Rhesus Protein Research Articles

Characterisation of the Rh50 protein from the ammonia-oxidising bacterium Nitrosomonas europaea

The transport of ammonium through the cell membranes, an essential process in all kingdoms of life, is accomplished by the ubiquitous Amt/Mep/Rh superfamily of proteins. The functional context of Amt/Mep and Rh transporters is diverse: bacteria, fungi, and plants use Amt/Mep proteins to scavenge ammonium for biosynthetic assimilation, whereas mammals use the Rh proteins for ammonium detoxification in erythrocytes, kidney, and liver tissues. While RH50 genes are widespread in eukaryotes they are present in some prokaryotes: an example is a chemolithoautotroph Nitrosomonas europaeawhich gains all its energy from the oxidation of ammonia to nitrate. While Amt/Mep/Rh proteins have divergent physiological functions, they are structurally very similar, which raises the important question about the universality of the transport mechanism. We have recently proposed an elegant new model for the mechanism of electrogenic ammonium transport in bacteria Amt protein: after deprotonation of NH4+ at the periplasmic side of the transporter, a previously undiscovered polar conduction route enables H+ transfer into the cytoplasm. A parallel pathway, lined by hydrophobic groups within the protein core, facilitates the simultaneous transfer of uncharged NH3. In this context, we propose to elucidate at the molecular level the mechanism of ammonium translocation through rhesus protein from Nitrosomonas europaeaand establish whether there is a universal mechanism for biological ammonium transport. Beyond the elucidation of a central biological process, this work has important medical implications, as some Rh mutations have been associated with human pathologies. We propose to demonstrate how specific Rh mutations affect the activity of the protein to establish the relationship between Rh malfunction and the associated diseases.

The Effects of Co‐Expression of Rhbg with Human CA‐IV on NH 3 /NH 4 + and CO 2 Transport

Background Rhbg is a member of the mammalian glycosylated rhesus (Rh) protein family expressed in multiple tissues including the basolateral membrane of the renal collecting duct. Rhbg was found to transport NH4+ and NH3 in the collecting duct. Carbonic anhydrase IV (CA-IV) is one of several carbonic anhydrases expressed in the mammalian kidney where it catalyzes the reversible hydration of CO2. It is anchored at both apical and basolateral membranes of renal proximal tubules and the collecting duct. Objective and Hypothesis Our previous studies showed that Rhbg and CA-IV proteins co-immunoprecipitated, suggesting that there is a physical interaction between Rhbg and CA-IV. This study is to detect if there is any functional effect related to this interaction. Methods We measured transport of NH4+, NH3, and CO2 in 4 groups of Xenopus oocytes expressing the following: In the first group, we co-expressed the mouse Rhbg with human CA-IV (by injecting oocytes with Rhbg and CA-IV mRNA) and compared the measurements to 3 groups of oocytes expressing either Rhbg or CA-IV, or injected with H2O. We used ion-selective microelectrodes to measure changes in surface pH (pHs), to monitor NH3 transport, and changes in intracellular pH (pHi) to monitor NH4+ and CO2 transport. We also used two-electrode voltage-clamp to measure current change (ΔI) caused by electrogenic NH4+ transport. In each oocyte, we monitored one of the three parameters indicated above (pHs, pHi or ΔI), in response to five experimental conditions: 1) increased CO2 (1.5%CO2/10mM HCO3-); 2) NH3/NH4+ (5mM NH4Cl in HEPES); 3) Methylamine/Methylammonium, often used as a substitute for ammonium (MA/MA+, 5mM in HEPES); 4) 5mM NH3/NH4+ in 1.5%CO2; 5) 5mM MA/MA+ in 1.5% CO2. Results Our results indicate that co-expressing CA-IV with Rhbg: 1) inhibited electrogenic NH4+ transport by Rhbg in the presence and absence of CO2; 2) reduced MA and NH3 transport by Rhbg only in the presence of CO2; 3) had no detectable effect on CO2 transport by Rhbg. Conclusions We demonstrated for the first time that co-expression of Rhbg and CA-IV inhibited electrogenic transport of NH4+ by Rhbg and reduced NH3 and MA transport in the presence of CO2. The function of Rhbg as a CO2 gas channel was demonstrated as an increase in the rate of CO2-induced pHiacidification in Rhbg expressing oocytes and was not altered by expression of CA-IV. The interaction of Rhbg and CA-IV is important to explain their role in renal acid-base homeostasis. We propose that NH3/NH4+ transport by Rhbg is modified in the presence of CO2.

