Transport of arachidonic acid across the neutrophil plasma membrane via a protein-facilitated mechanism

Arachidonic acid is the rate-limiting substrate in the biosynthesis of leukotrienes in activated neutrophils. Liberation of arachidonate from intracellular membranes and uptake of exogenous arachidonate are the two principal mechanisms by which the cell can increase the level of this substrate. We investigated arachidonate uptake and export by using intact polymorphonuclear neutrophils and inside-out plasma membrane vesicles thereof. Here we show that the cellular uptake of arachidonate is energy dependent with an energy of activation (EA) of 10.0 kcal/mol and half-saturated at an arachidonate concentration of 4.8 nmol/mg of cell protein. Protein-facilitated transport of arachidonate across the plasma membrane in either direction is sensitive to proteases, chemical protein modifying reagents, anion transport inhibitors, and, most notably, toward several structurally unrelated leukotriene B4 receptor antagonists with IC50 values in the range of 16-44 microM. The inhibitors did not inhibit the diffusional uptake of methyl arachidonate into neutrophils and inside-out plasma membrane vesicles, indicating that a transport protein is required for the rapid uptake of the free acid but not for the uptake of the ester. Other long-chain fatty acids did compete with the uptake of arachidonate in both assay systems, whereas leukotriene B4 did not. This study documents a novel protein-facilitated transport mechanism for arachidonate in neutrophils, potentially involved in transcellular eicosanoid biosynthesis and sPLA2-mediated arachidonate signaling in neutrophils.     

Ascorbic acid flux across mucosal border of guinea pig and human ileum

The unidirectional influx of L-[14C]ascorbic acid (vitamin C) across the mucosal border of guinea pig and human ileum was determined. Influx follows saturation kinetics, indicating that a carrier mechanism is operative. The maximal influx in guinea pig ileum bathed in Ringer is 140 nmol/cm2-h and the ascorbic acid concentration greater than 6 mM , or when the tissue is bathed in Na-free media, influx is approximately linearly related to the ascorbic acid concentration, and absorption by simple diffusion may predominate. With mucosal ascorbic acid concentration of 0.28 mM, influx is reduced by at least 70% when Na-free media is used; however, influx is not dependent on the intracellular Na concentration. The brush border mechanism appears to be stereospecific and not closely coupled to cellular metabolism. A model of transport is favored that features a carrier-mediated transport mechanism for simultaneous entry of ascorbic acid and Na across the brush border. This model is similar in nature to, but functionally distinct from, the sodium-gradient mechanism postulated to effect sugar and amino acid transport in mammalian ileal mucosa.     

Arachidonic acid activates a proton current in the rat glutamate transporter EAAT4

The excitatory amino acid transporter EAAT4 is expressed predominantly in Purkinje neurons in the rat cerebellum (1-3), and it participates in postsynaptic reuptake of glutamate released at the climbing fiber synapse (4). Transporter-mediated currents in Purkinje neurons are increased more than 3-fold by arachidonic acid, a second messenger that is liberated following depolarization-induced Ca2+ activation of phospholipase A2 (5). In this study we demonstrate that application of arachidonic acid to oocytes expressing rat EAAT4 increased glutamate-induced currents to a similar extent. However, arachidonic acid did not cause an increase in the rate of glutamate transport or in the chloride current associated with glutamate transport but rather activated a proton-selective conductance. These data reveal a novel action of arachidonate on a glutamate transporter and suggest a mechanism by which synaptic activity may decrease intracellular pH in neurons where this transporter is localized.     

Intestinal ascorbic acid transport following diets of high or low ascorbic acid content

Active transport of ascorbic acid in ileum is mediated by a carrier mechanism at the brush border membrane. This mechanism may show compensatory changes in activity in response to alterations of dietary ascorbic acid content. The unidirectional influx of ascorbic acid across the brush border into epithelial cells of guinea pig ileum was determined in vitro. Influx was significantly reduced in scorbutic animals and following 14 or 28 days of high doses (5 or 25 times normal) of ascorbic acid. The transport rate was reduced by intramuscular administration of ascorbic acid, suggesting that the transport mechanism may respond to circulating levels of the vitamin.     

