Forskolin, phosphodiesterase inhibitors, and cyclic AMP analogs inhibit proliferation of cultured bovine aortic endothelial cells

The role of cyclic AMP on endothelial cell proliferation was investigated, since these cells can be exposed to high concentrations of physiological and pharmacological agents that alter cyclic AMP metabolism. Cloned bovine aortic endothelial cells were plated at 25,000 cells/35mm dish and grown for 5 days in the presence of phosphodiesterase (PDE) inhibitors, forskolin, or cyclic AMP analogs. The PDE inhibitors dipyridamole, ZK 62 711, isobutylmethylxanthine (IBMX) and theophylline inhibited cell growth in a concentration-dependent manner. Dipyridamole produced a 30% and a 50% inhibition at 5 microM and 12.5 microM, while higher concentrations were cytotoxic. At its therapeutic plasma concentration range (50-100 microM) theophylline inhibited cell proliferation by 15-25%, while IBMX and the highly specific cyclic AMP phosphodiesterase inhibitor, ZK 62 711 inhibited growth by 60-80% and 40-50%, respectively. Forskolin (5 microM) increased cyclic AMP levels and cyclic AMP-kinase activity ratios by 2.5-fold and 2-fold. In the absence of PDE inhibitors forskolin produced a 20% growth inhibition at 0.5 microM and a 60% inhibition at 10 microM. The forskolin dose-response curve was not altered by theophylline, but was shifted to the left by approximately 10-fold with dipyridamole and ZK 62 711 and 5-fold with IBMX. Forskolin (5 microM), by itself produced a 1.8-fold increase in cyclic AMP. In the presence of 5 microM theophylline, dipyridamole, IBMX, and ZK 62 711, cyclic AMP was increased by forskolin 2.0, 2.6, 3.5, and 6.6-fold, respectively. 8-Bromo cyclic AMP and dibutyryl cyclic AMP produced a 55% and 60% growth inhibition at 100 microM. The cyclic GMP analogs were less effective inhibitors of growth (15-30%). Our results demonstrate that cyclic AMP analogs and pharmacological agents that elevate intracellular cyclic AMP levels inhibit cell growth and suggest that cyclic AMP may be an important endogenous regulator of endothelial cell proliferation.     

Glycodeoxycholate transport in brush border membrane vesicles isolated from rat jejunum and ileum.

The transport of the bile salt, glycodeoxycholate, was studied in vesicles derived from rat jejunal and ileal brush border membranes using a rapid filtration technique. The uptake was osmotically sensitive, linearly related to membrane protein and resembled D-glucose transport. In ileal, but not jejunal, vesicles glycodeoxycholate uptake showed a transient vesicle/medium ratio greater than 1 in the presence of an initial sodium gradient. The differences between glycodeoxycholate uptake in the presence and absence of a Na+ gradient yielded a saturable transport component. Kinetic analysis revealed a Km value similar to that described previously in everted whole intestinal segments and epithelial cells isolated from the ileum. These findings support the existence of a transport system in the brush border membrane that: (1) reflects kinetics and characteristics of bile salt transport in intact intestinal preparations, and (2) catalyzes the co-transport of Na+ and bile salt across the ileal membrane in a manner analogous to D-glucose transport.     

Characterization, cDNA cloning, and functional expression of mouse ileal sodium-dependent bile acid transporter

Mouse ileal sodium dependent bile acid transporter (ISBT) was characterized using isolated enterocytes. Only enterocytes from the most distal portion showed Na+-dependent [3H]taurocholate uptake. Northern blot analysis using a probe against mouse ISBT revealed the expression of mouse ISBT mRNA to be restricted to the distal ileum. The Km and Vmax for Na+-dependent [3H]taurocholate transport into isolated ileocytes were calculated as 27 microM and 360 pmol/mg protein/min, respectively. Uptake of [3H]taurocholate was inhibited by N-ethylmaleimide. We have cloned ISBT cDNA from mouse ileum. The cDNA included the entire open reading frame coding 348 amino acid protein with seven hydrophobic segments and two N-glycosylation sites. COS-7 cells transfected with the expression vector containing this cDNA expressed Na+-dependent [3H]taurocholate uptake activity with a Km of 34 microM.     

