Enterohepatic circulation - Physiological, pharmacokinetic and clinical implications

Enterohepatic recycling occurs by biliary excretion and intestinal reabsorption of a solute, sometimes with hepatic conjugation and intestinal deconjugation. Cycling is often associated with multiple peaks and a longer apparent half-life in a plasma concentration-time profile. Factors affecting biliary excretion include drug characteristics (chemical structure, polarity and molecular size), transport across sinusoidal plasma membrane and canniculae membranes, biotransformation and possible reabsorption from intrahepatic bile ductules. Intestinal reabsorption to complete the enterohepatic cycle may depend on hydrolysis of a drug conjugate by gut bacteria. Bioavailability is also affected by the extent of intestinal absorption, gut-wall P-glycoprotein efflux and gut-wall metabolism. Recently, there has been a considerable increase in our understanding of the role of transporters, of gene expression of intestinal and hepatic enzymes, and of hepatic zonation. Drugs, disease and genetics may result in induced or inhibited activity of transporters and metabolising enzymes. Reduced expression of one transporter, for example hepatic canalicular multidrug resistance-associated protein (MRP) 2, is often associated with enhanced expression of others, for example the usually quiescent basolateral efflux MRP3, to limit hepatic toxicity. In addition, physiologically relevant pharmacokinetic models, which describe enterohepatic recirculation in terms of its determinants (such as sporadic gall bladder emptying), have been developed. In general, enterohepatic recirculation may prolong the pharmacological effect of certain drugs and drug metabolites. Of particular importance is the potential amplifying effect of enterohepatic variability in defining differences in the bioavailability, apparent volume of distribution and clearance of a given compound. Genetic abnormalities, disease states, orally administered adsorbents and certain coadministered drugs all affect enterohepatic recycling.

Age does not influence the bone response to treadmill exercise in female rats

Purpose: Because it is believed that bone may respond to exercise differently at different ages, we compared bone responses in immature and mature rats after 12 wk of treadmill running. Methods: Twenty-two immature (5-wk-old) and 21 mature (17-wk-old) female Sprague Dawley rats were randomized into a running (trained, N = 10 immature, 9 mature) or a control group (controls, N 12 immature, 12 mature) before sacrifice 12 wk later. Rats ran on a treadmill five times per week for 60-70 min at speeds up to 26 m.min(-1). Both at baseline and after intervention, we measured total body, lumbar spine, and proximal femoral bone mineral, as well as total body soft tissue composition using dual-energy x-ray absorptiometry (DXA) in vivo. After sacrificing the animals, we measured dynamic and static histomorphometry and three-point bending strength of the tibia. Results: Running training was associated with greater differences in tibial subperiosteal area, cortical cross-sectional area, peak load, stiffness, and moment of inertia in immature and mature rats (P < 0.05). The trained rats had greater periosteal bone formation rates (P < 0.01) than controls, but there was no difference in tibial trabecular bone histomorphometry. Similar running-related gains were seen in DXA lumbar spine area (P = 0.04) and bone mineral content (BMC; P = 0.03) at both ages. For total body bone area and BMC, the immature trained group increased significantly compared with controls (P < 0.05), whereas the mature trained group gained less than did controls (P < 0.01). Conclusion: In this in vivo model, where a similar physical training program was performed by immature and mature female rats, we demonstrated that both age groups were sensitive to loading and that bone strength gains appeared to result more from changes in bone geometry than from improved material properties.

Sustained high levels of foliar herbivory of the mangrove Rhizophora stylosa by a moth larva Doratifera stenosa (Limacodidae) in north-eastern Australia

The moth larva, Doratifera stenosa (Lepidoptera: Limacodidae), was observed feeding voraciously in great numbers on mature leaves of Rhizophora stylosa in mangroves at Port Curtis in Central Queensland, NE Australia. This behaviour was considered unusual since mangroves, and the Rhizophora species in particular, reportedly harbour few herbivores and have relatively low levels of herbivory, less than 10%. During a two year period (1996-1998), larvae were observed consuming around 30-40% of leaves in the canopy each year, and the mangroves appeared able to sustain these high levels of herbivory. The impact on trees was assessed in conjunction with a study of the herbivore, its behaviour and life history, in an attempt to explain the occurrence. Larvae were 1-2 cm in length, bright green and gregarious, with numerous small, stinging hairs along their upper bodies. Feeding was in small cohort groups of 5-70 individuals that broke up immediately prior to each moult after which they regrouped in much larger numbers of mixed cohorts to form single-file processions across branches, stems and prop roots. In this way, they moved to neighbouring trees with less affected foliage. One of the outstanding characteristics of this herbivore was its ability to desist from killing host trees although it appeared quite capable of doing so had it remained on individual trees. By moving from tree to tree, the herbivore was able to heavily crop Rhizophora foliage in an apparently sustainable manner. These findings demonstrate the role and importance of foliar herbivory in severely affected forests and how such instances best not be ignored or treated as curiosities in future assessments of herbivory and forest turnover in mangrove ecosystems.

