Sustained upregulation in embryonic spinal neurons of a Kv3.1 potassium channel gene encoding a delayed rectifier current.

Differentiation of electrical excitability entails changes in the currents that generate action potentials in spinal neurons of Xenopus embryos, resulting in reduced calcium entry during impulses generated at later stages of development. A dramatic increase in delayed rectifier current (I(Kv)) during the first day of development plays the major role in this process. Identification of potassium channel genes responsible for the increase in I(Kv) is critical to understanding the molecular mechanisms involved. Several members of the Shaw Kv3 gene subfamily encode delayed rectifier currents, indicating that they could contribute to the upregulation of I(Kv) that reduces the duration of action potentials. We isolated a Xenopus (x) Kv3.1 gene whose expression is restricted to the central nervous system, which is upregulated throughout the period during which I(Kv) develops in vivo. The fraction of neurons in which transcripts of this gene are detected by single-cell RT-PCR increases to 40% with time in culture, paralleling the development of I(Kv) in neurons in vitro. Expression of xKv3.1 mRNA generates a delayed rectifier potassium current in oocytes, suggesting that xKv3. 1 contributes to the maturation of I(Kv) and shortening of the action potential.

Tumor necrosis factor-alpha upregulates angiotensin II type 1 receptors on cardiac fibroblasts.

Angiotensin II (Ang II) plays an important role in post-myocardial infarction (MI) remodeling. Most Ang II effects related to remodeling involve activation of the type 1 receptor (AT(1)). Although the AT(1) receptor is upregulated on cardiac fibroblasts post-MI, little is known about the mechanisms involved in the process. Consequently, we tested whether growth factors known to be present in the remodeling heart increased AT(1) mRNA levels. Using quantitative competitive reverse transcription-polymerase chain reaction, we found that norepinephrine, endothelin, atrial natriuretic peptide, and bradykinin had no significant effect on AT(1) mRNA levels. Ang II, transforming growth factor-beta(1), and basic fibroblast growth factor reduced AT(1) mRNA levels (P<0.02). Tumor necrosis factor-alpha (TNF-alpha), however, produced a marked increase in AT(1) mRNA. After 24 hours of TNF-alpha incubation, AT(1) mRNA increased by 5-fold above control levels (P<0.01). The EC(50) for the TNF-alpha effect was 4.6 ng/mL (0.2 nmol/L). Interleukin (IL)-1beta caused a 2.4-fold increase, whereas IL-2 and IL-6 had no significant effect. Studies of TNF-alpha enhancement of AT(1) mRNA levels demonstrate that the increase was not due to a change in transcript stability. TNF-alpha treatment for 48 hours also resulted in a 3-fold increase in AT(1) surface receptor and a 2-fold increase in Ang II-induced production of inositol phosphates. The present findings provide evidence for TNF-alpha regulation of AT(1) receptor density on cardiac fibroblasts. Because TNF-alpha concentration and AT(1) receptor density increase in the myocardium after MI, these results raise the possibility that TNF-alpha modulates post-MI remodeling by enhancing Ang II effects on cardiac fibroblasts.

Temporal regulation of Shaker- and Shab-like potassium channel gene expression in single embryonic spinal neurons during K+ current development.

A developmental increase in density of delayed rectifier potassium current (IKv) in embryonic Xenopus spinal neurons shortens action potential durations and limits calcium influx governing neuronal differentiation. Although previous work demonstrates that maturation of IKv depends on general mRNA synthesis, it is not known whether increases in K+ channel gene transcripts direct maturation of the current. Accordingly, the developmental appearance of specific Kv potassium channel genes was determined using single-cell reverse transcription-PCR techniques after whole-cell recording of IKv during the period of its development. Detection of a coexpressed housekeeping gene along with the potassium channel gene controlled for successful aspiration of cellular mRNA and allowed scoring of cells in which Kv gene transcripts were not detected. Diverse types of Xenopus spinal neurons exhibit homogeneous development of IKv both in vivo and in culture. In contrast, transcripts of two genes encoding delayed rectifier current, Kv1.1 (Shaker) and Kv2.2 (Shab), are expressed heterogeneously during the period in which the current develops. Kv1.1 mRNA achieves maximal appearance in approximately 30% of cells, while IKv is immature; Kv2.2 mRNA appears later in approximately 60% of mature neurons. Kv1.1 and 2.2 are thus candidates for generation of IKv, and spinal neurons are a heterogeneous population with respect to potassium channel gene expression. Moreover, correlation of gene expression with current properties shows that neurons lacking Kv2.2 have a characteristic voltage dependence of activation of IKv.