Characterization and chemical modification of the Na(+)-dependent bile-acid transport system in brush-border membrane vesicles from rabbit ileum.

The Na(+)-dependent uptake system for bile acids in the ileum from rabbit small intestine was characterized using brush-border membrane vesicles. The uptake of [3H]taurocholate into vesicles prepared from the terminal ileum showed an overshoot uptake in the presence of an inwardly-directed Na(+)-gradient ([Na+]out > [Na+]in), in contrast to vesicles prepared from the jejunum. The Na(+)-dependent [3H]taurocholate uptake was cis-inhibited by natural bile acid derivatives, whereas cholephilic organic compounds, such as phalloidin, bromosulphophthalein, bilirubin, indocyanine green or DIDS - all interfering with hepatic bile-acid uptake - did not show a significant inhibitory effect. Photoaffinity labeling of ileal membrane vesicles with 3,3-azo- and 7,7-azo-derivatives of taurocholate resulted in specific labeling of a membrane polypeptide with apparent molecular mass 90 kDa. Bile-acid derivatives inhibiting [3H]taurocholate uptake by ileal vesicles also inhibited labeling of the 90 kDa polypeptide, whereas compounds with no inhibitory effect on ileal bile-acid transport failed to show a significant effect on the labeling of the 90 kDa polypeptide. The involvement of functional amino-acid side-chains in Na(+)-dependent taurocholate uptake was investigated by chemical modification of ileal brush-border membrane vesicles with a variety of group-specific agents. It was found that (vicinal) thiol groups and amino groups are involved in active ileal bile-acid uptake, whereas carboxyl- and hydroxyl-containing amino acids, as well as tyrosine, histidine or arginine are not essential for Na(+)-dependent bile-acid transport activity. The irreversible inhibition of [3H]taurocholate transport by DTNB or NBD-chloride could be partially reversed by thiols like 2-mercaptoethanol or DTT. Furthermore, increasing concentrations of taurocholate during chemical modification with NBD-chloride were able to protect the ileal bile-acid transporter from inactivation. These findings suggest that a membrane polypeptide of apparent M(r) 90,000 is a component of the active Na(+)-dependent bile-acid reabsorption system in the terminal ileum from rabbit small intestine. Vicinal thiol groups and amino groups of the transport system are involved in Na(+)-dependent transport activity, whereas other functional amino acids are not essential for transport activity.     

Glycodeoxycholate transport in brush border membrane vesicles isolated from rat jejunum and ileum

The transport of the bile salt, glycodeoxycholate, was studied in vesicles derived from rat jejunal and ileal brush border membranes using a rapid filtration technique. The uptake was osmotically sensitive, linearly related to membrane protein and resembled d-glucose transport. In ileal, but not jejunal, vesicles glycodeoxycholate uptake showed a transient vesicle/medium ratio greater than 1 in the presence of an initial sodium gradient. The differences between glycodeoxycholate uptake in the presence and absence of a Na⁺ gradient yielded a saturable transport component. Kinetic analysis revealed a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value similar to that described previously in everted whole intestinal segments and epithelial cells isolated from the ileum. These findings support the existence of a transport system in the brush border membrane that: (1) reflects kinetics and characteristics of bile salt transport in intact intestinal preparations, and (2) catalyzes the co-transport of Na⁺ and bile salt across the ileal membrane in a manner analogous to d-glucose transport.     

Expression of the hepatocyte Na+/bile acid cotransporter in Xenopus laevis oocytes

The expression of the basolateral Na+/bile acid (taurocholate) cotransport system of rat hepatocytes has been studied in Xenopus laevis oocytes. Injection of rat liver poly(A)+ RNA into the oocytes resulted in the functional expression of Na+ gradient stimulated taurocholate uptake within 3-5 days. This Na(+)-dependent portion of taurocholate uptake exhibited saturation kinetics (apparent Km approximately 91 microM) and could be inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene. Furthermore, the expressed taurocholate transport activity demonstrated similar substrate inhibition and stimulation by low concentrations of bovine serum albumin as the basolateral Na+/bile acid cotransport system previously characterized in intact liver, isolated hepatocytes, and isolated plasma membrane vesicles. Finally, a 1.5- to 3.0-kilobase size-class of mRNA could be identified that was sufficient to express the basolateral Na+/taurocholate uptake system in oocytes. These results demonstrate that "expression cloning" represents a promising approach to ultimately clone the gene and to further characterize the molecular properties of this important hepatocellular membrane transport system.     

