Expression of the mammalian calcium signaling response to Trypanosoma cruzi in Xenopus laevis oocytes.

Infective stages of the protozoan parasite Trypanosoma cruzi contain a soluble factor that induces elevation in the intracellular free Ca2+ concentration ([Ca2+]i) of mammalian cells. The process is pertussis toxin (PTx)-sensitive, and involves phospholipase C (PLC) activation, inositol 1,4,5-trisphosphate (IP3) formation and Ca2+ release from intracellular stores (Tardieux I, et al. J Exp Med 1994;179:1017-1022; Rodriguez A, et al. J Cell Biol 1995;129:1263-1273). We now report that a molecule exposed on the surface of the target cells is required to trigger the signaling cascade, and that a response with identical characteristics can be induced in Xenopus laevis oocytes injected with mRNA from normal rat kidney (NRK) fibroblasts. Xenopus oocytes do not show an endogenous response to the trypomastigote Ca2+ signaling factor, but a vigorous response in the form of a propagating Ca2+ wave is expressed after injection of NRK cell mRNA. As previously demonstrated for mammalian cells, the response is inhibited when injected oocytes are pretreated with PTx, implicating Galphai or Galphao trimeric G-proteins, and with thapsigargin, which depletes intracellular Ca2+ stores. Moreover, the [Ca2+]i transients triggered by the T. cruzi soluble factor in mRNA-injected oocytes are blocked by the same inhibitors of the parasite oligopeptidase B that abolish the [Ca2+]i response in NRK cells (Burleigh B, Andrews NW. J Biol Chem 1995;270:5172-5180; Burleigh BA et al. J Cell Biol 1997;136:609-620). The NRK mRNA fraction that induces expression of the [Ca2+]i response to the T. cruzi signaling factor contains messages from 1.5 to 2.0 kb, a size range consistent with the family of seven-transmembrane G-protein-coupled receptors.

Cloning and characterization of a novel peptidase from rat and human ileum.

A novel 100-kDa ileal brush border membrane protein (I100) has been purified by anionic glycocholate affinity chromatography. Polyclonal antibodies raised against this protein were utilized to clone and characterize I100 in rats. A partial length human I100 cDNA was identified by hybridization screening. In the rat, the I100 protein is a 746-amino acid glycosylated (calculated core molecular mass of 80 kDa) type II integral membrane protein found on the apical surface of ileal villus enterocytes. Its 2.6-kilobase mRNA is expressed in distal small intestine in rats and in humans. The I100 cDNA is homologous to but distinct from human prostate-specific membrane antigen and rat brain N-acetylaspartylglutamate peptidase. It is expressed on both the basolateral and apical surfaces of stably transfected Madin Darby canine kidney cells. Analysis of these stably transfected Madin Darby canine kidney cells and I100 immunoprecipitates of rat ileal brush border membrane vesicles reveals that it has dipeptidyl peptidase IV activity. Future invesitgations will need to determine the exact substrate specificity of this novel peptidase.

A midregion parathyroid hormone-related peptide mobilizes cytosolic calcium and stimulates formation of inositol trisphosphate in a squamous carcinoma cell line.

