Essential role for collectrin in renal amino acid transport.

Angiotensin -converting enzyme 2 (ACE2) is a regulator of the renin angiotensin system involved in acute lung failure, cardiovascular functions and severe acute respiratory syndrome (SARS) infections in mammals. A gene encoding a homologue to ACE2, termed collectrin (Tmem27), has been identified in immediate proximity to the ace2 locus. The in vivo function of collectrin was unclear. Here we report that targeted disruption of collectrin in mice results in a severe defect in renal amino acid uptake owing to downregulation of apical amino acid transporters in the kidney. Collectrin associates with multiple apical transporters and defines a novel group of renal amino acid transporters. Expression of collectrin in Xenopus oocytes and Madin-Darby canine kidney (MDCK) cells enhances amino acid transport by the transporter B(0)AT1. These data identify collectrin as a key regulator of renal amino acid uptake.

Reduced intestinal and renal amino acid transport in PDK1 hypomorphic mice.

The phosphoinositide-dependent kinase PDK1 activates the serum- and glucocorticoid-inducible kinase isoforms SGK1, SGK2, and SGK3 and protein kinase B, which in turn are known to up-regulate a variety of sodium-coupled transporters. The present study was performed to explore the role of PDK1 in amino acid transport. As mice completely lacking functional PDK1 are not viable, mice expressing 10-25% of PDK1 (pdk1(hm)) were compared with their wild-type (WT) littermates (pdk1(wt)). Body weight was significantly less in pdk1(hm) than in pdk1(wt) mice. Despite lower body weight of pdk1(hm) mice, food and water intake were similar in pdk1(hm) and pdk1(wt) mice. According to Ussing chamber experiments, electrogenic transport of phenylalanine, cysteine, glutamine, proline, leucine, and tryptophan was significantly smaller in jejunum of pdk1(hm) mice than in pdk1(wt) mice. Similarly, electrogenic transport of phenylalanine, glutamine, and proline was significantly decreased in isolated perfused proximal tubules of pdk1(hm) mice. The urinary excretion of proline, valine, guanidinoacetate, methionine, phenylalanine, citrulline, glutamine/glutamate, and tryptophan was significantly larger in pdk1(hm) than in pdk1(wt) mice. According to immunoblotting of brush border membrane proteins prepared from kidney, expression of the Na+-dependent neutral amino acid transporter B(0)AT1 (SLC6A19), the glutamate transporter EAAC1/EAAT3 (SLC1A1), and the transporter for cationic amino acids and cystine b(0,+)AT (SLC7A9) was decreased but the Na+/proline cotransporter SIT (SLC6A20) was increased in pdk1(hm) mice. In conclusion, reduction of functional PDK1 leads to impairment of intestinal absorption and renal reabsorption of amino acids. The combined intestinal and renal loss of amino acids may contribute to the growth defect of PDK1-deficient mice.

An amino acid transporter involved in gastric acid secretion.

Gastric acid secretion is regulated by a variety of stimuli, in particular histamine and acetyl choline. In addition, dietary factors such as the acute intake of a protein-rich diet and the subsequent increase in serum amino acids can stimulate gastric acid secretion only through partially characterized pathways. Recently, we described in mouse stomach parietal cells the expression of the system L heteromeric amino acid transporter comprised of the LAT2-4F2hc dimer. Here we address the potential role of the system L amino acid transporter in gastric acid secretion by parietal cells in freshly isolated rat gastric glands. RT-PCR, western blotting and immunohistochemistry confirmed the expression of 4F2-LAT2 amino acid transporters in rat parietal cells. In addition, mRNA was detected for the B(0)AT1, ASCT2, and ATB(0+) amino acid transporters. Intracellular pH measurements in parietal cells showed histamine-induced and omeprazole-sensitive H+-extrusion which was enhanced by about 50% in the presence of glutamine or cysteine (1 mM), two substrates of system L amino acid transporters. BCH, a non-metabolizable substrate and a competitive inhibitor of system L amino acid transport, abolished the stimulation of acid secretion by glutamine or cysteine suggesting that this stimulation required the uptake of amino acids by system L. In the absence of histamine glutamine also stimulated H+-extrusion, whereas glutamate did not. Also, phenylalanine was effective in stimulating H+/K+-ATPase activity. Glutamine did not increase intracellular Ca2+ levels indicating that it did not act via the recently described amino acid modulated Ca2+-sensing receptor. These data suggest a novel role for heterodimeric amino acid transporters and may elucidate a pathway by which protein-rich diets stimulate gastric acid secretion.