Stimulation-Evoked Ca2+ Fluxes in Cultured Bovine Adrenal Chromaffin Cells Are Enhanced by Forskolin

Forskolin, 1 microM, increased acetylcholine (ACh)-stimulated 45Ca uptake by chromaffin cells. The stimulatory effects of forskolin decreased with increasing concentration of ACh. The attenuation of the effect of forskolin on 45Ca uptake as a function of ACh concentration correlated well with changes in the forskolin effect on ACh-evoked catecholamine (CA) release. Forskolin increased excess KCl- and veratrine-evoked CA release and 45Ca uptake. Forskolin by itself stimulated 45Ca efflux and enhanced ACh-, excess KCl-, and veratrine-stimulated 45Ca efflux. High doses of forskolin inhibited both ACh-evoked 45Ca uptake and CA release. The inhibitory action of forskolin was specific to receptor-mediated response because excess KCl- and veratrine-stimulated 45Ca uptake and CA release were not inhibited. Forskolin, 0.3-30 microM, dose-dependently increased caffeine-stimulated CA release and 45Ca efflux in the absence of Ca2+ in the medium, and the effects were mimicked by dibutyryl cyclic AMP. These results suggest that cyclic AMP increases stimulation-induced CA release by enhancing calcium uptake across the plasma membrane and/or altering calcium flux in an intracellular calcium store.     

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.     

Postnatal development of intestinal bile salt transport. Relationship to membrane physico-chemical changes

The postnatal development of intestinal bile salt transport in the rat was examined using the villus technique. Jejunal uptake of taurocholate was linear with respect to incubation concentration at all study ages. Ileal uptake was linear with taurocholate concentration during the first 2 postnatal weeks; a curvilinear relationship indicating the presence of saturable transport appeared during the third week. With the appearance of ileal active transport at age 3 weeks, the Km (app) was constant at 0.49 mM, 0.59 mM, and 0.50 mM in 3-week, 4-week, and adult animals, respectively. The V(app) was 14.65 nmol X mg-1 (dry wt) X min-1 at 3 weeks and declined with age to 11.40 and 10.51 nmol X mg-1 (dry wt) X min-1 in 4-week and adult animals, respectively. The role of physico-chemical changes in the microvillus membrane in the development of ileal active transport was examined. With increasing postnatal age, microvillus membrane cholesterol content rose while the phospholipid content remained unchanged in both ileum and jejunum. Corresponding rises in the cholesterol/phospholipid ratio were observed in both sites. Simultaneously, the microvillus membrane fatty acid composition was changing from predominantly saturated to unsaturated species in both ileum and jejunum. The microvillus membrane fluorescence anisotropy (r) increased with postnatal age in jejunum when measured at 25 degrees C and 37 degrees C and ileum when measured at 25 degrees C; however, no change was noted in ileum when measured at 37 degrees C. Ileal active bile salt transport develops during the third postnatal week, and is associated with concurrent changes in membrane lipid composition and fluidity when measured at 25 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of 3-O-methylglucose transport in human erythrocytes by forskolin