AQP1 and RhAG proteins play roles on oxygen offloading from murine red blood cells during aging

The diffusion of O2 across the red blood cell (RBC) membrane is critical for life. Although the traditional view had been that all O2 moves through RBC membranes by dissolving in and diffusing through the membrane lipid, our recent work (bioRxiv. doi:10.1101/2020.08.28.265066) indicates that this is not the case. Using stopped-flow absorbance spectroscopy to monitor the hemoglobin (Hb) absorbance spectrum in intact RBCs, we found that, compared with RBCs from wild-type (WT) mice, those from double knockout (dKO) mice genetically deficient in both aquaporin-1 (AQP1) and the rhesus (Rh) protein exhibit a 1/3 decrease in the rate constant for O2 offloading from Hb (kHbO2). Mathematical simulations show that this 1/3 decrease in kHbO2 corresponds to a ~55% decrease in the membrane permeability to O2 (PM,O2). In previous abstracts, we reported that the kHbO2 of RBCs from WT mice are indistinguishable from age 2–3 months through 12 months, but begins to fall at 18 months, and drops by ~13% at 29 months—the lifespan of our mice. However, the relatively low kHbO2 of RBCs from dKO mice remains stable through 29 months. In addition to increasing the number of observations for aging WT and dKO mice, we now report the effect of aging on the kHbO2 of RBCs from mice genetically deficient in just RhAG, studying mice at six ages (2–3 months, 6 months, 12 months, 18 months, 24 months, and 29 months). The kHbO2 of RBCs from RhAG-KO mice begins ~17% lower than that of WT mice at 2–3 months (see above bioRxiv ref.), and remains stable through 29 months (N = 5 to 8 at 6–24 months, 4 at 29 months), similar to that of dKO mice. Changes in hematological parameters or RBC size and shape cannot explain the observed decreases in kHbO2 in WT mice. RBC proteomics data on a single WT mouse at 29 months suggests decreases in AQP1 (11%), RhAG (19%), and mRh (27%), and AE1 (22%). We conclude from our dKO and RhAG-KO data that the non-AQP1/RhAG component of RBC membrane O2 permeability is independent of aging, and that the age-related fall in kHbO2 in WT mice is likely to be due, at least in part, to age-related decreased functional expression (i.e., combination of protein number and activity of individual proteins) of AQP1 and RhAG —which function asO2 channels. We continue to build our number of observations on WT, dKO, and RhAG-KO mice of various ages, and are now extending our work to RBCs from mice genetically deficient in just AQP1.

Acclimation to prolonged aquatic hypercarbia or air enhances hemoglobin‑oxygen affinity in an amphibious fish

When the amphibious mangrove rivulus (Kryptolebias marmoratus) leaves water for extended periods, hemoglobin-O2 binding affinity increases. We tested the hypothesis that the change in affinity was a consequence of hemoglobin isoform switching driven by exposure to environments associated with increased internal CO2 levels. We exposed K. marmoratus to either water (control, pH 8.1), air, aquatic hypercarbia (5.1 kPa CO2, pH 6.6–6.8), or aquatic acid (isocarbic control, pH 6.6–6.8), for 7 days, and measured hemoglobin-O2 affinity spectrophotometrically. We found that mangrove rivulus compensated for elevated CO2 and aquatic acid exposure by shifting hemoglobin-O2 affinity back to aquatic (control) levels when measured at an ecologically-relevant high CO2 level that would be experienced in vivo. Using proteomics, we found that the hemoglobin subunits present in the blood did not change between treatments, but air and aquatic acid exposure altered the abundance of cathodic hemoglobin subunits. We therefore conclude that hemoglobin isoform switching is not a primary strategy used by mangrove rivulus to adjust P50 under these conditions. Abundances of other RBC proteins also differed between treatment groups relative to control fish (e.g. Rhesus protein type A, band 3 anion exchanger). Overall, our data indicate that both aquatic hypercarbia and aquatic acidosis create similar changes in hemoglobin-O2 affinity as air exposure. However, the protein-level consequences differ between these groups, indicating that the red blood cell response of mangrove rivulus can be modulated depending on the environmental cue received.