Nomenclature of the GLUT/SLC2A family of sugar/polyol transport facilitators

The recent identification of several additional members of the family of sugar transport facilitators (gene symbol SLC2A, protein symbol GLUT) has created a heterogeneous and, in part, confusing nomenclature. Therefore, this letter provides a summary of the family members and suggests a systematic nomenclature for SLC2A and GLUT symbols.

Conformationally constrained macrocycles that mimic tripeptide beta-strands in water and aprotic solvents

The beta-strand conformation is unknown for short peptides in aqueous solution, yet it is a fundamental building block in proteins and the crucial recognition motif for proteolytic enzymes that enable formation and turnover of all proteins. To create a generalized scaffold as a peptidomimetic that is preorganized in a beta-strand, we individually synthesized a series of 15-22-membered macrocyclic analogues of tripeptides and analyzed their structures. Each cycle is highly constrained by two trans amide bonds and a planar aromatic ring with a short nonpeptidic linker between them. A measure of this ring strain is the restricted rotation of the component tyrosinyl aromatic ring (DeltaG(rot) 76.7 kJ mol(-1) (16-membered ring), 46.1 kJ mol(-1) (17-membered ring)) evidenced by variable temperature proton NMR spectra (DMF-d(7), 200-400 K). Unusually large amide coupling constants ((3)J(NH-CHalpha) 9-10 Hz) corresponding to large dihedral angles were detected in both protic and aprotic solvents for these macrocycles, consistent with a high degree of structure in solution. The temperature dependence of all amide NH chemical shifts (Deltadelta/T7-12 ppb/deg) precluded the presence of transannular hydrogen bonds that define alternative turn structures. Whereas similar sized conventional cyclic peptides usually exist in solution as an equilibrium mixture of multiple conformers, these macrocycles adopt a well-defined beta-strand structure even in water as revealed by 2-D NMR spectral data and by a structure calculation for the smallest (15-membered) and most constrained macrocycle. Macrocycles that are sufficiently constrained to exclusively adopt a beta-strand-mimicking structure in water may be useful pre-organized and generic templates for the design of compounds that interfere with beta-strand recognition in biology.

Antagonists of protein kinase C inhibit rat retinal glutamate transport activity in situ

Neuronal and glial high-affinity transporters regulate extracellular glutamate concentration, thereby terminating synaptic transmission and preventing neuronal excitotoxicity. Glutamate transporter activity has been shown to be modulated by protein kinase C (PKC) in cell culture. This is the first study to demonstrate such modulation in situ, by following the fate of the non-metabolisable glutamate transporter substrate, D-aspartate. In the rat retina, pan-isoform PKC inhibition with chelerythrine suppressed glutamate uptake by GLAST (glutamate/aspartate transporter), the dominant excitatory amino acid transporter localized to the glial Muller cells. This effect was mimicked by rottlerin but not by Go6976, suggesting the involvement of the PKCdelta isoform, but not PKCalpha, beta or gamma. Western blotting and immunohistochemical labeling revealed that the suppression of glutamate transport was not due to a change in transporter expression. Inhibition of PKCdelta selectively suppressed GLAST but not neuronal glutamate transporter activity. These data suggest that the targeting of specific glutamate transporters with isoform-specific modulators of PKC activity may have significant implications for the understanding of neurodegenerative conditions arising from compromised glutamate homeostasis, e.g. glaucoma and amyotrophic lateral sclerosis.

Functional significance of a highly conserved glutamate residue of the human noradrenaline transportere

The aim of the study was to investigate the role of glutamate residue 113 in transmembrane domain 2 of the human noradrenaline transporter in determining cell surface expression and functional activity. This residue is absolutely conserved in all members of the Na+- and Cl--dependent transporter family. Mutations to alanine (hE113A), aspartate (hE113D) and glutamine (hE113Q) were achieved by site-directed mutagenesis and the mutants were expressed in transfected COS-7 or HEK-293 cells. Cell surface expression of IIE113A and hE113D, but not hE113Q, was markedly reduced compared with wild type, and functional noradrenaline uptake was detected only for the hE113Q mutant. The pharmacological properties of the hE113Q mutant showed very little change compared with wild type, except for a decrease in V-max values for noradrenaline and dopamine uptake of 2-3-fold. However, the hE113D mutant showed very marked changes in its properties, compared with wild type, with 82-260-fold decreases in the affinities of the substrates, noradrenaline, dopamine and MPP+, and increased Na+ affinity for stimulation of nisoxetine binding. The results of the study show that the size and not the charge of the 113 glutamate residue of the noradrenaline transporter seems to be the most critical factor for maintenance of transporter function and surface expression.

