Examination of bile acid negative feedback regulation in rats

Recent data obtained using cultured rat hepatocytes showed that bile acids do not inhibit bile acid synthesis, whereas cholesterol concentrations vary in parallel with bile acid synthesis (Davis et al. (1983. J. Biol. Chem. 258: 4079-4082). This led us to re-evaluate in vivo experiments upon which the consensus that bile acid synthesis is primarily regulated by bile acid "negative feedback" is based. Infusion of taurocholate into either the jugular vein or duodenum of bile-diverted rats stimulated biliary cholesterol secretion and bile flow, but it did not inhibit bile acid synthesis. The lack of an inhibitory effect was evident using several different infusion rates of taurocholate. Even at the greatest rate of taurocholate infusion (25 mumol/(100 g.hr] there was no significant inhibition of bile acid synthesis. In contrast, infusing mevinolin (1 mg/hr), a potent competitive inhibitor of HMG-CoA reductase, almost completely inhibited bile acid synthesis and biliary cholesterol secretion. Since mevinolin did not affect bile flow, these results cannot be ascribed to bile secretory failure. Thus, while these studies suggest that taurocholate may not regulate bile acid synthesis directly via negative feedback, cholesterol is likely to act as a positive effector of bile acid synthesis.     

Taurocholate is more potent than cholate in suppression of bile salt synthesis in the rat

Synthesis of bile salts is regulated through negative feedback inhibition by bile salts returning to the liver. Individual bile salts have not been distinguished with regard to inhibitory potential. We assessed inhibition of bile salt synthesis by either cholate or its taurine conjugate in bile fistula rats. After allowing synthesis to maximize, baseline synthesis was determined by measuring bile salt output in four consecutive 6-hr periods. Next, sodium cholate (+[(14)C]cholate) or taurocholate (+[(14)C]taurocholate) was infused into the jugular vein for 36 hr and bile was collected in 6-hr aliquots. Hepatic flux of exogenous bile salt was determined by measuring output of radioactivity in bile divided by specific activity of the infusate. Synthesis was determined during the last four 6-hr periods of infusion by subtracting exogenous bile salt secretion from the total bile salt output. Thirteen studies using cholate and 13 using taurocholate were performed. Hepatic flux of infused bile salt varied from 1 to 12 micro mol/100 g per rat per hr. Percent suppression of synthesis varied directly with hepatic flux of exogenous bile salt for both cholate and taurocholate in a linear fashion (r = 0.66, P < 0.01 and r = 0.87, P < 0.0005, respectively). Slope of the taurocholate line was 7.82 (% suppression/ micro mol per 100 g per hr), while slope of the cholate line was 3.66 (P < 0.05), indicating that taurocholate was approximately twice as potent as cholate in suppression of synthesis. At fluxes of 10-12 micro mol/100 g per hr, taurocholate suppressed synthesis 84 +/- 8 (SEM) % while cholate suppressed synthesis only 42 +/- 12% (P < 0.02). The x-intercept of the taurocholate line was 0.65 ( micro mol/100 g per hr), while that of the cholate line was -1.01 (NS) suggesting that the threshold for initial suppression of synthesis did not differ for these two bile salts. We conclude that taurocholate is a more effective inhibitor of hepatic bile salt synthesis than cholate, and that intestinal deconjugation of bile salts may play a role in the regulation of synthesis.-Pries, J. M., A. Gustafson, D. Wiegand, and W. C. Duane. Taurocholate is more potent than cholate in suppression of bile salt synthesis in the rat.     