Role of AQP1 and RhAG proteins in the deoxygenation of mouse red blood cells during aging

The traditional view had been that all O2 moves through red blood cell (RBC) membranes by dissolving in and diffusing through the membrane lipid. In a previous abstract, we reported data obtained using stopped‐flow analysis of hemoglobin (Hb) absorbance spectra on RBCs from wild‐type (WT) vs. double‐knockout (dKO) mice genetically deficient in both aquaporin‐1 (AQP1) and the rhesus (Rh) protein RhAG. We found that the rate constant of Hb deoxygenation (kHbO2)—which includes intra/extracellular diffusion and membrane permeability—was ~31% lower for RBCs from dKO than for WT mice. From these data, we computed that, together, AQP1 and the Rh complex account for ~60% of membrane O2 permeability. We now examine the effect of aging on kHbO2 of RBCs from WT vs. dKO mice, studying mice at six ages (2–3 months, 6 months, 12 months, 18 months, 24 months, and 29 months). The kHbO2 of RBCs from WT mice are indistinguishable from age 2–3 months through 12 months. However, compared to 2–3 months, kHbO2 is 9% lower at 18 months (p<0.05, N = 6), 12% lower at 24 months (p<0.05, N = 5), and 13% lower at 29 months (p<0.05, N = 3). For RBCs from dKO mice, kHbO2 starts off ~30% lower than that of WT mice at 2–3 months (see above), and remains at this same depressed level through 29 months (N = 5 to 6 at 6–24 months, 4 at 29 months). In the case of WT mice at 24 months, our hematology data—which show decreases for both mean corpuscular volume and mean corpuscular hemoglobin—cannot explain the observed decreases in kHbO2. DIC microscopy of living RBCs and microscopic observation of stained blood smears with RBCs from 3 months and 29 months with both WT and dKO groups confirm that RBCs are normal biconcave disks. Preliminary data from mass spectroscopy of RBC ghosts from WT mice (N = 1 each for 2–3 months and 29 months) suggests that, compared to 2–3 months, membrane protein levels at 29 months are lower by 11% for AQP1, 19% for RhAG, 27% for mRh, and 22% for AE1. We conclude from our dKO data that the non‐AQP1/RhAG component of RBC membrane O2 permeability is independent of aging. We hypothesize that the 12–13% decrease of kHbO2 at 24–29 months for WT mice is due in part to decreased expression of AQP1 and the Rh complex (i.e., the AQP1 and RhAG components of O2 membrane permeability decline with aging).Support or Funding InformationN00014‐15‐1‐2060 Gas Channels, ONRand N00014‐16‐1‐2535 ONR‐MURI

Ion uptake pathways in European sea bass Dicentrarchus labrax

Ion uptake mechanisms are diverse in fish species, certainly linked to duplication events that have led to the presence of a multitude of paralogous genes. In fish, Na+ uptake involves several ion transporters expressed in different ionocyte subtypes. In the European sea bass Dicentrarchus labrax, several key transporters potentially involved in Na+ uptake have been investigated in seawater (SW) and following a 2 weeks freshwater (FW) acclimation. Using gel electrophoresis, we have shown that the Na+/H+-exchanger 3 (nhe3, slc9a3) is expressed in gills and kidney at both salinities. Quantitative realtime PCR analysis showed a significantly higher nhe3 expression in fresh water (FW) compared to SW. Its apical localization in a subset of gill ionocytes in freshwater-acclimated fish supports the role of NHE3 in Na+ uptake. Interestingly, NHE3-immunopositive cells also express basolateral Na+/K+/2Cl− cotransporter 1 (NKCC1) and are mainly localized in gill lamella. Among the three nhe2 (slc9a2) paralogs, only nhe2c shows differential branchial expression levels with higher mRNA levels in SW than in FW. The increased branchial expression of the ammonia transporter rhcg1 (Rhesus protein), nhe3 and cytoplasmic carbonic anhydrase (cac) in FW could indicate the presence of a functional coupling between ion transporters to form a Na+/NH4+ exchange complex. Acid-sensing ion channel 4 (asic4) seems not to be expressed in sea bass gills. Na+/Cl- cotransporter (ncc2a or ncc-like) is about three times more expressed in FW compared to SW suggesting coupled Na+ and Cl− uptake in a subset of gill ionocytes. Besides the main pump Na+/K+-ATPase, branchial NCC2a and NHE3 may be key players in ion uptake in sea bass following a long-term freshwater challenge.