VIP modulates neuronal nicotinic acetylcholine receptor function by a cyclic AMP-dependent mechanism.

Neuronal nicotinic ACh receptors (AChRs) mediate synaptic transmission throughout the nervous system, and are regulated by cellular processes and interactions that include second messenger signaling pathways. In the case of chick ciliary ganglion neurons, activation of the cAMP-dependent signaling pathway with cAMP analogs enhances ACh sensitivity in a manner consistent with an increase in the number of functional nicotinic receptors. We have now identified vasoactive intestinal peptide (VIP) as a neuromodulator or "first messenger" in the cAMP-mediated pathway that regulates neuronal AChRs. Using cAMP imaging and biochemical detection assays, we find that bath application of VIP elevates intracellular cAMP in freshly isolated ciliary ganglion neurons within minutes. The VIP treatment also enhances neuronal ACh sensitivity assessed with whole-cell recording. The enhanced ACh sensitivity produced by VIP appears with a short latency, similar to that associated with the increase in cAMP, and is not additive with the enhanced ACh sensitivity produced by bath application of a cAMP analog. In contrast, calcitonin gene-related peptide (CGRP), known to regulate muscle nicotinic AChRs via a cAMP-dependent pathway, has no detectable effect on levels of either cAMP or ACh sensitivity in the neurons. The results indicate that VIP enhances the ACh sensitivity of ciliary ganglion neurons via a cAMP-dependent signaling pathway, presumably by interaction with a specific receptor. Since VIP-like immunoreactivity is present in the presynaptic nerve terminals of avian ciliary ganglia, a VIP-like peptide could modulate AChRs in vivo.

Modulation by albumin of neuronal cholinergic sensitivity.

Bovine serum albumin greatly enhanced the cholinergic response mediated by neuronal nicotinic acetylcholine receptors in chick ciliary ganglion neurons. The enhancement exceeded 5-fold in some experiments (mean +/- standard error, 3.26 +/- 0.43-fold) and was rapid, was dose dependent, and occurred without changes in the unitary conductance or the mean open time of the acetylcholine receptor channel. This lack of detectable change in permeation or kinetic properties suggests that bovine serum albumin might increase acetylcholine responses by increasing the number of functional receptors. The enhancement appears to be specific to the albumin molecule, because activity could not be removed by detergent extraction, gel filtration, or dialysis. Acetylcholine responses in these cells are known to be enhanced by a cAMP-dependent mechanism that converts existing acetylcholine receptors from a nonfunctional to a functional state. We found that the enhancement by bovine serum albumin occurred without an increase in cAMP and that pretreatment with membrane-permeable cAMP analogs prevented any additional enhancement of the cholinergic response by bovine serum albumin. These observations are consistent with a cAMP-dependent modulation of the enhancement produced by bovine serum albumin or a convergence of the two enhancement mechanisms onto a single pathway.

Hypercholeresis induced by unconjugated bile acid infusion correlates with recovery in bile of unconjugated bile acids.