A midregion fragment of PTH-related protein (PTHrP), which is intensively conserved across species, has been identified as a secretory product of several different cell types, including keratinocytes and squamous carcinomas. As recent data suggest that a midregion PTHrP fragment may be biologically active, we hypothesized that midregion PTHrPs interact with unique cell surface receptors that mediate autocrine or paracrine action. Dose-dependent transient elevations in intracellular calcium ([Ca2-]i) were observed in fura-2-loaded SqCC/Y1 squamous carcinoma cells exposed to human (h) PTHrP-(67-86)NH2, [Tyr36]hPTHrP-(1-36)NH2, and hPTHrP-(1-141) at concentrations ranging from 1 pM to 1 microM. The effects of maximal stimulatory concentrations of [Tyr36]PTHrP-(1-36)NH2 and PTHrP-(67-86)NH2 on [Ca2+]i were additive. The inhibitory PTH analog, [D-Trp12,Tyr34]bovine PTH-(7-34)NH2, attenuated the [Ca2+]i response to [Tyr36]hPTHrP-(1-36)NH2, but not that to PTHrP-(67-86)NH2. These data suggest that PTHrP-(67-86)NH2 activates a different receptor pathway in SqCC/Y1 cells from the one activated by [Tyr36]hPTHrP-(1-36)NH2. Radiolabeled PTHrP-(67-86)NH2 did not bind to SqCC/Y1 cells, and PTHrP-(67-86)NH2 did not compete for binding of 125I-labeled [Tyr36]PTHrP-(1-36)NH2 to PTH/PTHrP receptors on SaOS-2 osteosarcoma cells. Activation of the phospholipase C pathway by PTHrP-(67-86)NH2 was confirmed by exposing SqCC/Y1 cells to peptide for 1 min and measuring the accumulation of inositol trisphosphates. PTHrP-(67-86)NH2 treatment (100 nM) resulted in maximal stimulation of inositol trisphosphates of 3.1 +/- 0.1-fold over the control value, with an EC50 of 1.5 +/- 1.2 nm. In contrast, PTHrP-(67-86)NH2 (0.1 nM to 1 microM) did not stimulate adenylyl cyclase in SqCC/Y1 cells despite vigorous stimulation of cAMP formation by isoproterenol (1 microM) to 66-fold over the basal value. To determine whether messenger RNA (mRNA) prepared from SqCC/Y1 cells would direct the translation of a receptor protein that mediated a [Ca2+]i response to PTHrP-(67-86)NH2, we performed expression studies in Xenopus oocytes. Fluo-3 fluorescence in Xenopus oocytes expressing SqCC/Y1 mRNA was visualized by confocal video microscopy after exposure to 1 microM PTHrP-(67-86)NH2. Clear increases in [Ca2+]i were detected in mRNA-injected, but not in sham-injected, oocytes. Finally, we examined the effect of PTHrP-(67-86)NH2 treatment on fibronectin secretion from SqCC/YN1 cells. A significant 3.5-fold increase in fibronectin secretion into conditioned medium was observed when SqCC/Y1 cells were exposed to 100 nM PTHrP-(67-86)NH2, and this effect was dose dependent, with an EC50 of 0.1 nM. We conclude that PTHrP-(67-86)NH2 activates phospholipase C-dependent pathways in SqCC/Y1 cells through a receptor distinct from that activated by PTHrP-(1-36) in the same cells. As a midregion secretory fragment of PTHrP has been partially purified from several different cell types, this receptor may have broad biological significance.

Pancreas divisum in a family with hereditary pancreatitis.

Hereditary pancreatitis is characterized by an autosomal-dominant mode of inheritance with incomplete penetrance, onset of symptoms in childhood or early adolescence (mean age of onset approximately 13 years), and an approximately equal sex incidence. Pancreas divisum is a congenital variant of pancreatic ductal anatomy in which the ventral and dorsal pancreatic ductal systems fail to fuse, so that two functional papillae drain the exocrine secretions of the pancreas. In recent years, several reports of pancreatitis associated with pancreas divisum in children have appeared. We now report a family in which the mother, son, and daughter all had presented with recurrent pancreatitis from an early age. Both the mother and son have endoscopic retrograde cholangiopancreatography-documented pancreas divisum, whereas the daughter has a stricture in her distal pancreatic duct. To our knowledge, this is the first such report of "familial" pancreas divisum. The implications of these findings in the setting of hereditary pancreatitis highlight the controversial issues of the clinical significance of pancreas divisum and the appropriateness of surgical therapy.

Mechanism of desensitization of the cloned vasopressin V1a receptor expressed in Xenopus oocytes.

The vasopressin V1a receptor exerts its effects by G protein-mediated increases in cytosolic Ca2+ (Cai2+) and activation of protein kinase C. The V1a receptor also undergoes autologous desensitization. To clarify the mechanism of this desensitization, we expressed the cloned receptor in Xenopus oocytes, and vasopressin-induced Cai2+ waves were examined as an index of V1a activation using confocal microscopy. Pretreatment of oocytes with a minimal concentration of vasopressin inhibited further generation of Cai2+ waves upon maximal stimulation. Such pretreatment did not abolish Cai2+ waves induced by subsequent microinjection of inositol trisphosphate, suggesting that this phenomenon represents receptor desensitization rather than depletion of inositol trisphosphate-sensitive Cai2+ stores. Pretreatment with phorbol dibutyrate, ionomycin, or 8-bromoadenosine 3',5'-cyclic monophosphate had no effect on vasopressin-induced Cai2+ waves. Oocytes recovered from desensitization within 1 h, but the microtubule inhibitor methyl-5-[2-thienylcarbonyl]-1H-benzimiidazol-2-yl)-carbamate (nocodazole) inhibited this recovery. Receptor binding sites were reduced by over 50% within 10 min of exposure to vasopressin, with no associated change in the Kd for the V1a receptor. These findings indicate that 1) expression of the cloned V1a receptor in Xenopus oocytes, coupled with subcellular Cai2+ imaging, provides a useful system to examine mechanisms of V1a desensitization, 2) the V1a receptor undergoes autologous desensitization in this experimental system, and 3) protein kinase C, Cai2+, and adenosine 3',5'-cyclic monophosphate do not appear responsible for this desensitization, but 4) microtubule-dependent recycling of the receptor is preserved in this system and may be important for receptor desensitization.