Characterization of a diamine exporter in Chinese hamster ovary cells and identification of specific polyamine substrates

Export of the diamine putrescine was studied using inside-out plasma membrane vesicles prepared from Chinese hamster cells. Putrescine uptake into vesicles was a saturable and an ATP- and antizyme-independent process. Excess amounts of a series of diamines or monoacetyl spermidine, but not monoacetyl putrescine, spermidine, or spermine, inhibited putrescine transport. Putrescine uptake into vesicles prepared at pH 7.4 was suppressed at pH 5, compared with pH 7.4; was stimulated approximately 2.5-fold at pH 7.4 in vesicles prepared at pH 6.25, compared with vesicles prepared at pH 7.4; and was not inhibited by valinomycin in the presence of potassium ions. Reserpine and verapamil blocked [3H]putrescine uptake into inverted vesicles. Verapamil treatment caused an increase in intracellular contents of putrescine, cadaverine, and N8-acetylspermidine, in unstressed proliferating cells, or of N1-acetylspermidine, in cells subjected to heat shock to induce acetylation of spermidine at N1. These data indicate that putrescine export in Chinese hamster cells is mediated by a non-electrogenic antiporter capable of using protons as the counter ion. Physiological substrates for this exporter include putrescine, cadaverine, and monoacetyl spermidine and have the general structure NH3+-(CH2)n-NH2 + R at acidic or neutral pH.     

Effects of vanadyl sulfate on carbohydrate and lipid metabolism in patients with non-insulin-dependent diabetes mellitus

Mechanisms for the cell-free activation of NADPH oxidase by sodium dodecyl sulfate (SDS) and arachidonate were compared in relation to their responsiveness to short chain diacylglycerols. The plasma membrane and cytosol prepared from guinea pig neutrophils were used for the cell-free system. The activation of NADPH oxidase by SDS was enhanced about 5- to 10-fold by 1,2-dioctanoylglycerol (diC8), but not by either 1,2-dihexanoylglycerol (diC6) or 1,2-didecanoylglycerol (diC10). However, none of these diacylglycerols potentiated the NADPH oxidase activation by arachidonate. The maximal extent of activation by the combination of SDS and diC8 was similar to that by arachidonate alone. In the presence of sufficient amounts of diC8 and SDS, GTP gamma S potentiated the activation of NADPH oxidase. The potentiating activity of diC8 was preserved in the membrane fraction, not in the cytosol fraction. These results suggest that arachidonate may possess the functions of both SDS and diC8 in the activation. In addition, diC8 and GTP gamma S seem to independently enhance the NADPH oxidase activation.     

Congenital malaria in a hyperendemic area: a preliminary study

When inverted vesicles prepared from the inner membrane of rat liver mitochondria were incubated with prepro-rat serum albumin, considerable amounts of prepro-albumin and pro-albumin were recovered with the inverted vesicles re-isolated by centrifugation. Pro-albumin was resistant to trypsin, but prepro-albumin was completely digested by trypsin, indicating that prepro-albumin was transported into the vesicles and concomitantly converted to pro-albumin. This transport process required ATP, but not a membrane potential. These results suggest that some export machinery for a protein having an amino acid sequence in its N-terminal portion similar to the signal sequence of secretory protein exists in the inner mitochondrial membrane.     

Arachidonic acid up-regulates and prostaglandin E2 down-regulates the expression of pancreatic-type phospholipase A2 and prostaglandin-endoperoxide synthase 2 in uterine stromal cells