The effect of forskolin, an activator of adenylate cyclase, was investigated on glucose transport in human erythrocytes. Forskolin was found to be a potent inhibitor of 3-O-methylglucose (3-O-MG) influx in human erythrocytes. The inhibition of 3-O-MG transport was instantaneous and reversible. The inhibitory effect of forskolin was concentration-dependent, having an IC50 value of 7.5 microM. Forskolin caused a decrease in Vmax of carrier-mediated 3-O-MG transport from 35.32 to 1.56 mumol/ml of cell X min in the presence of 50 microM forskolin. Inhibition of influx was not reversed at high concentrations of 3-O-MG. In addition, forskolin inhibited the influx of other carbohydrates including galactose, ribose, and fructose. In contrast, forskolin was without effect on adenosine transport. To unravel the underlying mechanism responsible for the inhibitory action of forskolin, the possible involvement of cyclic AMP in controlling glucose transport was examined. Erythrocytes treated with 50 microM forskolin exhibited an increase in cyclic AMP content from the basal levels of 258 fmol/ml of cell to 334 fmol/ml of cell within 10 s after forskolin exposure. However, erythrocytes in which cyclic AMP was allowed to accumulate in excess of 10,000 times the basal level, by means of preincubation with exogenous cyclic AMP, displayed 3-O-MG transport indistinguishable from that of cyclic AMP-poor control cells. In view of the finding that cyclic AMP plays no discernible role in the erythrocyte 3-O-MG transport, it is suggested that the forskolin inhibition is mediated by a mechanism other than by stimulating adenylate cyclase activity. Moreover, forskolin appears to directly inactivate the 3-O-MG transport system since glucose-sensitive cytochalasin B binding to erythrocyte membranes is virtually abolished by 50 microM forskolin.     

Regulation of adenosine 3':5'-monophosphate-dependent protein kinase in cerebral cortex

Alterations in the cyclic AMP-dependent protein kinase activity ratio in response to putative neurotransmitters and other cyclic AMP-elevating agents in intact cerebral cortical slices and Krebs-Ringer particulate preparations from cerebral cortex were examined. Both norepinephrine (30 microM) and forskolin (20 microM) produced a time-dependent increase in intracellular levels of cyclic AMP in cerebral cortical slices which was paralleled by an increase in both cyclic AMP and the protein kinase activity ratio. The increases were maximal at 5 min. and remained elevated for at least 15 min. Forskolin, norepinephrine, adenosine and isoproterenol produced a concentration-dependent increase in both cyclic AMP and the protein kinase activity ratio, however, the degree of increase observed was dissimilar. Thus, a 5-fold change in intracellular cyclic AMP resulted in only a 2-fold increase in the activity ratio. Of the agents examined, forskolin produced the most marked change in the activity ratio (from 0.23 to 0.78 at 100 microM) while isoproterenol at 100 microM produced only a 50% increase in the activity ratio. The half-time for the decline in forskolin elicited elevations of either the activity ratio or cyclic AMP was about 4-6 min. In the presence of the phosphodiesterase inhibitor, Ro 20-1724, both were significantly prolonged being 60-70% of the maximum observed immediately after forskolin stimulation, at 15 min. Potentiation of forskolin elicited increases in the activity ratio by Ro 20-1724 were also observed but the increase in the activity ratio was maximal at 7.5 min. while cyclic AMP accumulations continued to rise during the entire 15 min. incubation. Particulate preparations from cerebral cortex were found to contain a cyclic AMP-dependent protein kinase which could be activated 2 to 3-fold with either forskolin, norepinephrine, or adenosine. Unlike the intact brain slice the changes in protein kinase activity ratio and intracellular levels of cyclic AMP in cell-free particulate preparations were similar in both time and degree.     

Characterization of an organic anion transport system in a placental cell line

Transporters within the placenta play a crucial role in the distribution of nutrients and xenobiotics across the maternal-fetal interface. An organic anion transport system was identified on the apical membrane of the rat placenta cell line HRP-1, a model for the placenta barrier. The apical uptake of 3H-labeled organic anion estrone sulfate in HRP-1 cells was saturable (Km = 4.67 microM), temperature and Na+ dependent, Li+ tolerant, and pH sensitive. The substrate specificity of the transport system includes various steroid sulfates, such as beta-estradiol 3,17-disulfate, 17 beta-estradiol 3-sulfate, and dehydroepiandrosterone 3-sulfate (DHEAS) but does not include taurocholate, p-aminohippuric acid (PAH), and tetraethylammonium. Preincubation of HRP-1 cells with 8-bromo-cAMP (a cAMP analog) and forskolin (an adenylyl cyclase activator) acutely stimulated the apical transport activity. This stimulation was further enhanced in the presence of IBMX (a phosphodiesterase inhibitor). Together these data show that the apical membrane of HRP-1 cells expresses an organic anion transport system that is regulated by cellular cAMP levels. This transport system appears to be different from the known taurocholate-transporting organic anion-transporting polypeptides and PAH-transporting organic anion transporters, both of which also mediate the transport of estrone sulfate and DHEAS.     