Using the isolated perfused rat and hamster liver, the relationship between bile flow, bile acid secretion rate and bile acid biotransformation after the injection of a small, bolus dose of radioactive ursodeoxycholate or of its C23 homolog, norursodeoxycholate, was examined. Ursodeoxycholate was promptly secreted into bile mostly as amino acid conjugates; less than 3% was secreted in unchanged form in the rat and less than 2% in the hamster. In contrast, norursodeoxycholate was secreted slowly, and biotransformed into glucuronide conjugates and unconjugated trihydroxy derivatives; it was also secreted in part in unchanged form. In the rat, 7% was secreted in unconjugated trihydroxy derivatives and 3% in unchanged form; in the hamster, 7% was secreted as unconjugated trihydroxy derivatives and 4% in unchanged form. The secreted bile acid species that showed the highest correlation with bile flow by far was always the unconjugated form in both rat and hamster. By multiple regression analysis, the apparent choleretic activity (microliters of induced bile flow per micromoles recovered bile acid molecules) indicated marked hypercholeresis for the unconjugated bile acid marked hypercholeresis for the unconjugated bile acid with values ranging from 100 to 300 microliters/mumol. Bile flow also correlated with total bile acid recovery for ursodeoxycholate in rat and norursodeoxycholate in hamster, but in all studies the apparent choleretic activity was far lower. Other calculations indicated that most bile flow during the first 30 min was induced by secretion of the unconjugated bile acid species in all experiments, the proportion ranging from 50% to 90%. The results indicate that when a bolus of ursodeoxycholate or norursodeoxycholate is presented to the perfused rodent liver, the secretion of the unchanged bile acid appears to be responsible for most of the bile flow, probably by a cholehepatic shunting mechanism.

Changes in the number, function, and regulation of nicotinic acetylcholine receptors during neuronal development.

Acetylcholine receptor (AChR) development was examined in neurons freshly isolated from chick ciliary ganglia. Between Embryonic Day 8 (E8) and 16, both the ACh response per unit membrane and the density of surface AChRs increased, while the apparent affinity for ACh decreased. AChRs had single-channel conductances of 25 and 40 pS. The distribution of single-channel events shifted during development; at E8 events of both conductances were equally rare, while by E14 there were many events and most were 40 pS. The open durations of 25 and 40 pS events had two mean values. The open lifetimes of the 25 pS events did not change between E8 and E14, while the lifetimes of the 40 pS events increased, and by E14 most were long. The ACh response of the neurons also became sensitive to regulation by a cAMP-dependent mechanism at about E10. The observed changes may reflect developmental control over processing required for receptor regulation and differential expression of AChR subtypes.

Role of bile acid conjugation in hepatic transport of dihydroxy bile acids.

The effect of conjugation and side chain length on dihydroxy bile acid unidirectional hepatic uptake and efflux was studied using the isolated perfused rat liver which was perfused prograde or retrograde in single pass fashion. Deoxycholic acid (DC) and its C23 (nor) derivative nor-DC, as well as the synthetically prepared taurine conjugate of DC, were administered at a constant dose of 1 mumol/min/kg (body weight), upon which a bolus tracer dose of labeled bile acid was superimposed. Analysis of radioactivity recovery in perfusate indicated that unidirectional uptake of all three bile acids was equally rapid, but that only nor-DC showed considerable and continuing efflux into the perfusate; this involved mostly the unchanged acid. Nor-DC was not amidated but was metabolized to mostly ester glucuronides and hydroxylated derivatives; the biotransformation products did not reflux and were secreted into bile; similarly, DC was amidated with taurine; its taurine conjugate did not efflux and was secreted into bile. When nor-DC-taurine was infused, it did not efflux and was secreted rapidly into bile. When the liver was perfused retrograde fashion to increase concentrations of bile acids pericentral cells, only nor-DC showed efflux, which again involved only the unchanged acid. All bile acids were partly 7 alpha-hydroxylated, the magnitude being greater during retrograde perfusion presumably because slower cellular transport exposed bile acid to hydroxylation enzymes for a longer period. It is concluded that bile acid conjugation, whether by esterification with CoA formation adn subsequent amidation or by esterification with glucuronate, restricts the movement of lipophilic dihydroxy bile acids to the hepatocyte and canalicular lumen.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of Na+/H+ exchange in the rat is associated with decreased ursodeoxycholate hypercholeresis, decreased secretion of unconjugated urodeoxycholate, and increased ursodeoxycholate glucuronidation.