Bile secretory characteristics of beta-muricholate and its taurine conjugate are similar to those of ursodeoxycholate in the rat

Ursodeoxycholate (UDC) has very high biliary transport maxima values (Tm) for its conjugates as well as the capability of inducing choleresis rich in bicarbonate concentration in the bile in rats. We examined in the present study whether these properties are shared by beta-muricholate (beta-MC), using beta-MC, alpha-muricholate (alpha-MC) and tauro-beta-MC (T beta-MC) in the rat. Bile samples were collected every 20 min for 2 hr in male rats under the infusion of alpha- or beta-MC (1.2 mumol/min/100g). The choleretic response was quicker in beta-MC infused rats than in rats infused with alpha-MC. Bile salt excretion rates increased radically in both experiments. However, in beta-MC infused rats, the bile salt excretion rate began to decrease after 40 min, whereas in alpha-MC infused rats, it continued to increase after 1 hr. Bile bicarbonate concentration significantly increased in beta-MC infused rats but not in alpha-MC infused rats. The Tm of T beta-MC was 2 times higher than the Tm value for taurocholate and was comparable to that of tauroursodeoxycholate (TUDC) which was previously found by the authors. The bile flow (Y, microliter/min/100 g) was significantly correlated with the bile salt excretion rate (X, mumol/min/100 g) [Y = (6.90 +/- 0.24) X + (5.5 + 1.06), n = 41, -0.98, P less than 0.01)], the slope value being higher than that found for TUDC. The results suggest that UDC and beta-MC (and their conjugates) have very similar bile secretory characteristics and may probably share the same transport system in the rat.     

Determinants of biliary secretory pressure: the effects of two different bile acids

Biliary secretory pressure represents the force generated to deliver bile through the biliary system. Bile acid-induced toxicity may decrease canalicular bile formation and (or) induce back diffusion causing cholestasis. To determine if biliary secretory pressure is a sensitive indicator of bile toxicity, taurocholate was compared with a less cytotoxic bile acid, tauroursodeoxycholate. In fasted male Sprague-Dawley rats, the common bile duct was cannulated and the endogenous bile salt pool was removed by enteroclysis. Taurocholate (n = 35) or tauroursodeoxycholate (n = 35) in saline was infused for 1 h. Maximal biliary secretory pressure was then measured by attaching the biliary cannula to a column monometer and recording the maximum height to which bile rose. With taurocholate administration, bile flow and bile salt secretion linearly rose to a maximum infusion of 0.5 mumol/(min.g liver), above which hemolysis and death occurred. In contrast, tauroursodeoxycholate could be infused at higher rates with bile salt secretion plateauing at 1.25 mumol/(min.g liver] Both had similar choleretic potencies. Mean biliary secretory pressure at low (less than 0.15 mumol/(min.g liver] infusions was lower with taurocholate (22.5 cm bile) than tauroursodeoxycholate (25.2 cm). Further, increasing the taurocholate infusion decreased the biliary secretory pressure; yet for taurousodeoxycholate, pressure remained unchanged even at higher infusions. Thus, taurocholate but not tauroursodeoxycholate decreases biliary secretory pressure at high infusion rates, likely a reflection of its toxicity to the hepatobiliary epithelium.     

Feedback regulation of bile acid biosynthesis in the rat

The hepatic biosynthesis of bile salts in the rat has been shown to be controlled homeostatically by the quantity of bile salt returning to the liver via the portal circulation. The feedback mechanism was demonstrated in two kinds of experiments. In the first, rats with bile fistulas were infused intraduodenally with sodium taurocholate 12 hr after surgery. If the rate of infusion was greater than 10 mg per 100 g rat per hr, the increase in bile acid output normally observed in bile fistula rats was prevented. In the second type of experiment, the rats were infused with taurocholate 48-72 hr after biliary diversion, when bile acid output had reached a maximal value. Provided the rate of infusion exceeded 10 mg per 100 g rat per hr, bile acid secretion returned to the low levels observed in intact rats. Previous attempts to demonstrate the feedback control have been unsuccessful because too little bile salt was infused. The taurocholate pool of the experimental animals was measured as approximately 15 mg per 100 g rat; it was calculated from this and the above results that this pool circulated 10-13 times daily.     