It is well known that arachidonic acid, as a substrate of prostaglandin G/H synthase (PGHS), is converted into prostaglandins of the two-series. In this work, we attempted to determine whether arachidonic acid and prostaglandin E2 might regulate the expression of PGHS and the pancreatic-type phospholipase A2 (PLA2I), which may be involved in the liberation of arachidonic acid from membrane phospholipids. For this purpose, we used the uterine stromal cell line UIII, which produces prostaglandin E2 and expresses both the constitutive and inducible PGHS enzymes (PGHS1 and PGHS2) and PLA2 I. The results show that PGHS1, which is expressed at a high level in UIII cells, was not modified by arachidonic acid. The expression of PGHS2 and PLA2 I was up-regulated by increasing arachidonate concentrations (1-10 microM). The maximal response was obtained at 24 h, reaching a 2.3-fold and 2.6-fold increase for PGHS2 and PLA2 I expression, respectively, compared to the control level. To discriminate between the effect of arachidonic acid and that of prostaglandins, which are highly increased in the presence of exogenous arachidonic acid, we treated the cells with two inhibitors of PGHS activity, aspirin and meclofenamic acid. Both inhibitors failed to suppress the arachidonate-induced increase of PLA2 I and PGHS2 expression and even enhanced it either in the presence or absence of arachidonic acid. In contrast, the addition of prostaglandin E2 to the culture medium decreased the expression of both enzymes in a dose-dependent manner, the maximal response being reached at 1 microM. We conclude that arachidonic acid up-regulates the expression of PLA2 I and PGHS2 in the uterine stromal cells, independently of prostanoids, and that prostaglandin E2 is capable of down-regulating enzyme expression.     

Transport of tricarballylate by intestinal brush-border membrane vesicles from steers

Tricarballylic acid is a non-metabolizable rumen bacterial fermentation product of the naturally occurring tricarboxylic acid trans-aconitic acid. The aim of the present study was to investigate intestinal absorption of tricarballylate using brush-border membrane vesicles (BBMVs) isolated from the proximal jejunum of steers by a Ca2+ precipitation method with subsequent differential centrifugation. Transport of tricarballylate was investigated indirectly (influence of tricarballylate on the uptake of 14C-labelled citrate) as well as directly (uptake of 3H-labelled tricarballylate). Citrate as well as tricarballylate uptake (at a concentration of 0.05 mmol l-1) was strongly stimulated by an inwardly directed initial Na+ gradient. Furthermore, transport of both tricarboxylates under Na+ gradient conditions was clearly enhanced by lowering the extravesicular pH from 7.8 to 5.6. The imposition of an inwardly directed H+ gradient (pH(out)/pH(in) = 5.6/7.8) further enhanced the intravesicular accumulation of citrate as well as of tricarballylate compared with pH(out)/pH(in) = 5.6/5.6. Unequivocal evidence for a common transport site for tricarballylate and citrate was obtained from 'cis-inhibition' and 'trans-stimulation' of Na(+)-dependent citrate uptake by tricarballylate. In further experiments the influence of different substances on the uptake of 3H-labelled tricarballylate was evaluated. Unlabelled tricarballylate, citrate, succinate as well as trans- and cis-aconitate significantly inhibited the accumulation of 3H-labelled tricarballylate by BBMVs. Tricarballylate uptake as a function of the tricarballylate concentration revealed a Na(+)-dependent saturable component (apparent kinetic parameters: maximal transport capacity (Vmax) = 119 pmol (mg protein)-1 (3s)-1; affinity constant (Km) = 0.097 mmol l-1) and a Na(+)-independent diffusional component (diffusion constant: 169 nl (mg protein)-1 (3s)-1). It is concluded that tricarballylate and citrate are transported across the intestinal brush-border membrane by a common, Na(+)-dependent transport mechanism. The stimulatory influence of a low extravesicular pH most probably indicates that the protonated forms of tricarboxylates are better transported than the trivalent species.     