An anion exchanger mediates bile acid transport across the placental microvillous membrane.

Microvillous membrane vesicles from the term human placental syncytiotrophoblast were used to characterize further the properties of a transport mechanism for bile acids. Taurocholate (TC) uptake into an osmotically reactive intravesicular space was temperature dependent and independent of sodium. TC uptake (2 microM) was markedly inhibited by 250 microM taurine and glycine-conjugated cholate and chenodeoxycholate and unconjugated cholate but not by chenodeoxycholate, deoxycholate, etianic acid, bromosulfophthalein, pyruvate, lactate, alanine, or taurine. The initial rate of TC uptake was inhibited significantly by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) but was not inhibited significantly by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, amiloride, or furosemide. Preincubation of vesicles with DIDS in the presence of TC partially blocked the action of the inhibitor. Efflux of 5 microM TC from membrane vesicles was stimulated by the presence of 50 microM TC in the incubation media. Basal as well as transstimulated TC efflux was inhibited by DIDS. The initial rate of TC influx followed saturation kinetics with an apparent Michaelis constant of 112 +/- 23 microM and maximal velocity of 2.01 +/- 0.19 nmol.mg protein-1.min-1. When the transmembrane electrical potential difference across the brush-border membrane vesicles was altered by external anion replacement or by valinomycin-induced K+ diffusion potentials, TC uptake was not significantly affected. DIDS-sensitive TC uptake was stimulated two-to threefold by an outwardly directed hydroxyl gradient (pH 7.7in/5.5out) compared with TC influx under pH-equilibrated conditions (pH 7.7in/7.7out). These studies are consistent with an electroneutral anion-exchange mechanism that mediates transfer of conjugated bile acids across the microvillous membrane of the syncytiotrophoblast.

Characterization of endogenous carrier-mediated taurocholate efflux from Xenopus laevis oocytes.

Taurocholate efflux was studied in Xenopus laevis oocytes and is consistent with a carrier-mediated process. This carrier can be competitively inhibited and trans stimulated by glycocholate. Transport is also trans stimulated by taurochenodeoxycholate and S-hexylglutathione, but not taurolithocholate or daunomycin, reflecting a range of specificity including substrates of both the hepatic canalicular bile acid transporter and the multispecific organic anion transporter. In addition, ATP added to the outside of the oocyte results in an increase in the maximal velocity of this transport process. The physiologic function of this endogenous carrier is not known, but it may act as a generalized system for the efflux of potentially toxic organic anions.

Mechanisms of subcellular cytosolic Ca2+ signaling evoked by stimulation of the vasopressin V1a receptor.

Receptor activation may result in distinct subcellular patterns of Ca2+ release. To define the subcellular distribution of Ca2+i signals induced by stimulation of the vasopressin V1a receptor, we expressed the cloned receptor in Xenopus oocytes. Oocytes were then loaded with fluo-3 and observed using confocal microscopy. Vasopressin induced a single concentric wave of increased Ca2+ that radiated inward from the plasma membrane. With submaximal stimulation, however, regions of the Ca2+ wave spontaneously reorganized into repetitive (oscillatory) waves. Focal stimulation of a small part of the plasma membrane resulted in a Ca2+ wave which began at the point of stimulation, radiated toward the center of the cell, then reorganized into multiple foci of repetitive, colliding waves and spirals of increased Ca2+i. The pattern of Ca2+ signaling induced by focal or global stimulation was not altered in Ca(2+)-free medium, although signals did not propagate as fast. Finally, subcellular Ca2+ signaling patterns induced by vasopressin were inhibited by caffeine, while neither vasopressin nor microinjection of inositol trisphosphate blocked caffeine-induced increases in cytosolic Ca2+. Thus, stimulation of the V1a receptor in this cell system induces a complex pattern of Ca2+ signaling which is influenced by (1) the magnitude of the stimulus, (2) the distribution of the surface receptors that are stimulated, and (3) mobilization of Ca2+ from the extracellular space as well as from two distinct endogenous Ca2+ pools. The manner in which a single type of receptor is activated may represent an important potential mechanism for subcellular Ca2+i signaling.

The role of chloride in taurine transport across the human placental brush-border membrane.