The bile flow and biliary excretion of ursocholate in the rat

Several studies reported that ursodeoxycholate (but not its conjugates), when administered intravenously, increased the biliary bicarbonate concentration in the rat (1–3). At the same time, a complete dissociation between bile flow and the bile salt excretion rate was produced in the second hr of infusion (2). In order to examine whether this property was due to the 7β-hydroxy group in its molecular structure, the choleretic property of ursocholate (3α, 7β, 12α-trihydroxy-5β-cholanoic acid) was investigated in male Wistar rats. Immediately after the start of iv infusion of ursocholate at a rate of 1.2 μmole/min/100 g b. wt., both the bile flow and bile salt excretion rate began to increase. However, unlike with ursodeoxycholate, the bile salt excretion rate continued to be high in the second and third hr of infusion, while the bile flow rate gradually increased. Furthermore, the bicarbonate concentration in the bile fell slightly 10 min after the start of ursocholate infusion. Although the concentration tended to return to the baseline value before the bile salt infusion in the later period of observation, no significant increase in bicarbonate concentration was observed during the whole observation period. These properties were quite similar to those of cholate rather than those of ursodeoxycholate. However, a cholate infusion at the same rate of 1.2 μmole/min/100 g b.wt. caused a cholestasis as early as 20 to 30 min after the start of an infusion. These results suggest that the previously reported properties of ursodeoxycholate (that it causes a complete dissociation between the bile flow and bile salt excretion rate in the second hr and that it increases the biliary bicarbonate concentration) were not due to the 7β-hydroxy group in its steroidal structure, and that the choleretic property of ursocholate is similar to its 7α-hydroxy epimar, cholate. However, the much lower cytotoxicity of ursocholate compared to cholate appears to be due to the 7β-hydroxy group that ursocholate has.     

Role of the hepatocyte microtubular system in the excretion of bile salts and biliary lipid: implications for intracellular vesicular transport

The role of the hepatocyte microtubular system in the transport and excretion of bile salts and biliary lipid has not been defined. In this study the effects of microtubule inhibition on biliary excretion of micelle- and non-micelle-forming bile salts and associated lipid were examined in rats. Low-dose colchicine pretreatment had no effect on the baseline excretion of biliary bile salts and phospholipid in animals studied 1 hr after surgery (basal animals), but slightly retarded the excretion of tracer [14C]taurocholate relative to that of lumicolchicine-pretreated (control) rats. However, colchicine pretreatment resulted in a marked reduction in the excretion of 2 mumol/100 g doses of a series of four micelle-forming bile salts of differing hydrophilicity, but had no significant effect on the excretion of the non-micelle-forming bile salt, taurodehydrocholate. Continuous infusion of 0.2 mumol of taurocholate/(100 g.min) following 24 hr of biliary drainage (depleted/reinfused animals) resulted in physiologic bile flow with biliary excretion rates of bile salts, phospholipid, and cholesterol that were markedly inhibited (mean 33, 39, and 42%, respectively) by colchicine or vinblastine pretreatment. Excretion of tracer [14C]taurocholate also was markedly delayed by colchicine in these bile salt-depleted/reinfused animals. In contrast, colchicine did not inhibit bile salt excretion in response to reinfusion of taurodehydrocholate. Thus, under basal conditions, the microtubular system appears to play a minor role in hepatic transport and excretion of bile salts and biliary lipid. However, biliary excretion of micelle-forming bile salts and associated phospholipid and cholesterol becomes increasingly dependent on microtubular integrity as the transcellular flux and biliary excretion of bile salts increases, in both bile salt-depleted and basal animals. We postulate that cotransport of micelle-forming bile salts and lipids destined for biliary excretion, via an intracellular vesicular pathway, forms the basis for this microtubule dependence.     

Apolipoprotein B synthesis in rat small intestine: regulation by dietary triglyceride and biliary lipid