Heat shock proteins and arthritis--new readers start here

We have characterised G protein and fatty acid regulation of the Na+ conductance in purified apical membrane vesicles prepared from late gestation fetal guinea-pig lung. Addition of 100 microM GTP gamma S or beta gamma-methylene-GTP, irreversible G protein activators, stimulated conductive 22Na+ uptake (ratio of experimental to control 1.35 +/- 0.02 and 1.34 +/- 0.05, respectively). Conversely, the addition of GDP beta S, an irreversible G protein inhibitor, reduced conductive 22Na+ uptake from 1.00 (control) to 0.79 +/- 0.04. A range of saturated (myristic, palmitic, stearic), monounsaturated (elaidic, oleic) and polyunsaturated (linoleic, arachidonic) fatty acids all stimulated conductive 22Na+ uptake, by between 1.18 +/- 0.05 to 1.56 +/- 0.13 over the control. Both arachidonic acid and GTP gamma S-dependent stimulation were abolished in the presence of 10 microM amiloride. The non-metabolisable analogue of arachidonic acid, eicosa-5,8,11,14-tetraynoic acid also stimulated conductive 22Na+ uptake. Furthermore, addition of indomethacin and nordihydroguairetic acid, inhibitors of cyclooxygenase and lipoxygenase pathways of arachidonate metabolism respectively, did not affect the arachidonic acid stimulation suggesting a direct effect of fatty acid upon the Na+ channel Since mepacrine (50 microM), a phospholipase A2 inhibitor, did not affect the GTP gamma S-stimulated conductive 22Na+ uptake, and inhibition of G protein turnover by GDP beta S did not attenuate the arachidonic acid response we conclude that these two regulatory pathways modulate alveolar Na+ transport directly and independently of each other.     

Glass fragility and the stability of pharmaceutical preparations--excipient selection

L-lactate transport mechanism across rat jejunal enterocyte was investigated using isolated membrane vesicles. In basolateral membrane vesicles L-lactate uptake is stimulated by an inwardly directed H+ gradient; the effect of the pH difference is drastically reduced by FCCP, pCMBS and phloretin, while furosemide is ineffective. The pH gradient effect is strongly temperature dependent. The initial rate of the proton gradient-induced lactate uptake is saturable with respect to external lactate with a K(m) of 39.2 +/- 4.8 mM and a Jmax of 8.9 +/- 0.7 nmoles mg protein-1 sec-1. A very small conductive pathway for L-lactate is present in basolateral membranes. In brush border membrane vesicles both Na+ and H+ gradients exert a small stimulatory effect on lactate uptake. We conclude that rat jejunal basolateral membrane contains a H(+)-lactate cotransporter, whereas in the apical membrane both H(+)-lactate and Na(+)-lactate cotransporters are present, even if they exhibit a low transport rate.     

Regulation of capacitative Ca2+ influx in human neutrophil granulocytes. Alterations in chronic granulomatous disease

Ca2+ entry through the capacitative (store-regulated) pathway was shown to be inhibited in neutrophil granulocytes by the protein kinase C activator phorbol 12-myristate 13-acetate and the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) by a hitherto unknown mechanism. Measuring both Ca2+ and Mn2+ entry into store-depleted cells we show in the present study that inhibition of the capacitative pathway is absent in various forms of chronic granulomatous disease. To establish the possible relationship between inhibition of the capacitative pathway and ability of O-2 production and consequent membrane depolarization, gradual changes of the membrane potential were evoked in neutrophils of healthy individuals. This was accomplished by pharmacological manipulation of the membrane potential and by variations of the concentration and type of the stimulant. Close relationship was observed between membrane depolarization and inhibition of Mn2+ entry through the capacitative transport route. Our results provide an explanation for the inhibitory action of fMLP and phorbol 12-myristate 13-acetate on capacitative cation influx and reveal that upon physiological stimulation, Ca2+ entry into neutrophils is restricted by the depolarization accompanying O-2 production.     