Regulation of the mouse Nas1 promoter by vitamin D and thyroid hormone

The renal sodium-sulfate cotransporter, NaSi-1, a protein implicated to control serum sulfate levels, has been shown to be regulated in vivo by 1,25-dihydroxyvitamin D-3 (1,25-(OH)(2)D-3) and tri-iodothyronine (T-3). Recently, we cloned the mouse NaSi-1 gene (Nas1) and in the present study identified a 1,25-(OH)(2)D-3- and T-3-responsive element located within the Nas1 promoter. Mutational analysis of the Nas1 promoter resulted in identification of a direct repeat 6-type vitamin-D-responsive element (DR6 VDRE) at -525 to -508 and an imperfect inverted repeat 0-type T-3-responsive element (IR0 T3RE) at -436 to -425 which conferred 1,25(OH)(2)D-3 and T3 responsiveness, respectively. In summary, we have identified responsive elements that mediate the enhanced transcription of Nas1 by 1,25-(OH)(2)D-3 and T-3, and these mechanisms may provide important clues to the physiological control of sulfate homeostasis.

Transcriptional regulation of the sodium-sulfate cotransporter NaSi-1 gene

Inorganic sulfate is one of the most abundant anions in mammalian plasma and is essential for proper cell growth and development, as well as detoxification and activation of many biological compounds. To date, little is understood how physiological levels of sulfate are maintained in the body. Our studies, and of others, have identified the NAS(i)-1 protein to be a functional sulfate transporter in the kidney and intestine, and due to this localization, constitutes a strong candidate gene for maintaining body sulfate homeostasis. Several factors, including hormones and metabolic conditions, have been shown to alter NAS(i)-1 mRNA and protein levels in vivo. In this study, we describe the transcriptional regulation of NaSi-1, with a focus on the mouse NaSi-1 gene (Nas1) that was recently cloned in our laboratory. Vitamin D (1,25-(OH)(2)D-3) and thyroid hormone (T-3) led to an increase in Nas1 promoter activity in OK cells. Mutational analysis of the Nas1 promoter resulted in identification of a direct repeat 6-type vitamin-D-responsive element (DR6 VDRE) at -525 to -508 and an imperfect inverted repeat 0-type T-3 responsive element (IRO T3RE) at -426 to -425 which conferred 1,25-(OH)(2)D-3 and T-3 responsiveness respectively. These findings suggest for vitamin D and thyroid hormone regulation of NaSi-1, may provide important clues to the physiological control of sulfate homeostasis.

The lateral line canal sensory organs of the Epaulette Shark (Hemiscyllium ocellatum)

Examination of the lateral line canals in the Epaulette Shark reveals a much more differentiated sensory system than previously reported from any elasmobranch. Two main types of lateral line canals are found. In one type rounded patches of sensory epithelia are separated by elevations of the canal floor. The other type is a straight canal without restrictions and with an almost continuous sensory epithelium. In addition, we found epithelia (type A) with very long apical microvilli on the supporting cells. These microvilli reach beyond the stereovilli of the hair cells. Another type (B) of sensory epithelium has short microvilli on the supporting cells. In this latter type of epithelium the stereovilli of the hair cells are comparatively tall and reach out beyond the supporting cell microvilli. New hair cells are found widely in both types of sensory epithelia. These always occur as single cells, unlike those described in teleost lateral line canal sensory epithelia where new hair cells seem to form in pairs. Dying hair cells are also widespread, indicating a continuous turnover of hair cells.

Localization of taurine transporters, taurine, and H-3 taurine accumulation in the rat retina, pituitary, and brain

The nervous system contains an abundance of taurine, a neuroactive sulfonic acid. Antibodies were generated against two cloned high-affinity taurine transporters, referred to in this study as TAUT-1 and TAUT-2. The distribution of such was compared with the distribution of taurine in the rat brain, pituitary, and retina. The cellular pattern of [H-3] taurine uptake in brain slices, pituitary slices, and retinas was examined by autoradiography. TAUT-2 was predominantly associated with glial cells, including the Bergmann glial cells of the cerebellum and astrocytes in brain areas such as hippocampus. Low-level labeling for TAUT-2 was also observed in some neurones such as CA1 pyramidal cells. TAUT-1 distribution was more limited; in the posterior pituitary TAUT-1 was associated with the pituicytes but was absent from glial cells in the intermediate and anterior lobes. Conversely, in the brain TAUT-1 was associated with cerebellar Purkinje cells and, in the retina, with photoreceptors and bipolar cells. Our data suggest that intracellular taurine levels in glial cells and neurons may be regulated in part by specific high-affinity taurine transporters. The heterogeneous distribution of taurine and its transporters in the brain does not reconcile well with the possibility that taurine acts solely as a ubiquitous osmolyte in nervous tissues. (C) 2002 Wiley-Liss, Inc.