Apolipoprotein B (apoB) synthesis rates have been determined, in vivo, in rat enterocytes. Following intralumenal administration of a pulse of [3H]leucine, newly synthesized apoB was quantitated by specific immunoprecipitation and compared to [3H]leucine incorporation into total, trichloroacetic acid-insoluble protein. ApoB synthesis rates were determined after acute administration of either 0.1 or 1 g of triglyceride to fasting animals. No differences were found at any time from 90 min to 6 hr after challenge and values were not different from the basal values established in fasted controls. Animals rechallenged with triglyceride after 8 days' intake of fat-free chow also failed to demonstrate a change in intestinal apoB synthesis rate. By contrast, enterocyte content of apoB appeared to fall, temporarily, with the onset of active triglyceride flux. Groups of animals were then subjected to external bile diversion for 48 hr, a maneuver designed to remove all lumenal sources of lipid. Jejunal apoB synthesis rates fell by 43% (from 0.76% +/- 0.14 to 0.43% +/- 0.12, P less than 0.001), a change that was completely prevented by continuous replacement with 10 mM Na taurocholate. The suppression of jejunal apoB synthesis, induced by prolonged bile diversion, was reversed after 14 hr, but not 8 hr, of intralumenal perfusion with 10 mM Na taurocholate. The addition of micellar fatty acid-monoolein to the perfusate for 4 hr produced no further change in apoB synthesis. Ileal apoB synthesis rates fell by 70% (from 0.61% +/- 0.15 to 0.18% +/- 0.10, P less than 0.001) following 48 hr external bile diversion, a change that was only partially prevented by continuous bile salt replacement. These results suggest that jejunal apoB synthesis demonstrates bile salt dependence but not regulation by acute triglyceride flux. The data further suggest that key aspects of the regulation of apoB synthesis by cellular lipid flux may be mediated independently in jejunal and ileal enterocytes.     

Effect of ursodeoxycholate on the bile flow in the rat

The relationship between the bile flow and biliary excretion rate of bile salt was studied by a continuous infusion of ursodeoxycholate and its glycine conjugate in rats. Infusion of glycoursodeoxycholate produced a higher flow rate and higher bile salt concentration than previously reported values for taurocholate. The estimated biliary transport maximum value was 2.21±0.15 μmole/min/100g body weight (mean±SD, N=13). Furthermore, a linear relation was found between the bile flow and bile salt excretion rate for a wide range of bile salt excretion with a slope value of 4.10±0.64 μl/μmole (N=10). These values were close to values previously reported for tauroursodeoxycholate. In contrast, when free ursodeoxycholate was infused, a bile salt excretion rate increased at first to a level of around 1.0 μmole/min/100g body weight with a concomitant bile flow increase, but after one hr, the bile salt excretion dropped sharply and a lower plateau of about half of the initial maximum level was established in the following hr. On the other hand, the bile flow further increased even in the second hr. Consequently, the linear relationship initially observed between the bile flow and bile salt excretion rate became gradually distorted and after one hr even the positive correlation between the two parameters was completely lost. The sharp drop in the bile salt excretion rate was found to be due to the decrease in the taurine conjugate of ursodeoxycholate in the bile. The excretion rate of free ursodeoxycholate remained at a very low level (about 0.1 μmole/min/100g body weight) throughout the experiments. The concentration of ursodeoxycholate in the liver increased sharply in the second hr corresponding to the decrease in the bile salt excretion rate. These results appear to be most easily explained by the thesis that there is a fraction of bile independent of bile salt excretion but dependent on the bile salt concentration in the hepatocyte.     

The effect of oleate on pancreatic and bile secretion in the conscious rat

The effects of sodium oleate infused into either the duodenum or the terminal ileum on bile and pancreatic secretion were examined in the conscious rat. Rats were prepared with cannulae draining pure bile and pancreatic juice separately, and with an ileal and two duodenal cannulae. A 40 mM taurocholate solution containing 7 mg/ml bovine trypsin was infused into the duodenum throughout the experiment to replace diverted bile-pancreatic juice to maintain the normal regulation of pancreatic secretion. The intraduodenal infusion of sodium oleate significantly increased pancreatic juice flow, protein, and bicarbonate outputs, whereas it did not affect bile secretion. Intravenous infusion of proglumide (300 mg/kg/hr) did not inhibit pancreatic secretion stimulated by intraduodenal infusion of sodium oleate. An intravenous infusion of atropine (100 micrograms/kg/hr) attenuated protein and fluid secretions but not that of bicarbonate in response to intraduodenal oleate. In contrast, the intraileal infusion of oleate had no effect on pancreatic secretion, whereas it decreased bile flow, bicarbonate, and bile salt outputs. In conclusion, sodium oleate introduced in the duodenum stimulates pancreatic secretion but oleate in the terminal ileum inhibits bile secretion.     