ATP-dependent transport of reduced glutathione on YCF1, the yeast orthologue of mammalian multidrug resistance associated proteins

The transport systems involved in the export of cellular reduced glutathione (GSH) have not been identified, although recent studies implicate a role for some of the multidrug resistance associated proteins (MRP), including MRP1 and MRP2. The present study examined the hypothesis that the yeast orthologue of MRP, Ycf1p, mediates ATP-dependent GSH transport. [3H]GSH transport was measured in vacuolar membrane vesicles isolated from a control strain of Saccharomyces cerevisiae (DTY165), the isogenic DTY167 strain that lacks a functional Ycf1p, and in DTY167 transformed with a 2-micrometer plasmid vector containing YCF1. GSH transport in control vacuolar membrane vesicles was mediated largely by an ATP-dependent, low affinity pathway (Km = 15 +/- 4 mM). ATP-dependent [3H]GSH transport was cis-inhibited by substrates of the yeast Ycf1p transporter and inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, probenecid, and sulfinpyrazone, inhibitors of MRP1 and MRP2, but was minimally affected by membrane potential or pH gradient uncouplers. In contrast, ATP-dependent GSH transport was not seen in vacuolar membrane vesicles isolated from the DTY167 yeast strain without a functional Ycf1p but was restored to near wild-type levels in the DTY167 strain transformed with YCF1 and expressing the vacuolar Ycf1p transporter. On the other hand, expression and functional activity of a bile acid transporter, Bat1p, and of the V-type ATPase were similar in all three yeast strains. These results provide direct evidence for ATP-dependent low affinity transport of GSH by the yeast Ycf1p transporter. Because of the structural and functional homology between Ycf1p and MRP1 and MRP2, these data support the hypothesis that GSH efflux from mammalian cells is mediated by these membrane proteins.     

Interrelationship between the transport of L-aspartate and potassium ions into the cell (author's transl)

The mechanisms involved in active transport and intracellular accumulation of amino acids have been reviewed. In particular, the frequently observed interrelationship between the transport of acidic amino acids and potassium ions was discussed. Kinetic studies on the uptake of radioactive L-aspartate and K+ in the microorganism Streptomyces hydrogenans were performed. The following results were obtained:1. L-Aspartate was actively transported into the cells. However, only a part of the aspartate taken up from the medium remained in the pool as free amino acid. Within 60 min, up to 35% of the label was incorporated into protein. By thin-layer chromatography of cell extracts several radioactive metabolites of aspartate were detected. 2. Aspartate was transported by a t least two different uptake systems exhibiting moderate specificity. At neutral pH , the amino acid was transported as anion; its uptake was inhibited by L-glutamate as well as by dicarboxylic acids, whereas neutral amino acids did not have a significant effect. 3. The influx of aspartate into K+-rich cells was stimulated specifically by extracellular Rb+ and K+, whereas Ki+ and Na+ inhibited aspartate transport. 4. Kinetic analysis of the aspartate influx showed that extracellular K+ increased the affinity of the transport systems for aspartate by a factor of three. These results suggest that K+ is bound by the aspartate carrier and is cotransported together with the amino acid across the membrane. 5. Kinetic measurements of the uptake of 42K+ revealed that the influx of K+ as well was stimulated by extracellular aspartate. Likewise the rate of 28Mg2+ uptake was increased by aspartate.     

Characterization of L-arginine uptake by plasma membrane vesicles isolated from cultured pulmonary artery endothelial cells

We investigated the mechanisms of [3H]-L-arginine transport via System Y+ using plasma membrane vesicles derived from cultured pulmonary artery endothelial cells. [3H]-L-arginine uptake into plasma membrane vesicles was Na-independent, sensitive to trans-stimulation, unaffected by proton-conducting ionophores, and selectively inhibited by cationic amino acids. Kinetic experiments performed over a wide range of substrate concentrations revealed only one population of L-arginine transporters with Km = 130 microM. To elucidate the driving force for L-arginine transport, we measured [3H]-L-arginine uptake by plasma membrane vesicles at different transmembrane ion gradients. Plasma membrane vesicles accumulated [3H]-L-arginine only when a membrane potential was imposed across the vesicles, and the velocity of uptake was linearly related to the magnitude of the created membrane potential. The presence of potassium ions inside the vesicles was not essential for uptake of L-arginine into vesicles, but it was essential for trans-stimulation of L-arginine transport. [3H]-L-arginine accumulated in plasma membrane vesicles can be released by agents that dissipate transmembrane potassium gradients (e.g. saponin, gramicidin, and nigericin). Diazoxide and pinacidil, activators of K(+)-channels, had no significant effect on [3H]-L-arginine uptake, whereas tetraethylammonium chloride, 4-aminopyridine, and glibenclamide, inhibitors of K(+)-channels, caused decreases in [3H]-L-arginine transport by plasma membrane vesicles. This study demonstrates for the first time a specific role for potassium ions in the mechanism of L-arginine transport, particularly in the phenomenon of trans-stimulation.     

