A Reduction in Intestinal Cell pH Due to Loss of the Caenorhabditis elegans Na+/H+ Exchanger NHX-2 Increases Life Span

Abstract

Na؉ /H ؉ exchangers are involved in cell volume regulation, fluid secretion and absorption, and pH homeostasis. NHX-2 is a Caenorhabditis elegans Na ؉ /H ؉ exchanger expressed exclusively at the apical membrane of intestinal epithelial cells. The inactivation of various intestinal nutrient transport proteins has been shown previously to influence aging via metabolic potential and a mechanism resembling caloric restriction. We report here a functional coupling of NHX-2 activity with nutrient uptake that results in long lived worms. Gene inactivation of nhx-2 by RNAi led to a loss of fat stores in the intestine and a 40% increase in longevity. The NHX-2 protein was coincidentally expressed with OPT-2, an oligopeptide transporter that is driven by a transmembrane proton gradient and that is also known to be involved in fat accumulation. Gene inactivation of opt-2 led to a phenotype resembling that of nhx-2, although not as severe. In order to explore this potential functional interaction, we combined RNA interference with a genetically encoded, fluorescence-based reagent to measure intestinal intracellular pH (pH i ) in live worms under physiological conditions. Our results suggest first that OPT-2 is the main dipeptide uptake pathway in the nematode intestine, and second that dipeptide uptake results in intestinal cell acidification, and finally that recovery following dipeptide-induced acidification is normally a function of NHX-2. The loss of NHX-2 protein results in decreased steady-state intestinal cell pH i , and we hypothesize that this change perturbs proton-coupled nutrient uptake processes such as performed by OPT-2. Our data demonstrate a functional role for a Na ؉ /H ؉ exchanger in nutrient absorption in vivo and lays the groundwork for examining integrated acid-base physiology in a non-mammalian model organism.

RAB-10 Is Required for Endocytic Recycling in theCaenorhabditis elegansIntestine

“…Because all exogenous tracers that we have identified for studying endocytosis in C. elegans are sent to lysosomes when internalized apically but not recycled back to the intestinal lumen, we have not been able to determine if rab-10 or rme-1 mutations also affect apical recycling in the worm intestine. The only currently available transmembrane marker for the apical intestinal membrane is OPT-2, but it is not known if OPT-2 cycles through the apical endosomal system (Nehrke, 2003). In preliminary studies we find OPT-2-GFP to be normally localized in the intestine of rab-10 mutant worms (S. Vashist and B. D. Grant, unpublished results).…”

An autonomous DNA nanomachine maps spatiotemporal pH changes in a multicellular living organism

“…S10 ), showing that this DNA scaff old is non-toxic in this model system. Many pH-sensitive probes are limited by their fi xed wavelengths 33,34 ; as the I-switch is FRET-based, it can be modulated to use FRET pairs at wavelengths that are compatible with the background of fl uorescent protein-expressing transgenics. It can also be used in a range of mutant backgrounds, because it functions independently of the organismal milieu.…”

“…Worms were incubated at 22 ° C for 1 h and then immersed in clamping buff ers (120 mM KCl, 5 mM NaCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 20 mM HEPES) of varying pH, containing 100 μ M nigericin and 100 μ M monensin 23 . To facilitate entry of the buff er into the body, the cuticle was perforated at three regions of the body using a microinjection needle 33 . Aft er 75-min incubation in the clamping buff er, coelomocytes were imaged using wide-fi eld microscopy.…”

Deletion of the Intestinal Peptide Transporter Affects Insulin and TOR Signaling in Caenorhabditis elegans

“…Both parameters are indistinguishable in pep-2 and wild type animals (data not shown; Ref. 31). Therefore, malabsorption of dipeptides and the associated lower intake of amino acids appear to be the sole determinants of the developmental defects.…”