Difference in Nitrogen Starvation-Inducible Expression Patterns among Phylogenetically Diverse Ammonium Transporter Genes in the Red Seaweed <i>Pyropia yezoensis</i>

Nitrogen deficiency induces senescence and the expression of genes encoding ammonium transporters (AMTs) in terrestrial plants where the AMT family is subdivided into AMT1 and AMT2 subfamilies. Nitrogen starvation in the red seaweed Pyropia yezoensis causes senescence-like discoloration. In this study, we identified five genes in P. yezoensis encoding AMT domain-containing proteins, which were phylogenetically categorized into the AMT1 subfamily. We also found a gene encoding a Rhesus protein (Rh) that was related to, but diverged from, AMTs. Moreover, our phylogenetic analysis showed that AMT domain-containing proteins from micro- and macro-algae belonged to either the AMT1 or Rh subfamily, indicating the absence of AMT2 in algae. Gene expression analyses revealed the presence of gametophyte- and sporophyte-specific AMT1 genes that were up-regulated transiently and continually, respectively, under nitrogen-deficient conditions. In addition, up-regulated sporophyte-specific gene expression was suppressed when nitrogen was resupplied. Accordingly, an expansion of the ancient AMT gene has produced AMT1 functional variants differing in temporal and nitrogen starvation-inducible expression patterns during the life cycle of P. yezoensis. These findings help elucidate the unique nutrition starvation responses involving functionally diverse AMT1 and Rh subfamilies in red seaweed.

Perfused Gills Reveal Fundamental Principles of pH Regulation and Ammonia Homeostasis in the Cephalopod Octopus vulgaris.

In contrast to terrestrial animals most aquatic species can be characterized by relatively higher blood concentrations despite its potential toxicity to the central nervous system. Although many aquatic species excrete via specialized epithelia little information is available regarding the mechanistic basis for NH3/ homeostasis in molluscs. Using perfused gills of Octopus vulgaris we studied acid-base regulation and ammonia excretion pathways in this cephalopod species. The octopus gill is capable of regulating ammonia (NH3/) homeostasis by the accumulation of ammonia at low blood levels (<260 μM) and secretion at blood ammonia concentrations exceeding in vivo levels of 300 μM. transport is sensitive to the adenylyl cyclase inhibitor KH7 indicating that this process is mediated through cAMP-dependent pathways. The perfused octopus gill has substantial pH regulatory abilities during an acidosis, accompanied by an increased secretion of . Immunohistochemical and qPCR analyses revealed tissue specific expression and localization of Na+/K+-ATPase, V-type H+-ATPase, Na+/H+-exchanger 3, and Rhesus protein in the gill. Using the octopus gill as a molluscan model, our results highlight the coupling of acid-base regulation and nitrogen excretion, which may represent a conserved pH regulatory mechanism across many marine taxa.

(Uncommon) Mechanisms of Branchial Ammonia Excretion in the Common Carp (Cyprinus carpio) in Response to Environmentally Induced Metabolic Acidosis.

Freshwater fishes generally increase ammonia excretion in acidic waters. The new model of ammonia transport in freshwater fish involves an association between the Rhesus (Rh) protein Rhcg-b, the Na(+)/H(+) exchanger (NHE), and a suite of other membrane transporters. We tested the hypothesis that Rhcg-b and NHE3 together play a critical role in branchial ammonia excretion in common carp (Cyprinus carpio) chronically exposed to a low-pH environment. Carp were exposed to three sequential environmental treatments-control pH 7.6 water (24 h), pH 4.0 water (72 h), and recovery pH 7.6 water (24 h)-or in a separate series were simply exposed to either control (72 h) or pH 4.0 (72 h) water. Branchial ammonia excretion was increased by ∼2.5-fold in the acid compared with the control period, despite the absence of an increase in the plasma-to-water partial pressure NH3 gradient. Alanine aminotransferase activity was higher in the gills of fish exposed to pH 4 versus control water, suggesting that ammonia may be generated in gill tissue. Gill Rhcg-b and NHE3b messenger RNA levels were significantly elevated in acid-treated relative to control fish, but at the protein level Rhcg-b decreased (30%) and NHE3b increased (2-fold) in response to water of pH 4.0. Using immunofluorescence microscopy, NHE3b and Rhcg-b were found to be colocalized to ionocytes along the interlamellar space of the filament of control fish. After 72 h of acid exposure, Rhcg-b staining almost disappeared from this region, and NHE3b was more prominent along the lamellae. We propose that ammoniagenesis within the gill tissue itself is responsible for the higher rates of branchial ammonia excretion during chronic metabolic acidosis. Unexpectedly, gill Rhcg-b does not appear to be important in gill ammonia transport in low-pH water, but the strong induction of NHE3b suggests that some NH4(+) may be eliminated directly in exchange for Na(+). These findings contrast with previous studies in larval zebrafish (Danio rerio) and medaka (Oryzias latipes), underlining the importance of species comparisons.