Taurine, a sulfated beta-amino acid, is conditionally essential during development. A maternal supply of taurine is necessary for normal fetal growth and neurologic development, suggesting the importance of efficient placental transfer. Uptake by the brush-border membrane (BBM) in several other tissues has been shown to be via a selective Na(+)-dependent carrier mechanism which also has a specific anion requirement. Using BBM vesicles purified from the human placenta, we have confirmed the presence of Na(+)-dependent, carrier-mediated taurine transport with an apparent Km of 4.00 +/- 0.22 microM and a Vmax of 11.72-0.36 pmol mg-1 protein 20 s-1. Anion dependence was examined under voltage-clamped conditions, in order to minimize the contribution of membrane potential to transport. Uptake was significantly reduced when anions such as thiocyanate, gluconate, or nitrate were substituted for Cl-. In addition, a Cl(-)-gradient alone (under Na(+)-equilibrated conditions) could energize uphill transport as evidenced by accelerated uptake (3.13 +/- 0.8 pmol mg-1 protein 20 s-1) and an overshoot compared to Na+, Cl- equilibrated conditions (0.60 +/- 0.06 pmol mg-1 protein 20 s-1). A Cl(-)-gradient (Na(+)-equilibrated) also stimulated uptake of [3H]taurine against its concentration gradient. Analysis of uptake in the presence of varying concentrations of external Cl- suggested that 1 Cl- ion is involved in Na+/taurine cotransport. We conclude that Na(+)-dependent taurine uptake in the placental BBM has a selective anion requirement for optimum transport. This process is electrogenic and involves a stoichiometry of 2:1:1 for Na+/Cl-/taurine symport.

Sulfasalazine hypersensitivity with hepatotoxicity, thrombocytopenia, and erythroid hypoplasia.

Sulfasalazine, a commonly prescribed drug for the treatment of inflammatory bowel disease, can cause an allergic reaction with hepatotoxicity, lymphadenopathy, and rash. In addition, many hematologic complications have been observed with sulfasalazine, including aplastic anemia, thrombocytopenia, and erythroid hypoplasia. However, thrombocytopenia in association with sulfasalazine hypersensitivity has not been previously reported. We report a child who developed severe thrombocytopenia and anemia during a hypersensitivity reaction to sulfasalazine.

Ontogenesis of intestinal taurine transport: evidence for a beta-carrier in developing rat jejunum.

Taurine, a sulfur-containing beta-amino acid, may be conditionally essential during development. However, the existence of a carrier system for beta-amino acids has not been demonstrated in brush-border membrane vesicles (BBMV) from adult rat jejunum. We studied the uptake of [3H]taurine in BBMV prepared from the jejunum of developing and adult rats using a cation-precipitation technique. Uptake of 10 microM [3H]taurine by adult BBMV was slightly enhanced in the presence of an inwardly directed 100 mM Na+ gradient compared with a K+ gradient, and there was no intravesicular accumulation of isotope above the equilibrium concentration ("overshoot"). In contrast, taurine transport by BBMV from 10-day-old rat pups was markedly accelerated in the presence of a Na+ gradient compared with a K+ gradient and a twofold overshoot was observed. Na+-dependent taurine uptake was inhibited by the structural analogues hypotaurine and beta-alanine but not by alpha-alanine or glutamine, which are amino acids served by other transport systems. By computer analysis, Na+-dependent taurine uptake (2-1,000 microM) was saturable with an apparent Km of 74.80 +/- 11.87 microM and a Vmax of 53.55 +/- 2.76 pmol.mg protein-1.min-1. With increasing postnatal age, there was a marked decrease in the initial rate and peak intravesicular accumulation of taurine with disappearance of the overshoot by 21 days of age. We conclude 1) a Na+-dependent carrier mechanism for taurine transport is present in the brush-border membrane of suckling rat jejunum and 2) the activity of this carrier decreases after weaning.(ABSTRACT TRUNCATED AT 250 WORDS)

Taurocholate transport and Na+-K+-ATPase activity in fetal and neonatal rat liver plasma membrane vesicles.