Biochemical site of regulation of bile acid biosynthesis in the rat

The production of bile salts by rat liver is regulated by a feedback mechanism, but it is not known which enzyme controls endogenous bile acid synthesis. In order to demonstrate the biochemical site of this control mechanism, bile fistula rats were infused intravenously with (14)C-labeled bile acid precursors, and bile acid biosynthesis was inhibited as required by intraduodenal infusion of sodium taurocholate. The infusion of taurocholate (11-14 mg/100 g of rat per hr) inhibited the incorporation of acetate-1-(14)C, mevalonolactone-2-(14)C, and cholesterol-4-(14)C into bile acids by approximately 90%. In contrast, the incorporation of 7alpha-hydroxycholesterol-4-(14)C into bile acids was reduced by less than 10% during taurocholate infusion. These results indicate that the regulation of bile acid biosynthesis is exerted via cholesterol 7alpha-hydroxylase provided that hepatic cholesterol synthesis is adequate.     

Pancreatic cholesterol esterases. 3. Kinetic characterization of cholesterol ester resynthesis by the pancreatic cholesterol esterases

The ability of cholesterol esterase to catalyze the synthesis of cholesterol esters has been considered to be of limited physiological significance because of its bile salt requirements for activity, though detailed kinetic studies have not been reported. This study was performed to determine the taurocholate, pH, and substrate requirements for optimal cholesterol ester synthesis catalyzed by various pancreatic lipolytic enzymes, including the bovine 67- and 72-kDa cholesterol esterases, human 100-kDa cholesterol esterase, and human 52-kDa triglyceride lipase. In contrast to current beliefs, cholesterol esterase exhibits a bile salt independent as well as a bile salt dependent synthetic pathway. For the bovine pancreatic 67- and 72-kDa cholesterol esterases, the bile salt independent pathway is optimal at pH 6.0-6.5 and is stimulated by micromolar concentrations of taurocholate. For the bile salt dependent synthetic reaction for the 67-kDa enzyme, increasing the taurocholate concentration from 0 to 1.0 mM results in a progressive shift in the pH optimum from pH 6.0-6.5 to pH 4.5 or lower. In contrast, cholesterol ester hydrolysis by the 67-, 72-, and 100-kDa enzymes was characterized by pH optima from 5.5 to 6.5 at all taurocholate concentrations. Optimum hydrolytic activity for these three enzyme forms occurred with 10 mM taurocholate. Since hydrolysis is minimal at low taurocholate concentrations, the rate of synthesis actually exceeds hydrolysis when the taurocholate concentration is less than 1.0 mM. The 52-kDa enzyme exhibits very low cholesterol ester synthetic and hydrolytic activities, and for this enzyme both activities are bile salt independent. Thus, our data show that cholesterol esterase has both bile salt independent and bile salt dependent cholesterol ester synthetic activities and that it may catalyze the net synthesis of cholesterol esters under physiological conditions.     

Ileal absorption of tyrosine-conjugated bile acids in Wistar rats

We recently reported that tyrosine-conjugated bile acids, when injected intravenously into bile-fistula rats, are extracted by the liver and secreted intact into bile with an efficiency similar to that seen for taurocholate. Now the effect of tyrosine and glycyltyrosine conjugation of bile acids on ileal absorption has been studied in Wistar rats. 125I-labelled tyrosine- and glycyltyrosine-conjugated bile acid or [14C]taurocholate was injected in 400 microliters aliquots of physiological saline buffered to pH 7.8 into the ileal lumen of bile-fistula rats. Recovery of bile salts in bile was taken as proof of ileal absorption. In comparison with taurocholate, ileal absorption was about 10% less for cholyltyrosine and chenodeoxycholyltyrosine and about 50% less for deoxycholyltyrosine. Thus, tyrosine-conjugated bile acids are absorbed by the ileum and excreted into bile and may undergo enterohepatic circulation. Low recoveries of deoxycholyltyrosine relative to deoxycholylglycine suggested that side chain structure was important for ileal absorption of 3 alpha,12 alpha-dihydroxy bile acids. Elongation of cholic acid to form cholylglycyltyrosine markedly reduced 90-min cumulative ileal absorption relative to cholyltyrosine. Although initial rates of recovery of cholylglycyltyrosine were comparable to those of the other bile acids, very little further absorption was seen in the last hour of the experiment, suggesting that this compound was rapidly degraded within the intestinal lumen.     