Functional analysis of amino acid residues essential for activity in the Na+/H+ exchanger of fission yeast

We identified amino acid residues important for activity of sod2, the Na+/H+ antiporter of Schizosaccharomyces pombe. We mutated all eight His residues of sod2 into Arg. Only His367-->Arg affected function and resulted in complete inability of sod2 to allow growth of S. pombe in LiCl-containing medium. Mutant S. pombe (H367R) could not expel sodium in acidic (pH 4.0) medium and were defective in their ability to alkalinize external medium. When His367 was replaced by Asp, sodium export of S. pombe was suppressed at acidic pH while the sodium-dependent proton influx at pH 6.1 was increased compared to wild type. We also mutated three residues conserved in putative membrane regions of various eukaryotic and prokaryotic Na+/H+ exchangers. S. pombe containing Asp241-->Asn and Asp266, 267-->Asn mutations had greatly impaired growth in LiCl-containing medium. In addition, sodium-dependent proton influx at external pH 6. 1 was impaired. Sodium export from S. pombe cells at external pH 4.0 was also almost completely abolished by the D266,267N mutation; however, the D241N mutant protein retained almost normal Na+ export. The results demonstrate that His367, Asp241, and Asp266,267 are important in the function of the eukaryotic Na+/H+ exchanger sod2.     

Changes in internal pH caused by movement of fatty acids into and out of clonal pancreatic beta-cells (HIT)

Cells require a constant influx of free fatty acids for lipid resynthesis and metabolic energy. Fatty acids also act as second messengers and modulate channel activities. In the pancreatic beta-cell, fatty acids have both acute and chronic effects on insulin secretion. We show that the addition of fatty acid to pancreatic beta-cells in vitro produces a persistent decrease in intracellular pH, which begins immediately after the addition of fatty acid and has an exponential time course with t1/2 approximately 60 s. The pH drop can be largely reversed by the addition of albumin. The observed pH effect can be explained by passive diffusion ("flip-flop") of un-ionized fatty acid across the plasma membrane. Acidification by a fatty acid dimer and alkalinization by an alkylamine also favor the flip-flop mechanism of transport rather than a protein-mediated mechanism. Our method provides for the first time a real-time measurement of fatty acid import into cells. The significant pH change may be important in mediating some of the regulatory effects of fatty acid, such as inhibition of glycolysis.     

Distinct effects of fatty acids on translocation of gamma- and epsilon-subspecies of protein kinase C

Effects of fatty acids on translocation of the gamma- and epsilon-subspecies of protein kinase C (PKC) in living cells were investigated using their proteins fused with green fluorescent protein (GFP). gamma-PKC-GFP and epsilon-PKC-GFP predominated in the cytoplasm, but only a small amount of gamma-PKC-GFP was found in the nucleus. Except at a high concentration of linoleic acid, all the fatty acids examined induced the translocation of gamma-PKC-GFP from the cytoplasm to the plasma membrane within 30 s with a return to the cytoplasm in 3 min, but they had no effect on gamma-PKC-GFP in the nucleus. Arachidonic and linoleic acids induced slow translocation of epsilon-PKC-GFP from the cytoplasm to the perinuclear region, whereas the other fatty acids (except for palmitic acid) induced rapid translocation to the plasma membrane. The target site of the slower translocation of epsilon-PKC-GFP by arachidonic acid was identified as the Golgi network. The critical concentration of fatty acid that induced translocation varied among the 11 fatty acids tested. In general, a higher concentration was required to induce the translocation of epsilon-PKC-GFP than that of gamma-PKC-GFP, the exceptions being tridecanoic acid, linoleic acid, and arachidonic acid. Furthermore, arachidonic acid and the diacylglycerol analogue (DiC8) had synergistic effects on the translocation of gamma-PKC-GFP. Simultaneous application of arachidonic acid (25 MicroM) and DiC8 (10 microM) elicited a slow, irreversible translocation of gamma-PKC- GFP from the cytoplasm to the plasma membrane after rapid, reversible translocation, but a single application of arachidonic acid or DiC8 at the same concentration induced no translocation. These findings confirm the involvement of fatty acids in the translocation of gamma- and epsilon-PKC, and they also indicate that each subspecies has a specific targeting mechanism that depends on the extracellular signals and that a combination of intracellular activators alters the target site of PKCs.     