In the perfused rat liver, ursodeoxycholate in high dose produces an HCO3- -rich hypercholeresis which we have shown previously to be inhibited by replacement of perfusate Na+ with Li+ or by addition of amiloride (or amiloride analogues). In the present studies, we have determined whether such inhibition is associated with altered ursodeoxycholate biotransformation. Under control conditions, ursodeoxycholate infusion produced a 3.7-fold increase in bile flow and a 9.2-fold increase in biliary HCO3- output. By thin-layer chromatography, ursodeoxycholate radioactivity in bile was present in unconjugated form (15%) or as glycine or taurine amidates. Glucuronide conjugates of ursodeoxycholate accounted for less than 1% of biliary bile acids. Li+/Na+ substitution decreased ursodeoxycholate-stimulated bile flow and HCO3- secretion by greater than 90%, but decreased recovery of ursodeoxycholate and metabolites by only 25%. Amiloride or amiloride analogues decreased ursodeoxycholate-stimulated choleresis and HCO3- output by 38%-76%, yet did not cause decreased recovery of ursodeoxycholate and metabolites. Inhibition of the hypercholeresis was associated with a decrease in unconjugated ursodeoxycholate to less than 2% of total biliary bile acids, a striking increase in ursodeoxycholate glucuronides, and a reciprocal decrease in glycine and taurine amidates. With Li+/Na+ substitution, the predominant metabolites were a mixture of the 24-ester and the 3-aketal (ethereal) glucuronide (29%), and amidation with glycine appeared to be selectively inhibited; with amiloride or its analogues, only the 3-ethereal glucuronide was formed (20%-60% of biliary bile acids), and both taurine and glycine amidation were inhibited. Thus, maneuvers that decrease Na+/H+ exchange inhibit ursodeoxycholate hypercholeresis and cause replacement of unconjugated ursodeoxycholate in bile by its glucuronide. The secretion of unconjugated ursodeoxycholate, a lipophilic bile acid, appears to be necessary for hypercholeresis induced by high-dose ursodeoxycholate infusion.

Hypercholeresis induced by norchenodeoxycholate in biliary fistula rodent.

The biliary recovery and effect on bile flow and biliary bicarbonate secretion of infused norchenodeoxycholate (nor-CDC), the synthetically prepared C23 homologue of chenodeoxycholate (CDC), were defined in the anesthetized biliary fistula hamster, rat, and guinea pig and compared with those of its taurine conjugate as well as those of the natural C24 bile acid, CDC. In the hamster and rat, nor-CDC was recovered slowly in bile in contrast to its taurine conjugate or CDC. Hepatic biotransformation of nor-CDC was complex. Little amidation with glycine or taurine occurred and the compound was recovered in bile in unchanged form, in the form of hydroxylated derivatives as well as glucuronates and sulfates, the proportion varying in the different species. In contrast, CDC was efficiently amidated with glycine or taurine. The taurine conjugate of nor-CDC was secreted largely unchanged. Nor-CDC infusion caused a striking hypercholeresis in the hamster (108 microliters bile/mumol bile acid in bile) and in the rat (220 microliters/mumol); these values for bile acid-dependent flow far exceed those reported for any other natural bile acid to date in these species. The induced hypercholeresis was of canalicular origin and was accompanied by an enrichment in bicarbonate ion concentration as well as increased bicarbonate output. The taurine conjugate of nor-CDC did not display hypercholeretic properties in the hamster. In the guinea pig, whose native bile is bicarbonate-rich relative to other species, nor-CDC was only mildly hypercholeretic relative to CDC and caused no change in bicarbonate concentration. Thus shortening the side chain of a natural dihydroxy bile acid by a single carbon atom formed a compound that underwent a different hepatic biotransformation than that of most natural bile acids and induced a bicarbonate-rich canalicular choleresis far greater than that which can be explained by current theories of bile formation.

Biliary lipids support serum-free growth of Giardia lamblia.