Biliary transport maximum of tauroursodeoxycholate is twice as high as that of taurocholate in the rat

Biliary excretory transport maximum (Tm) and choleretic efficiency were compared for tauroursodeoxycholate and taurocholate in Wistar male rats. Under a continuous iv infusion of bile salts with a stepwise increase in infusion rate, the Tm of tauroursodeoxycholate was found to be two times higher (2.25±0.07 μmole/min/100 g body weight, n=8, mean±SD) than that of taurocholate (0.97±0.05 μmole/min/100 g body weight, n=14, p<0.001). On the other hand, the amount of bile water obligated by the excretion of 1μmole of tauroursodeoxycholate was significantly lower than that of taurocholate. (4.71±0.08 μl/μmole for tauroursodeoxycholate, vs. 9.27±0.76 μl/μmole for taurocholate, mean±SD, p<0.001). It was concluded that tauroursodeoxycholate can be excreted into the bile in male rats twice as efficiently as taurocholate. Furthermore, the higher efficiency in the choleretic property of ursodeoxycholate previously reported by Dumont et al. appears to be specific to free ursodeoxycholate and not to its taurine conjugate used in the present experiment.     

Effect of bile salts on the biliary excretion of glycodihydrofusidate in the rat

The biliary elimination of glycodihydrofusidate (GDHF), a structural analogue of bile salts, was studied in bile fistula rats. GDHF was excreted in bile with a maximal excretory rate (Tm = 0.80 mumol min-1 kg-1) which is much lower than bile salts Tm. The effects of dehydrocholate and taurocholate on GDHF biliary secretion suggest a stimulatory effect of bile salts on canalicular excretion of the drug. (a) When a bolus intravenous injection of 3 mumol of GDHF was followed after 2 min by a continuous dehydrocholate perfusion (10 mumol min-1 kg-1), biliary excretion of GDHF was increased in comparison with control rats. (b) Upon attaining the biliary Tm by continuous perfusion of GDHF at a rate of 1.35 mumol min-1 kg-1, infusion with either taurocholate or dehydrocholate increased its Tm to a similar degree. These results are similar to those previously obtained with the effects of bile salt infusions on the Tm of bromosulfophthalein. They suggest therefore that hepatic transport of GDHF and bile salts occurs by routes which are distinct for canalicular transport in spite of the striking structural similarities between GDHF and bile salts.     

Apolipoprotein A-I synthesis in rat small intestine: regulation by dietary triglyceride and biliary lipid