Fluorescein-methotrexate transport in dogfish shark (Squalus acanthias) choroid plexus

The vertebrate choroid plexus removes potentially toxic metabolites and xenobiotics from cerebrospinal fluid (CSF) to blood for subsequent excretion in urine and bile. We used confocal microscopy and quantitative image analysis to characterize the mechanisms driving transport of the large organic anion, fluorescein-methotrexate (FL-MTX), from bath (CSF-side) to blood vessels in intact lateral choroid plexus from dogfish shark, Squalus acanthias, an evolutionarily ancient vertebrate. With 2 microM FL-MTX in the bath, steady-state fluorescence in the subepithelium/vascular space exceeded bath levels by 5- to 10-fold, and fluorescence in the epithelial cells was slightly below bath levels. FL-MTX accumulation in both tissue compartments was reduced by NaCN, Na removal, and ouabain, but not by a 10-fold increase in medium K. Certain organic anions, e.g., probenecid, MTX, and taurocholate, reduced FL-MTX accumulation in both tissue compartments; p-aminohippurate and estrone sulfate reduced subepithelial/vascular accumulation, but not cellular accumulation. At low concentrations, digoxin, leukotriene C4, and MK-571 reduced fluorescence in the subepithelium/vascular space while increasing cellular fluorescence, indicating preferential inhibition of efflux over uptake. In the presence of 10 microM digoxin (reduced efflux, enhanced cellular accumulation), cellular FL-MTX accumulation was specific, concentrative, and Na dependent. Thus transepithelial FL-MTX transport involved the following two carrier-mediated steps: electroneutral, Na-dependent uptake at the apical membrane and electroneutral efflux at the basolateral membrane. Finally, FL-MTX accumulation in both tissue compartments was reduced by phorbol ester and increased by forskolin, indicating antagonistic modulation by protein kinase C and protein kinase A.     

Inhibition by forskolin of insulin-stimulated glucose transport in L6 muscle cells.

The cardioactive diterpene forskolin is a known activator of adenylate cyclase, but recently a specific interaction of this compound with the glucose transporter has been identified that results in the inhibition of glucose transport in several human and rat cell types. We have compared the sensitivity of basal and insulin-stimulated hexose transport to inhibition by forskolin in skeletal muscle cells of the L6 line. Forskolin completely inhibited both basal and insulin-stimulated hexose transport when present during the transport assay. The inhibition of basal transport was completely reversible upon removal of the diterpene. In contrast, insulin-stimulated hexose transport did not recover, and basal transport levels were attained instead. This effect of inhibiting (or reversing) the insulin-stimulated fraction of transport is a novel effect of the diterpene. Forskolin treatment also inhibited the stimulated fraction of transport when the stimulus was by 4 beta-phorbol 12,13-dibutyrate, reversing back to basal levels. Half-maximal inhibition of the above-basal insulin-stimulated transport was achieved with 35-50 microM-forskolin, and maximal inhibition with 100 microM. Forskolin did not inhibit 125I-insulin binding under conditions where it caused significant inhibition of insulin-stimulated hexose transport. Forskolin significantly elevated the cyclic AMP levels in the cells; however its inhibitory effect on the above basal, insulin-stimulated fraction of hexose transport was not mediated by cyclic AMP since: (i) 8-bromo cyclic AMP and cholera toxin did not mimic this effect of the diterpene, (ii) significant decreases in cyclic AMP levels caused by 2',3'-dideoxyadenosine in the presence of forskolin did not prevent inhibition of insulin-stimulated hexose transport, (iii) isobutylmethylxanthine did not potentiate forskolin effects on glucose transport but did potentiate the elevation in cyclic AMP, and (iv) 1,9-dideoxyforskolin, which does not activate adenylate cyclase, inhibited hexose transport analogously to forskolin. We conclude that forskolin can selectively inhibit the insulin- and phorbol ester-stimulated fraction of hexose transport under conditions where basal transport is unimpaired. The results are compatible with the suggestions that glucose transporters operating in the stimulated state (insulin or phorbol ester-stimulated) differ in their sensitivity to forskolin from transporters operating in the basal state, or, alternatively, that a forskolin-sensitive signal maintains the stimulated transport rate.     