Electrolyte transport by gallbladders of rabbit and guinea pig: effect of amphotericin B and evidence of rheogenic Na transport

Ion transport and electrical properties of rabbit and guinea pig gallbladders were investigated to gain further information about the active transport mechanism that mediates fluid absorption. The intracellular and transepithelial electrical potentials were measured simultaneously using the microelectrode technique. Exposure of the mucosal surface to Amphotericin B resulted in the prompt development of a serosa-positive electrical potential difference (PD) which could not be attributed to an alteration in ion diffusion potentials across either the cell membrane or across the tight junction. Because the Amphotericin B-induced PD was immediately dependent on warm temperatures and O2, and was independent of NA and K concentration gradients across the cell membrane, it is suggested that active ion transport is directly responsible for the PD. Since the PD was abolished in the absence of Na in the bathing solutions, a rheogenic Na pump is postulated; this pump also appears to be operative in tissues not exposed to Amphotericin B. The specific tissue properties altered by Amphotericin B to produce a serosa-positive PD remain incompletely defined. The results of the present study indicate that ion transport by rabbit gallbladder in vitro is a consequence of a rheogenic active Na transport mechanism at the basolateral membranes which, in conjunction with a coupled NaC1 influx process at the mucosal border, ultimately results in absorption of NaC1 and water.     

Cyclo-oxygenase products released by low pH have capsaicin-like actions on sensory nerves in the isolated guinea pig heart

OBJECTIVE: Previous work has shown that ischaemia releases calcitonin gene related peptide (CGRP) from capsaicin sensitive nerve terminals in the perfused heart. Prostacyclin (PGI2) is also released during ischaemia. The aim of this study was to investigate whether the release of CGRP by low pH and lactic acid was associated with PGI2 formation and if PGI2 mediated its effect through capsaicin receptors which could be inhibited by capsazepine. METHODS: The isolated Langendorff perfused guinea pig heart was used with a constant perfusion pressure of 70 cm H2O. Low pH was accomplished by changing the Tyrode solution to buffers with pH 7, 6, and 5, or lactic acid (5 mM with pH 6.9). The outflow of CGRP and the stable PGI2 metabolite 6-keto-PGF1 alpha was measured by radioimmunoassay. RESULTS: Low pH (pH 7, 6, 5) and lactic acid evoked release of CGRP. At moderate acidosis (pH 7 and 6) the CGRP release was dependent on extracellular Ca2+, while at pH 5 approximately half of the peptide release persisted in the absence of extracellular Ca2+. This release was attenuated by diclofenac or indomethacin, two inhibitors of prostaglandin formation, as well as by the capsaicin receptor antagonist capsazepine. Both arachidonic acid and PGI2, the predominant cyclo-oxygenase product formed during myocardial ischaemia, evoked a capsazepine sensitive release of CGRP, while capsazepine did not influence the formation of PGI2 evoked by low pH or arachidonic acid. CONCLUSIONS: In the isolated guinea pig heart, moderate acidosis is associated with CGRP release dependent on influx of extracellular Ca2+ and formation of PGI2, with subsequent stimulation of capsazepine sensitive receptors. With more severe acidosis there is an additional non-PGI2-linked CGRP release. Capsazepine represents a novel pharmacological principle for inhibiting the effects of prostanoids on sensory nerves without influencing their formation.