Techniques were developed to provide direct quantitation of apolipoprotein A-I (apoA-I) synthesis rates in rat small intestine. Following intralumenal administration of a pulse of [3H]leucine, newly synthesized enterocyte apoA-I was quantitated by specific immunoprecipitation and compared to [3H]leucine incorporation into total trichloroacetic acid-precipitable protein. ApoA-I synthesis rates (% total protein) were found to be significantly higher in jejunal enterocytes (1.84 +/- 0.20) compared to ileal enterocytes (0.91 +/- 0.25) from the same, fasting, animals, P less than 0.01. It was found that rats consuming regular (4.5% w/w fat) rodent chow had apoA-I synthesis rates, 30 to 240 min after receiving an intraduodenal bolus of 100 mg of triglyceride, that were indistinguishable from control animals receiving either saline or an isocaloric, but fat-free, enteral preparation. By contrast, animals consuming a fat-free chow for 8 days prior to study had a small but significant response to acute reintroduction of dietary triglyceride. Four hours after 100 mg of triglyceride was administered, jejunal apoA-I synthesis (% total protein) was 1.84 +/- 0.1 compared to 1.37 +/- 0.04 for animals exposed to an isocaloric, fat-free enteral preparation, P less than 0.01. External bile diversion for 48 hr, which effectively removed all lumenal sources of lipid, reduced apoA-I synthesis in jejunal enterocytes but produced no more depression than that found in sham-operated controls infused for 48 hr with dextrosesaline or control animals fasted for 30 hr. By contrast, apoA-I synthesis in ileal enterocytes was reduced significantly by external bile diversion (0.59 +/- 0.20) in comparison to sham-operated controls (1.19 +/- 0.32) P less than 0.01. Continuous infusion of 10 mM Na taurocholate for 48 hr or 10 mM Na taurocholate for 44 hr and 80 mg of micellar lipid for 4 hr produced results similar to those obtained by bile diversion alone (0.56 +/- 0.2 and 0.61 +/- 0.25, respectively) suggesting that bile salt deficiency alone was not responsible for the observed depression in ileal apoA-I synthesis. These results suggest that, under conditions of physiological dietary triglyceride intake, apoA-I synthesis in jejunal enterocytes is not actuely regulated by changes in triglyceride flux. After prolonged dietary triglyceride withdrawal, the reintroduction of fat produces a small, but significant, increase in jejunal apoA-I synthesis. The data further suggest that apoA-I synthesis in jejunal enterocytes is regulated in part by the availability of lumenal lipid, but that the presence of bile does not exert an additional level of control.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of verapamil on glycogenolysis and gluconeogenesis in the perfused rat liver

In perfused livers from fed rats, rates of glucose production (glycogenolysis) were 133 +/- 12 mumol/g/hr. Infusion of 2 microM verapamil into these livers decreased the rates of glucose production significantly to 97 +/- 15 mumol/g/hr within 10 min. Conversely, rates of production of lactate plus pyruvate (glycolysis) of 64 +/- 6 mumol/g/hr were not significantly altered by verapamil (60 +/- 3 mumol/g/hr). When 50 microM verapamil was infused, however, rates of both glycogenolysis and glycolysis were diminished to 56 +/- 11 and 43 +/- 5 mumol/g/hr, respectively. In perfused livers from fasted rats, infusion of 20 mM fructose increased the rates of production of glucose (gluconeogenesis) significantly from 11 +/- 7 to 121 +/- 17 mumol/g/hr. These rates reached 138 +/- 7 mumol/g/hr upon the simultaneous infusion of verapamil (2 microM). In these livers, fructose also increased rates of production of lactate from 6 +/- 2 to 132 +/- 11 mumol/g/hr, which were further increased to 143 +/- 8 mumol/g/hr when 2 microM verapamil was infused. The results show that calcium-dependent processes involved in hepatic carbohydrate metabolism respond differently to the calcium channel blocker verapamil. Low concentrations of verapamil inhibited glycogenolysis significantly while having no effect on either glycolysis or gluconeogenesis. These data suggest that these two processes have different sensitivities to changes in intracellular calcium concentrations and/or different sources of regulatory calcium.     

Regulation of cholesterol 7 alpha-hydroxylase by hepatic 7 alpha-hydroxylated bile acid flux and newly synthesized cholesterol supply