Treatment of Asymptomatic Carriers of Plasmodium falciparum with Artemether-Lumefantrine: Impact on the Prevalence of Anemia.

INTRODUCTION: An investigation of whether treatment of asymptomatic carriers of Plasmodium falciparum with artemether-lumefantrine (AL), in addition to the routine treatment of symptomatic cases with AL, could improve the prevalence of anemia in 18 villages in Burkina Faso. METHODS: This was a single-center, controlled, parallel, cluster-randomized study to evaluate the effect of systematic treatment of P. falciparum asymptomatic carriers at a community level on hemoglobin (Hb) levels and anemic status of children (<5 years) and adults during four screening campaigns carried out over a 12-month period, compared with no treatment of asymptomatic carriers. RESULTS: The change in Hb level in all asymptomatic carriers aged >6 months from Day 1 to Day 28 of the first campaign was +0.53 g/dl (from 11.81 to 12.33 g/dl) in the intervention arm vs. -0.21 g/dl (from 12.06 to 11.86 g/dl) in the control arm (P < 0.001). During the same period, the proportion of asymptomatic carriers aged >6 months to <5 years with anemia in the intervention arm decreased by 31.1% (from 75.7% to 44.6%), compared with a decrease of 4.7% (from 76.3% to 71.6%) in the control arm. Over 12 months, the proportion of asymptomatic carriers with anemia (mild, moderate, or severe) was reduced in both arms. CONCLUSION: Systematic screening and treatment of asymptomatic carriers of P. falciparum with AL at the community level can reduce the prevalence of anemia in children in the short term (28 days), although the effect was not maintained at 12 months.

Stimulatory pathways of the Calcium-sensing receptor on acid secretion in freshly isolated human gastric glands.

Gastric acid secretion is not only stimulated via the classical known neuronal and hormonal pathways but also by the Ca(2+)-Sensing Receptor (CaSR) located at the basolateral membrane of the acid-secretory gastric parietal cell. Stimulation of CaSR with divalent cations or the potent agonist Gd(3+) leads to activation of the H(+)/K(+)-ATPase and subsequently to gastric acid secretion. Here we investigated the intracellular mechanism(s) mediating the effects of the CaSR on H(+)/K(+)-ATPase activity in freshly isolated human gastric glands. Inhibition of heterotrimeric G-proteins (G(i) and G(o)) with pertussis toxin during stimulation of the CaSR with Gd(3+) only partly reduced the observed stimulatory effect. A similar effect was observed with the PLC inhibitor U73122. The reduction of the H(+)/K(+)-ATPase activity measured after incubation of gastric glands with BAPTA-AM, a chelator of intracellular Ca(2+), showed that intracellular Ca(2+) plays an important role in the signalling cascade. TMB-8, a ER Ca(2+)store release inhibitor, prevented the stimulation of H(+)/K(+)-ATPase activity. Also verapamil, an inhibitor of L-type Ca(2+)-channels reduced stimulation suggesting that both the release of intracellular Ca(2+) from the ER as well as Ca(2+) influx into the cell are involved in CaSR-mediated H(+)/K(+)-ATPase activation. Chelerythrine, a general inhibitor of protein kinase C, and Go 6976 which selectively inhibits Ca(2+)-dependent PKC(alpha) and PKC(betaI)-isozymes completely abolished the stimulatory effect of Gd(3+). In contrast, Ro 31-8220, a selective inhibitor of the Ca(2+)-independent PKCepsilon and PKC-delta isoforms reduced the stimulatory effect of Gd(3+) only about 60 %. On the other hand, activation of PKC with DOG led to an activation of H(+)/K(+)-ATPase activity which was only about 60 % of the effect observed with Gd(3+). Incubation of the parietal cells with PD 098059 to inhibit ERK1/2 MAP-kinases showed a significant reduction of the Gd(3+) effect. Thus, in the human gastric parietal cell the CaSR is coupled to pertussis toxin sensitive heterotrimeric G-Proteins and requires calcium to enhance the activity of the proton-pump. PLC, ERK 1/2 MAP-kinases as well as Ca(2+) dependent and Ca(2+)-independent PKC isoforms are part of the down-stream signalling cascade.