Weanling, but not adult, rabbit colon absorbs bile acids: flux is linked to expression of putative bile acid transporters

Intestinal handling of bile acids is age dependent; adult, but not newborn, ileum absorbs bile acids, and adult, but not weanling or newborn, distal colon secretes Cl(-) in response to bile acids. Bile acid transport involving the apical Na(+)-dependent bile acid transporter (Asbt) and lipid-binding protein (LBP) is well characterized in the ileum, but little is known about colonic bile acid transport. We investigated colonic bile acid transport and the nature of the underlying transporters and receptors. Colon from adult, weanling, and newborn rabbits was screened by semiquantitative RT-PCR for Asbt, its truncated variant t-Asbt, LBP, multidrug resistance-associated protein 3, organic solute transporter-alpha, and farnesoid X receptor. Asbt and LBP showed maximal expression in weanling and significantly less expression in adult and newborn rabbits. The ileum, but not the colon, expressed t-Asbt. Asbt, LBP, and farnesoid X receptor mRNA expression in weanling colon parallel the profile in adult ileum, a tissue designed for high bile acid absorption. To examine their functional role, transepithelial [(3)H]taurocholate transport was measured in weanling and adult colon and ileum. Under short-circuit conditions, weanling colon and ileum and adult ileum showed net bile acid absorption: 1.23 +/- 0.62, 5.53 +/- 1.20, and 11.41 +/- 3.45 nmol x cm(-2) x h(-1), respectively. However, adult colon secreted bile acids (-1.39 +/- 0.47 nmol x cm(-2) x h(-1)). We demonstrate for the first time that weanling, but not adult, distal colon shows net bile acid absorption. Thus increased expression of Asbt and LBP in weanling colon, which is associated with parallel increases in taurocholate absorption, has relevance in enterohepatic conservation of bile acids when ileal bile acid recycling is not fully developed.     

Alteration of the enterohepatic recirculation of bile acids in rats after exposure to ionizing radiation

The aim of this work was to study acute alterations of the enterohepatic recirculation (EHR) of bile acids 3 days after an 8-Gy radiation exposure in vivo in the rat by a washout technique. Using this technique in association with HPLC analysis, the EHR of the major individual bile acids was determined in control and irradiated animals. Ex vivo ileal taurocholate absorption was also studied in Ussing chambers. Major hepatic enzyme activities involved in bile acid synthesis were also measured. Measurements of bile acid intestinal content and intestinal absorption efficiency calculation from washout showed reduced intestinal absorption with significant differences from one bile acid to another: absorption of taurocholate and tauromuricholate was decreased, whereas absorption of the more hydrophobic taurochenodeoxycholate was increased, suggesting that intestinal passive diffusion was enhanced, whereas ileal active transport might be reduced. Basal hepatic secretion was increased only for taurocholate, in accordance with the marked increase of CYP8B1 activity in the liver. The results are clearly demonstrate that concomitantly with radiation-induced intestinal bile acid malabsorption, hepatic bile acid synthesis and secretion are also changed. A current working model for pathophysiological changes in enterohepatic recycling after irradiation is thus proposed.     

Interactions of cationic bile salt derivatives with the ileal bile salt transport system.