Mechanism of ammonia excretion in the freshwater leech Nephelopsis obscura: characterization of a primitive Rh protein and effects of high environmental ammonia.

Remarkably little is known about nitrogenous excretion in freshwater invertebrates. In the current study, the nitrogen excretion mechanism in the carnivorous ribbon leech, Nephelopsis obscura, was investigated. Excretion experiments showed that the ribbon leech is ammonotelic, excreting 166.0 ± 8.6 nmol·grams fresh weight (gFW)(-1)·h(-1) ammonia and 14.7 ± 1.9 nmol·gFW(-1)·h(-1) urea. Exposure to high and low pH hampered and enhanced, respectively, ammonia excretion rates, indicating an acid-linked ammonia trapping mechanism across the skin epithelia. Accordingly, compared with body tissues, the skin exhibited elevated mRNA expression levels of a newly identified Rhesus protein and at least in tendency the Na(+)/K(+)-ATPase. Pharmacological experiments and enzyme assays suggested an ammonia excretion mechanism that involves the V-ATPase, Na(+)/K(+)-ATPase, and carbonic anhydrase, but not necessarily a functional microtubule system. Most importantly, functional expression studies of the identified Rh protein cloned from leech skin tissue revealed an ammonia transport capability of this protein when expressed in yeast. The leech Rh-ammonia transporter (NoRhp) is a member of the primitive Rh protein family, which is a sister group to the common ancestor of vertebrate ammonia-transporting Rh proteins. Exposure to high environmental ammonia (HEA) caused a new adjustment of body ammonia, accompanied with a decrease in NoRhp and Na(+)/K(+)-ATPase mRNA levels, but unaltered ammonia excretion rates. To our knowledge, this is only the second comprehensive study regarding the ammonia excretion mechanisms in a freshwater invertebrate, but our results show that basic processes of ammonia excretion appear to also be comparable to those found in freshwater fish, suggesting an early evolution of ionoregulatory mechanisms in freshwater organisms.

Branchial NH 4 + ‐dependent Acid‐base Transport Mechanisms and Energy Metabolism of Squid ( Sepioteuthis lessoniana ) Affected by Seawater Acidification

CO2 induced pH fluctuation in the extracellular compartments are a unifying physiological phenomenon that all animals are confronted with. Cephalopods have evolved acid-base regulatory abilities to accommodate temporal pH fluctuations via highly pH sensitive hemocyanins. Interestingly, it has been suggested that metabolic energetic limitations may be a critical factor when exposed to acidified seawater. Here we show the effects of short-term (few hours) to medium-term (up to 168 h) seawater acidification on pelagic squids, Sepioteuthis lessoniana. Routine metabolic rates, NH4+ secretion and extracellular acid-base balance were monitored during exposure to control (pH 8.1) and acidified conditions of pH 7.7 and 7.3 along a period of 168h. Metabolic rates were significantly depressed by 40% after 168 hours in pH 7.3 conditions exposure. The extracellular pH is fully restored within 20 h and is maintained along the period of 168 h, and the accompanied acid-base transporter transcripts were up-regulated including V-type H+-ATPase (VHA), Rhesus protein (RhP), Na/HCO3- cotransporter (NBC) and cystolic carbonic anhydrase (CAc). Immunohistochemical analyses demonstrated co-localization of Na+/K+-ATPase (NKA), VHA in basolateral and Na+/H+-exchanger 3 (NHE3), RhP in apical membranes of the branchial epithelium. Protein levels of branchial VHA and RhP were increased in response to acidified conditions. The present work demonstrated the NH4+ based proton secretion via RhP may contribute to a well-developed acid-base regulatory machinery in branchial epithelia of cephalopods.

