importance of the diversity in BA species was recovered earlier in SLR than in NLR. In summary, these results indicate that profound changes in BA secretion during rat liver regeneration do occur. Most of them are probably related to the existence of retro-differentiation/re-differentiation processes which are speeded up by hydroxyurea-induced synchronization of the wave of hepatocyte proliferation.

Sensitivity of bile acid transport by organic anion-transporting polypeptides to intracellular pH.

We investigated the influence of intracellular pH (pHi) on [14C]-glycocholate (GC) uptake by human hepatoblastoma HepG2 cells that express sodium-independent (mainly OATP-A and OATP-8), but not sodium-dependent, GC transporters. Replacement of extracellular sodium by choline (Chol) stimulated GC uptake but did not affect GC efflux from loaded cells. Amiloride or NaCl replacement by tetraethylammonium chloride (TeACl) or sucrose also increased GC uptake. All stimulating circumstances decreased pHi. By contrast, adding to the medium ammonium or imidazole, which increased pHi, had no effect on GC uptake. In Chinese hamster ovary (CHO) cells expressing rat Oatp1, acidification of pHi had the opposite effect on GC uptake, that is, this was reduced. Changes in extracellular pH (pHo) between 7.40 and 7.00 had no effect on GC uptake at pHi 7.30 or 7.45 when pHopHi. Inhibition was not proportional to the pHo-pHi difference. Intracellular acidification decreased V(max), but had no effect on K(m). In sum, sodium-independent GC transport can be affected by intracellular acidification, possibly due both to modifications in the driving forces and to the particular response to protonation of carrier proteins involved in this process.

Influence of hydroxylation and conjugation in cross-inhibition of bile acid transport across the human trophoblast basal membrane.

Taurocholate (TC) transport across the basal plasma membrane of the human trophoblast is a carrier-mediated process, whose specificity is probably not restricted to TC. The aim of this work was to gain further insight into the role of hydroxylation and conjugation in the behavior of the carrier system vs. bile acid (BA) species. Radiolabeled TC transport by basal plasma membrane (BPM) vesicles obtained from human term placenta was measured by a rapid filtration technique. Glycocholate (GC), taurochenodeoxycholate (TCDC) and taurodeoxycholate (TDC) inhibited TC binding to BPM. These bile acids compete with TC for the binding sites. Symmetry properties for GC- and TCDC-induced inhibition of TC transport was found in experiments where GC or TCDC were at the cis-side of the membrane (uptake and efflux experiments). GC and TCDC-induced inhibition seems to be of mixed type. By contrast, TDC was observed to affect TC transport differently, depending on whether the experiments addressed uptake or efflux. At the intracellular side of the membrane (uptake), TDC induced a marked increase in both Vmax and Kt. However, at the fetal side (efflux) a significant reduction in both Vmax and Kt was found. In spite of these peculiarities, the values for Ki were very close for GC, TCDC and TDC at the intracellular side but not at the fetal side, where the decreasing order for Ki was GC > TCDC > TDC. TC uptake by BPM vesicles was not modified in the presence of a wide range of estrone sulfate concentrations (0.002-1.0 mM). In summary, these results indicate that a particular bile acid molecular structure is necessary for steroid-related compounds to interact with the bile acid carrier located in BPM. They also suggest that changes in the number and position of hydroxy groups, as well as in the amino-acid moiety in amidated bile acids modify the behavior of the carrier, which may play an important role in the net vectorial transfer of bile acids across the placenta.

EFFECT OF STREPTOZOTOCIN-INDUCED DIABETES ON SEX DIFFERENCES IN BILIARY LIPID SECRETION IN THE RAT

Diabetes mellitus is often associated with lipid abnormalities that may differ with sex. In this work we studied biliary lipid secretion in male and female anaesthetized Wistar rats (250 g). Diabetes was induced by a single intraperitoneal injection of streptozotocin (6 mg/100 body weight) 6 days before carrying out the studies on bile secretion. Our results confirm the existence of sex differences in bile formation and composition, most of them probably due to a higher (+27%) bile acid output in the female animals. Diabetes induced profound alterations in these sex differences. (a) Bile flow was reduced in both sexes, but more markedly so in female diabetic rats; thus the difference observed in healthy animals was reduced (from 2.22 to 1.58 and from 1.84 to 1.40 microliters/min per g liver in female and male rats, respectively). (b) Bile acid and phosphatidylcholine outputs were increased to a similar extent (bile acid output: from 46.7 to 55.8 nmol/min per g liver, in females and from 36.8 to 50.7 nmol/min per g liver, in males; phosphatidylcholine output: from 3.3 to 13.1 nmol/min per g liver, in females and from 4.5 to 12.5 nmol/min per g liver, in males), and hence the sex differences were abolished. (c) Cholesterol output was increased in both sexes, but this enhancement was significantly higher in female rats (from 0.75 to 1.31 and from 0.65 to 0.89 nmol/min per g liver, in females and males, respectively). (d) The fractional pool of phospholipid species secreted into bile was different in female compared with male rats. The percentage of phosphatidylcholine was higher in female than in male healthy rats. Streptozotocin treatment reversed this proportion, which suggests that changes in the phospholipid composition of the canalicular plasma membrane may play a role in the observed alterations in biliary lipid secretion during diabetes mellitus. Most of the above-described streptozotocin-induced changes were prevented by insulin replacement from the 3rd to the 6th days after streptozotocin injection. In summary, the present study describes alterations in sex differences in biliary lipid secretion of streptozotocin-induced diabetes. These changes are dependent on the insulin deficiency state rather than on a direct hepatotoxicity of the diabetogenic drug.

Effect of insulin and glucose load on bile lactate secretion by the isolated rat liver. Role of hepatic parenchyma heterogeneity.

Parenchymal heterogeneity in lactate disposal by perivenous and periportal hepatocytes is believed to be an important factor affecting the overall lactate metabolism by the liver. The aim of this work was to investigate the possibility of the existence of a different role of both acinar zones 1 and 3 in lactate secretion into bile. The effect of insulin and glucose load was also studied using isolated in situ rat liver preparations. Perfusions with erythrocyte-free Krebs-Henseleit solutions were carried out in intact livers and after restricted damage of zone 1 or zone 3 by digitonin administration as a bolus through the portal or the hepatic vein, respectively. In intact livers lactate concentrations in bile were similar to those found in the perfusate. In both compartments lactate concentrations were observed to increase over 90 min of perfusion. During this time, bile lactate output increased from 5 to 8 nmol/min per g liver with no significant effect on bile flow. Replacement of perfusate by a fresh lactate-free one at 60 min failed to induce any reduction in lactate concentration in bile samples collected during the following 30 min which suggests the absence of easy equilibration of biliary lactate with the sinusoidal compartment. Insulin administration (bolus: 100 mU/100 g body weight, plus portal infusion: 5 mU/min per 100 g body weight) was found to markedly enhance bile lactate concentrations (+110%) and output (+139%). On the contrary, glucose load was found to have no effect on lactate output into bile. No significant difference in the increase in bile lactate output was observed during 90 min perfusion with either 0, 5, 10, 15, 25 or 35 mM initial glucose concentrations. After restricted damage of acinar zone 1 or 3, insulin-induced bile lactate secretion was significantly reduced. This effect was not different regardless the damaged acinar zone. In summary these results suggest that insulin plays an important role in the control of the output of lactate into bile and that the existence of acinar heterogeneity in this function seems unlikely. Moreover the quantitative contribution of bile lactate to overall lactate handling by the liver and to bile formation seems very low.