Previous structure-activity studies of the active ileal bile salt transport system have demonstrated that a single negative charge on the side chain is essential for active transport. Furthermore, mutual inhibition studies between different pairs of bile salt substrates indicated that dihydroxy bile salts had a greater apparent affinity for the transport system than the trihydroxylated compounds and triketo bile salts had the least such affinity. In this study, a series of cationic bile salt derivatives (cholamine conjugates) were prepared with one, two, and three alpha-hydroxyl groups on the steroid moiety. Based on the previous observations one would expect (1) no active transport of any of the cholamine conjugates by the ileal transport system; (2) interaction of these compounds with the transport system in such a way as to inhibit the transport of bile salts, with inhibition potency of the transport of any single bile salt inversely related to the number of hydroxyl groups present on the cholamine conjugate; and (3) transport of triketo anionic bile salts to be most readily inhibited, trihydroxy compounds less readily inhibited, and dihydroxy bile salts least inhibited. Using everted gut sac preparations it was demonstrated that all three aforementioned expectations did occur. Furthermore, reversible inhibition of ileal absorption of taurocholate and the bile salt derivative taurodehydrocholate could be demonstrated in vivo. The dihydroxy cholamine conjugates were better inhibitors than the trihydroxy compound. Relative specificity for the bile salt system of these cationic bile salt derivatives was demonstrated in the in vivo preparation by comparing its inhibition of taurodehydrocholate absorption with their lesser capacity to inhibit glucose transport.     

Mechanism of Neurotransmitter Release Elicited by the Preferential α1-Adrenergic Receptor Agonist Phenylephrine in Superfused Superior Cervical Ganglion Cells in Culture

Phenylephrine increased [3H]norepinephrine efflux and accumulation of cyclic AMP in cultured rat superior cervical ganglion cells superfused with Tyrode's solution. The purpose of this study was to determine the mechanism and relationship between these two events. Electrical stimulation (1-2 Hz), potassium chloride (50 mM), and the preferential alpha 1-adrenergic receptor agonist phenylephrine (1-100 microM) increased fractional tritium efflux, whereas methoxamine, cirazoline, and amidephrine were relatively ineffective. Phenylephrine, but not methoxamine and cirazoline, also increased cyclic AMP accumulation. Phenylephrine-induced tritium efflux was not altered by alpha- and beta-adrenergic receptor antagonists or by removal of extracellular calcium. Phenylephrine-induced cyclic AMP accumulation was blocked by the beta-adrenergic receptor antagonists propranolol and atenolol. Forskolin (10 microM) and the nonhydrolyzable cyclic AMP analogue 8-(4-chlorophenylthio)cyclic AMP (100 microM) had minimal effect on tritium efflux. However, phenylephrine-evoked increase in tritium efflux was dose dependently attenuated by the neuronal uptake blocker cocaine, and phenylephrine dose-dependently inhibited the incorporation of [3H]norepinephrine into neuronal stores. We conclude that the increase in tritium efflux induced by phenylephrine is independent of cyclic AMP accumulation and appears to be mediated by uptake of phenylephrine via the neuronal carrier-mediated amine transport process, which in turn promotes efflux of the adrenergic transmitter from its storage sites.     

Hepatocellular uptake of 3H-dihydromicrocystin-LR, a cyclic peptide toxin

The cellular uptake of microcystin-LR, a cyclic heptapeptide hepatotoxin from the cyanobacterium Microcystis aeruginosa, was studied by means of a radiolabelled derivative of the toxin. 3H-dihydromicrocystin-LR. The uptake of 3H-dihydromicrocystin-LR was shown to be specific for freshly isolated rat hepatocytes whereas the uptake in the human hepatocarcinoma cell line Hep G2 as well as the mouse fibroblast cell line NIH-3T3, and the human neuroblastoma cell line SH-SY5Y, was negligible. By means of a surface barostat technique it was shown that the membrane penetrating capacity (surface activity) of microcystin-LR was low, indicating that the toxin requires an active uptake mechanism. The hepatocellular uptake of microcystin-LR could be inhibited in the presence of bile acid transport inhibitors such as antamanide (5 microM), sulfobromophthalein (50 microM) and rifampicin (30 microM). The uptake was also reduced in a concentration dependent manner when the hepatocytes were incubated in the presence the bile salts cholate and taurocholate. A complete inhibition of the hepatocellular uptake was achieved by 100 microM of either bile salt. The overall results indicate that the uptake of microcystin-LR is through the multispecific transport system for bile acids. This mechanism of cell entry would explain the previously observed cell specificity and organotropism of microcystin-LR.