We measured hepatic cholesterol 7 alpha-hydroxylase activity, mass, and catalytic efficiency (activity/unit mass) in bile fistula rats infused intraduodenally with taurocholate and its 7 beta-hydroxy epimer, tauroursocholate, with or without mevalonolactone to supply newly synthesized cholesterol. Enzyme activity was measured by an isotope incorporation assay and enzyme mass by densitometric scanning of immunoblots using rabbit anti-rat liver cholesterol 7 alpha-hydroxylase antisera. Cholesterol 7 alpha-hydroxylase activity increased 6-fold, enzyme mass 34%, and catalytic efficiency 5-fold after interruption of the enterohepatic circulation for 48 h. When taurocholate was infused to the bile acid-depleted animals at a rate equivalent to the hepatic bile acid flux (27 mumol/100-g rat/h), cholesterol 7 alpha-hydroxylase activity and enzyme mass declined 60 and 61%, respectively. Tauroursocholate did not significantly decrease cholesterol 7 alpha-hydroxylase activity, mass and catalytic efficiency. The administration of mevalonolactone, which is converted to cholesterol, modestly increased cholesterol 7 alpha-hydroxylase activity and enzyme mass in the bile acid-depleted rats. However, when taurocholate was infused together with mevalonolactone, cholesterol 7 alpha-hydroxylase activity and catalytic efficiency were markedly depressed while enzyme mass did not change as compared with bile acid-depleted rats. These results show that (a) hepatic bile acid depletion increases bile acid synthesis mainly by activating cholesterol 7 alpha-hydroxylase with only a small rise in enzyme mass, (b) replacement with taurocholate for 24 h decreases both cholesterol 7 alpha-hydroxylase activity and mass proportionally, (c) when cholesterol is available (mevalonolactone supplementation), the infusion of taurocholate results in the formation of a catalytically less active cholesterol 7 alpha-hydroxylase, and (d) tauroursocholate, the 7 beta-hydroxy epimer of taurocholate, does not inhibit cholesterol 7 alpha-hydroxylase. Thus, bile acid synthesis is modulated by the catalytic efficiency and mass of cholesterol 7 alpha-hydroxylase. The enterohepatic flux of 7 alpha-hydroxylated bile acids and the formation of hepatic cholesterol apparently control cholesterol 7 alpha-hydroxylase by different mechanisms.     

The utilization of sodium taurocholate in excystation of Cryptosporidium parvum and infection of tissue culture.

The present work deals with optimization of excystation of Cryptosporidium parvum oocysts and the infection process of tissue culture cells by the parasite. It was shown that presence of the bile salt sodium taurocholate in the incubation medium expedited excystation of the tested GCH1 isolate and enhanced it, as compared with bleaching of the oocysts. This bile salt had no effect on the viability of tissue culture cell lines MDBK and HCT-8 at the tested concentration of 0.375% for up to 2 hr of coincubation. Infection studies conducted on tissue culture cells showed higher infection rates in the presence of sodium taurocholate than with bleached oocysts in the absence of this bile salt. It may be concluded that, at least as regards the GCH1 strain of C. parvum, the whole infection process can be performed in the presence of sodium taurocholate, and does not require separation and cleaning of the excysted sporozoites before their application to tissue culture cells.     

Absence of negative feedback control of bile acid biosynthesis in cultured rat hepatocytes

The effect of individual bile acids on bile acid synthesis was studied in primary hepatocyte cultures. Relative rates of bile acid synthesis were measured as the conversion of lipoprotein [4-14C]cholesterol into 4-14C-labeled bile acids. Additions to the culture media of cholate, taurocholate, glycocholate, chenodeoxycholate, taurochenodeoxycholate, glycochenodeoxycholate, deoxycholate, and taurodeoxycholate (10-200 microM) did not inhibit bile acid synthesis. The addition of cholate (100 microM) to the medium raised the intracellular level of cholate 10-fold, documenting effective uptake of added bile acid by cultured hepatocytes. The addition of 200 microM taurocholate to cultured hepatocytes prelabeled with [4-14C]cholesterol did not result in inhibition of bile acid synthesis. Taurocholate (10-200 microM) also failed to inhibit bile acid synthesis in suspensions of freshly isolated hepatocytes after 2, 4, and 6 h of incubation. Surprisingly, the addition of taurocholate and taurochenodeoxycholate (10-200 microM) stimulated taurocholate synthesis from [2-14C]mevalonate-labeled cholesterol (p less than 0.05). Neither taurocholate nor taurochenodeoxycholate directly inhibited cholesterol 7 alpha-hydroxylase activity in the microsomes prepared from cholestyramine-fed rats. By contrast, 7-ketocholesterol and 20 alpha-hydroxycholesterol strongly inhibited cholesterol 7 alpha-hydroxylase activity at low concentrations (10 microM). In conclusion, these data strongly suggest that bile acids, at the level of the hepatocyte, do not directly inhibit bile acid synthesis from exogenous or endogenous cholesterol even at concentrations 3-6-fold higher than those found in rat portal blood.