Giardia lamblia has been grown in vitro only in media containing serum or serum fractions. How this pathogen can grow in the human small intestinal lumen without serum is not known. We found that samples of human hepatic or gall bladder bile maintained G. lamblia survival for 24 to 48 h in medium without serum but did not support growth. By contrast, an artificial biliary lipid dispersion containing six bile salts, phosphatidylcholine (PC), and cholesterol, in the ratios characteristic of human bile, supported parasite growth in medium without serum or serum fractions. To define the requirements, we showed that 1-palmitoyl-2-linoleoyl-PC or 1-palmitoyl-2-oleoyl-PC (which predominate in human bile) satisfied the requirement for PC. Moreover, either glycocholate or glycodeoxycholate could be substituted for the bile salt mixture. The finding that biliary lipids can support serum-free growth of G. lamblia may help explain why this parasite colonizes the upper small intestine.

Effect of side-chain shortening on the physiologic properties of bile acids: hepatic transport and effect on biliary secretion of 23-nor-ursodeoxycholate in rodents.

To define whether side-chain length influences the physiologic properties of bile acids, nor-ursodeoxycholate (nor-UDC), the C23-nor derivative of ursodeoxycholate (UDC), was synthesized in both nonradioactive and radioactive forms (23-14C). Its hepatic translocation, hepatic biotransformation, and effect on bile flow, biliary bicarbonate, and biliary lipid secretion were compared with that of UDC and those of their respective glycine and taurine conjugates in anesthetized biliary fistula hamsters, rats, and guinea pigs, as well as the isolated perfused hamster liver. Hepatic uptake and biliary output of nor-UDC was slower than that of UDC or cholyltaurine in the isolated perfused hamster liver. In biliary fistula animals, nor-UDC was secreted only in bile. Biliary recovery of nor-UDC as compared to that of UDC was prolonged in the rat and hamster, although not in the guinea pig. Hepatic biotransformation, assessed by chromatography of bile, showed that conjugation of nor-UDC was inefficient, as unconjugated nor-UDC was present in bile; there was little amidation with glycine or taurine in any species, but sulfates and glucuronides, as well as other metabolites, were formed, with the pattern of biotransformation varying among species. When infused over a dosage range of 0.2-30 mumol/kg X min, nor-UDC induced a striking choleresis of canalicular origin. The bile acid-dependent flow was increased threefold in hamsters, ninefold in rats, and nearly twofold in guinea pigs when compared to that induced by UDC. The choleresis was associated with a linear increase in bicarbonate output and concentration in bile, and little phospholipid or cholesterol secretion was induced. A competition experiment in the bile fistula hamster indicated that nor-UDC or its metabolites, or both, appeared to compete for canalicular transport of ursocholyltaurine (a cholyltaurine epimer) when the latter was secreted under its Vmax conditions. Conjugates of nor-UDC and UDC were promptly and almost completely recovered in bile without appreciable hepatic biotransformation; the conjugates did not induce a hypercholeresis or increase biliary bicarbonate concentration. It is proposed that a fraction of nor-UDC is secreted into canalicular bile in the unconjugated form and is protonated by a hydrogen ion derived from carbonic acid that was generated by the hydration of luminal CO2 by carbonic anhydrase present in biliary ductular cells. The protonated bile acid is absorbed, thus generating a bicarbonate anion. The bile acid passes through the cholangiocyte, returns to the sinusoids via the periductular capillary plexus, and is resecreted into bile.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of bile acid structure on bile flow and biliary lipid secretion in the hamster.