5-Hydroxytryptamine and platelets: uptake and aggregation in hypoxic pulmonary hypertensive rats

Pulmonary hypertension is associated with various alterations in 5-hydroxytryptamine (5-HT) physiology. In this study in platelets from hypoxic pulmonary hypertensive rats (10% O-2; 1 week) and normoxic rats (room air), (i) initial rates of specific [H-3]5-HT uptake were measured and (ii) potentiation of collagen- and ADP-induced aggregation by 5-HT was quantified. The platelet count was almost halved in hypoxic rats. In uptake experiments, there was a decrease in 5-HT uptake in platelets from hypoxic compared with normoxic rats, due to a 36% reduction in the maximal initial rate of uptake. The aggregation experiments showed that 5-HT (1-100 muM) increased the magnitude of responses to collagen and the duration of responses to ADP, but there was no difference between hypoxic and normoxic rats. Abnormalities in platelet function may conceivably lead to increases in plasma 5-HT levels in hypoxic pulmonary hypertension, but are unlikely to aggravate pulmonary thromboembolism. (C) 2002 Elsevier Science B.V. All rights reserved.

Potentiation of 5-hydroxytryptamine (5-HT) responses by a 5-HT uptake inhibitor in pulmonary and systemic vessels: effects of exposing rats to hypoxia

The aim was to determine whether uptake of 5-hydroxytryptamine (5-HT) by the 5-HT transporter (SERT) modulates contractile responses to 5-HT in rat pulmonary arteries and whether this modulation is altered by exposure of rats to chronic hypoxia (10% oxygen; 8 h/day; 5 days). The effects of the SERT inhibitor, citalopram (100 nM), on contractions to 5-HT were determined in isolated ring preparations of pulmonary artery (intralobar and main) and compared with data obtained in systemic arteries. In intralobar pulmonary arteries citalopram produced a potentiation (viz. an increase in potency, pEC(50)) of 5-HT. The potentiation was endothelium-dependent in preparations from normoxic rats but endothelium-independent in preparations from hypoxic rats. In main pulmonary artery endothelium-independent potentiation was seen in preparations from hypoxic rats but no potentiation occurred in preparations from normoxic rats. In systemic arteries, citalopram caused endothelium-independent potentiation in aorta but no potentiation in mesenteric arteries; there were no differences between hypoxic and normoxic rats. It is concluded that SERT can influence the concentration of 5-HT in the vicinity of the vasoconstrictor receptors in pulmonary arteries. The data suggest that in pulmonary arteries from hypoxic rats, unlike normoxic rats, the SERT responsible for this effect is not in the endothelium and, hence, is probably in the smooth muscle. The data are compatible with reports that, in the pulmonary circulation, hypoxia induces/up-regulates SERT, and hence increases 5-HT uptake, in vascular smooth muscle. The findings may have implications in relation to the suggested use of SERT inhibitors in the treatment of pulmonary hypertension.

Increased expression of the SNARE accessory protein Munc18c in lipid-mediated insulin resistance

Fatty acids inhibit insulin-mediated glucose metabolism in skeletal muscle, an effect largely attributed to defects in insulin-mediated glucose transport. Insulin-resistant mice transgenic for the overexpression of lipoprotein lipase (LPL) in skeletal muscle were used to examine the molecular mechanism(s) in more detail. Using DNA gene chip array technology, and confirmation by RT-PCR and Western analysis, increases in the yeast Sec1p homolog Munc18c mRNA and protein were found in the gastrocnemius muscle of transgenic mice, but not other tissues. Munc18c has been previously demonstrated to impair insulin-mediated glucose transport in mammalian cells in vitro. Of interest, stably transfected C2C12 cells overexpressing LPL not only demonstrated increases in Munc18c mRNA and protein but also in transcription rates of the Munc18c gene. jlr To confirm the relevance of fatty acid metabolism and insulin resistance to the expression of Munc18c in vivo, a 2-fold increase in Munc18c protein was demonstrated in mice fed a high-fat diet for 4 weeks. Together, these data are the first to implicate in vivo increases in Munc18c as a potential contributing mechanism to fatty acid-induced insulin resistance.