The ontogenesis of Na+-K+-ATPase activity and Na+-taurocholate cotransport was studied in basolateral plasma membrane vesicles from fetal and neonatal rat liver. Membrane vesicles from each age group were 30-fold enriched in the basolateral marker enzyme Na+-K+-ATPase, 4- to 7-fold enriched in the bile canalicular membrane marker enzymes alkaline phosphatase and Mg2+ ATPase, and not significantly enriched in activities of marker enzymes for intracellular organelles. Na+-K+-ATPase activity was significantly lower in basolateral membranes from late fetal (day 21-22) and neonatal (day 1) rat liver. Kinetic analysis of Na+-K+-ATPase activity at various concentrations of ATP revealed that the maximum velocity of enzyme reaction (Vmax) for Na+-K+-ATPase was 70 and 90% of adult activity in the fetus and the neonate, respectively. The ATP Km was significantly lower in the neonate than the adult, suggesting a higher affinity of the neonatal enzyme for ATP. In contrast to the early maturation of Na+-K+-ATPase, transport of taurocholate was markedly lower in both fetal and neonatal vesicles compared with the adult. Taurocholate uptake on day 19 of gestation did not differ in the presence of a Na+ or K+ gradient, and uphill transport, as indicated by an overshoot, did not occur. On day 20 taurocholate uptake was stimulated by a Na+ compared with a K+ gradient, and accumulation of isotope above equilibrium was demonstrated. Na+-dependent transport of taurocholate by late fetal (day 22) and neonatal vesicles was saturable but the Vmax at each age was significantly lower and the apparent Km higher in developing compared with adult membrane vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

The immature rat small intestine exhibits an increased sensitivity and response to Escherichia coli heat-stable enterotoxin.

Escherichia coli which elaborate heat stable enterotoxin (ST) are a major cause of endemic diarrhea in infants. The reason(s) for this increased susceptibility of infants to ST-mediated diarrhea is unknown. We investigated the possibility that the immature (14 and 21 day old) rat small intestine is more sensitive to ST than is the adult. Initially we found there was a 600-fold increased jejunal sensitivity to ST in the immature animals as measured by dose required for half maximal secretion. Also there was a greater jejunal secretory response in the immature animals (14 greater than or equal to 21 days old greater than adult). To determine the cause for this increased sensitivity and secretory response to ST, we examined: 1) binding characteristics of 125I-ST to brush border membrane (BBM) receptors and 2) membrane bound guanylate cyclase activation by ST in both immature and adult rats. Our findings demonstrate that more ST receptors are present in jejunal BBM from 14- and 21-day-old rats than in jejunal BBM from adult rats (2.34 +/- 0.18, 2.85 +/- 0.82, and 0.79 +/- 0.13 X 10(12) receptors/mg BBM protein, respectively), while the affinity of the BBM receptor for ST is similar at all three ages in both jejunum and ileum. Furthermore, both the jejunum and ileum of the rats of all three ages revealed an equal sensitivity of guanylate cyclase to activation by ST. These findings suggest that the increased number of jejunal receptors in the immature rat may, in part, explain the increased sensitivity and secretory response observed in vivo.

Ontogeny of bile acid transport in brush border membrane vesicles from rat ileum.

We studied the postnatal development of bile acid transport in rat ileum, using brush border membrane vesicles prepared by a Ca2+ precipitation method. Membrane vesicles from developing (day 14-21) and adult Sprague-Dawley rats were enriched to a similar degree in brush border membrane marker enzyme activities (sucrase or lactase) compared with homogenate. Uptake of 25 microM [3H]taurocholate by adult membrane vesicles was markedly accelerated in the presence of an inwardly directed 100 mM Na+ gradient compared with a K+ gradient, and there was a transient intravesicular accumulation of isotope above equilibrium ("overshoot"). In contrast, at 14 and 16 days of age there was no difference in taurocholate uptake in the presence of a Na+ or a K+ gradient, and uptake was not saturable. The integrity of the vesicle preparation from 14- and 16-day-old rats was confirmed by the demonstration of Na+-dependent uphill transport of 100 microM L-[3H]alanine. Stimulation of taurocholate uptake by a Na+ compared with a K+ gradient ("sodium effect") was first observed at age 17 days, but an overshoot was not present until 18 days of age. The initial rate of Na+-dependent taurocholate (25 microM) uptake increased sixfold between 17 and 21 days of age (24.36 +/- 6.11 to 148.59 +/- 8.56 pmol X mg-1 protein X 5 s-1). Absent or decreased Na+-dependent taurocholate uptake was not due to increased permeability or "leakiness" of vesicles from younger animals to Na+. Ileal brush border membrane vesicles demonstrated saturable kinetics at 21 days, but the Vmax was significantly lower (10.15 +/- 0.44 vs. 13.42 +/- 0.59 nmol X mg-1 protein X min -1, p less than 0.001) and the apparent Km higher (130.6 +/- 18.9 vs. 70.1 +/- 12.6 microM, p less than 0.007) than the adult. We conclude that (a) saturable, Na+-bile acid coupled transport is absent in rat ileum throughout most of the suckling period and (b) kinetic analysis suggests that maturation occurs near weaning, primarily through an increase in functional bile acid carriers within the ileal brush border membrane.