A comprehensive study of the influence of bile acid structure on bile flow and biliary lipid secretion was carried out by infusing pure bile acids at a physiological rate into the proximal small intestine of a bile fistula hamster. Twelve individual bile acids, cholate (C), ursocholate (UC), chenodeoxycholate (CDC), and ursodeoxycholate (UDC) as their glycine (G), taurine (T), or unconjugated form, were studied so that influence of the hydroxy substituents as well as side-chain structure could be defined. The pattern of bile acid output was dependent on bile acid structure and reflected the site and rate of intestinal absorption. Conjugated bile acid output was delayed because of late ileal absorption, and TUC was poorly absorbed. Unconjugated trihydroxy bile acids, C and UC, also exhibited a delay in absorption, while CDC and UDC were absorbed immediately and achieved the highest bile acid output. Unconjugated bile acids were conjugated initially mostly with taurine and then mostly with glycine. The effect of glycine conjugates of each bile acid on bile flow and biliary lipid secretion was similar to that of their corresponding taurine conjugates. All conjugated bile acids induced a similar rate of bile flow (9-15 microliter bile/mumol bile acid), but unconjugated bile acids other than C induced more flow (20-25 microliter bile/mumol bile acid) than their corresponding conjugates. Conjugates of the dihydroxy bile acids induced a greater secretion of phospholipid and cholesterol than cholyl conjugates, whereas conjugates of UC were unique in inducing extremely low phospholipid and cholesterol secretion. For an increase of 1 mumol X min-1 X kg-1 in bile acid output, the increase in phospholipid secretion was 0.072 mumol X min X kg for GCDC and TCDC; 0.051 mumol X min-1 X kg-1 for GUDC and TUDC; and 0.030 mumol X min-1 X kg-1 for GC and TC. Increase in cholesterol output per mumol X min-1 X kg-1 of bile acid output was 0.013 mumol X min-1 X kg-1 for GCDC and TCDC, 0.011 mumol X min-1 X kg-1 for GUDC and TUDC, and 0.005 mumol X min-1 X kg-1 for GC and TC. In general, unconjugated bile acids induced more phospholipid and cholesterol than their corresponding conjugates; however, the rank-order effect of the steroid nucleus substituents was similar to that observed for the respective conjugates. These results indicate that both nuclear and side-chain structure influence the enterohepatic circulation and biliary secretory properties of bile acids.(ABSTRACT TRUNCATED AT 400 WORDS)

Differing effects of norcholate and cholate on bile flow and biliary lipid secretion in the rat.

The effects of norcholate (a C23 bile acid that differs from cholate in having a side chain containing four rather than five carbon atoms) on bile flow and biliary lipid secretion were compared with those of cholate, using the anesthetized rat with a bile fistula. Norcholate and cholate were infused intravenously over the range of 0.6-6.0 mumol X min-1 X kg-1. Both bile acids were quantitatively secreted into bile; norcholate was secreted predominantly in unconjugated form in contrast to cholate, which was secreted predominantly as its taurine or glycine conjugates. The increase in bile flow per unit increase in bile acid secretion induced by norcholate infusion [17 +/- 3.2 (SD) microliters/mumol, n = 8] was much greater than that induced by cholate infusion (8.6 +/- 0.9 microliters/mumol, n = 9) (P less than 0.001). Both bile acids induced phospholipid and cholesterol secretion. For an increase in bile acid secretion (above control values) of 1 mumol X min-1 X kg-1, the increases in phospholipid secretion [0.052 +/- 0.024 (SD) mumol X min-1 X kg-1, n = 9] and cholesterol secretion (0.0071 +/- 0.0033 mumol X min-1 X kg-1, n = 9) induced by norcholate infusion were much less than those induced by cholate infusion (0.197 +/- 0.05 mumol X min-1 X kg-1, n = 9, and 0.024 +/- 0.011 mumol X min-1 X kg-1, n = 9, respectively; P less than 0.001 for both phospholipid and cholesterol). The strikingly different effects of norcholate on bile flow and biliary lipid secretion were attributed mainly to its possessing a considerably higher critical micellar concentration than cholate.

Enzymatic measurement of choline-containing phospholipids in bile.

An enzymatic method, previously used to determine choline-containing phospholipids in serum, was applied to the estimation of phospholipid concentration in bile. The method combines three enzymatic reactions: a) release of choline by phospholipase D; b) choline oxidation by choline oxidase, a reaction which generates hydrogen peroxide; and c) formation of a red quinone dye by peroxidase. The method is an endpoint, spectrophotometric determination. It is simple, sensitive, and reproducible. Accuracy was demonstrated both by linear recoveries of the choline-containing phospholipids in bile and a good correlation with a chemical determination. No interferences by bile acids and/or cholesterol were observed; bile pigment interference was removed by bleaching with white light. This method which determines lipid choline is simpler than the widely used chemical determination of lipid phosphorus.