Impaired intestinal and renal glucose transport in PDK-1 hypomorphic mice.

The phosphoinositide-dependent kinase-1 (PDK-1) activates the serum- and glucocorticoid-inducible kinase and protein kinase B isoforms, which, in turn, are known to stimulate the renal and intestinal Na+-dependent glucose transporter 1. The present study has been performed to explore the role of PDK-1 in electrogenic glucose transport in small intestine and proximal renal tubules. To this end, mice expressing approximately 20% of PDK-1 (pdk1hm) were compared with their wild-type littermates (pdk1wt). According to Ussing chamber experiments, electrogenic glucose transport was significantly smaller in the jejunum of pdk1hm than of pdk1wt mice. Similarly, proximal tubular electrogenic glucose transport in isolated, perfused renal tubule segments was decreased in pdk1hm compared with pdk1wt mice. Intraperitoneal injection of 3 g/kg body wt glucose resulted in a similar increase of plasma glucose concentration in pdk1hm and in pdk1wt mice but led to a higher increase of urinary glucose excretion in pdk1hm mice. In conclusion, reduction of functional PDK-1 leads to impairment of electrogenic intestinal glucose absorption and renal glucose reabsorption. The experiments disclose a novel element of glucose transport regulation in kidney and small intestine.

The calcium-sensing receptor acts as a modulator of gastric acid secretion in freshly isolated human gastric glands.

Gastric acid secretion is activated by two distinct pathways: a neuronal pathway via the vagus nerve and release of acetylcholine and an endocrine pathway involving gastrin and histamine. Recently, we demonstrated that activation of H(+)-K(+)-ATPase activity in parietal cells in freshly isolated rat gastric glands is modulated by the calcium-sensing receptor (CaSR). Here, we investigated if the CaSR is functionally expressed in freshly isolated gastric glands from human patients undergoing surgery and if the CaSR is influencing histamine-induced activation of H(+)-K(+)-ATPase activity. In tissue samples obtained from patients, immunohistochemistry demonstrated the expression in parietal cells of both subunits of gastric H(+)-K(+)-ATPase and the CaSR. Functional experiments using the pH-sensitive dye 2',7'-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein and measurement of intracellular pH changes allowed us to estimate the activity of H(+)-K(+)-ATPase in single freshly isolated human gastric glands. Under control conditions, H(+)-K(+)-ATPase activity was stimulated by histamine (100 microM) and inhibited by omeprazole (100 microM). Reduction of the extracellular divalent cation concentration (0 Mg(2+), 100 microM Ca(2+)) inactivated the CaSR and reduced histamine-induced activation of H(+)-K(+)-ATPase activity. In contrast, activation of the CaSR with the trivalent cation Gd(3+) caused activation of omeprazole-sensitive H(+)-K(+)-ATPase activity even in the absence of histamine and under conditions of low extracellular divalent cations. This stimulation was not due to release of histamine from neighbouring enterochromaffin-like cells as the stimulation persisted in the presence of the H(2) receptor antagonist cimetidine (100 microM). Furthermore, intracellular calcium measurements with fura-2 and fluo-4 showed that activation of the CaSR by Gd(3+) led to a sustained increase in intracellular Ca(2+) even under conditions of low extracellular divalent cations. These experiments demonstrate the presence of a functional CaSR in the human stomach and show that this receptor may modulate the activity of acid-secreting H(+)-K(+)-ATPase in parietal cells. Furthermore, our results show the viability of freshly isolated human gastric glands and may allow the use of this preparation for experiments investigating the physiological regulation and properties of human gastric glands in vitro.