Studies of Neurohypophysial System on Epithelial Cells Functions in Cephalopod Embryos

Despite epithelial acid-base regulatory abilities described for cephalopods, the evo-devo basis for extracellular pH homeostasis is still poorly understood in these non-model organisms. In this study expressions of epidermal morphogenesis marker (ΔNp63) and neurohypophysial hormone (sepiatocin) receptors were examined by using embryos of cuttlefishSepia pharaonis. Using CO2 perturbation experiments, key players of epithelial acid-base regulation in different ontogenetic stages, including V-type H+-ATPase (VHA), Na+/HCO3- co-transporter (NBC), Na+/H+ exchanger3 (NHE3), Rhesus protein (RhP) and Na+/H+ exchanger3 (NHE3) as well as ΔNp63 and sepiatocin receptor 2 (str2) were apparently affected by ambient hypercapnic challenges.Those transcripts expressions inferred that the activations of ΔNp63 and STR2 might be involved in operating epidermal ion fluxes. Moreover in situ hybridization images showed that both ΔNp63 and STR2 were expressed on epithelium and gills in embryos of S. pharaonis supporting the central sites of acid-base regulatory mechanism. The present work suggests that in convergence to teleosts, cephalopod embryos have evolved sophisticated evolution pathway regarding epithelium differentiation and neurohypophysial hormones regulation to cope with acid-base disturbances during their oviparous development.

Ammonia excretion in Caenorhabditis elegans: mechanism and evidence of ammonia transport of the Rhesus protein CeRhr-1.

The soil-dwelling nematode Caenorhabditis elegans is a bacteriovorous animal, excreting the vast majority of its nitrogenous waste as ammonia (25.3±1.2 µmolgFW(-1)day(-1)) and very little urea (0.21±0.004 µmolgFW(-1)day(-1)). Although these roundworms have been used for decades as genetic model systems, very little is known about their strategy to eliminate the toxic waste product ammonia from their bodies into the environment. The current study provides evidence that ammonia is at least partially excreted via the hypodermis. Starvation reduced the ammonia excretion rates by more than half, whereas mRNA expression levels of the Rhesus protein CeRhr-2, V-type H(+)-ATPase (subunit A) and Na(+)/K(+)-ATPase (α-subunit) decreased correspondingly. Moreover, ammonia excretion rates were enhanced in media buffered to pH 5 and decreased at pH 9.5. Inhibitor experiments, combined with enzyme activity measurements and mRNA expression analyses, further suggested that the excretion mechanism involves the participation of the V-type H(+)-ATPase, carbonic anhydrase, Na(+)/K(+)-ATPase, and a functional microtubule network. These findings indicate that ammonia is excreted, not only by apical ammonia trapping, but also via vesicular transport and exocytosis. Exposure to 1 mmoll(-1) NH4Cl caused a 10-fold increase in body ammonia and a tripling of ammonia excretion rates. Gene expression levels of CeRhr-1 and CeRhr-2, V-ATPase and Na(+)/K(+)-ATPase also increased significantly in response to 1 mmoll(-1) NH4Cl. Importantly, a functional expression analysis showed, for the first time, ammonia transport capabilities for CeRhr-1 in a phylogenetically ancient invertebrate system, identifying these proteins as potential functional precursors to the vertebrate ammonia-transporting Rh-glycoproteins.

Branchial NH4+-dependent acid–base transport mechanisms and energy metabolism of squid (Sepioteuthis lessoniana) affected by seawater acidification

Cephalopods have evolved strong acid–base regulatory abilities to cope with CO2 induced pH fluctuations in their extracellular compartments to protect gas transport via highly pH sensitive hemocyanins. To date, the mechanistic basis of branchial acid–base regulation in cephalopods is still poorly understood, and associated energetic limitations may represent a critical factor in high power squids during prolonged exposure to seawater acidification. The present work used adult squid Sepioteuthis lessoniana to investigate the effects of short-term (few hours) to medium-term (up to 168 h) seawater acidification on pelagic squids. Routine metabolic rates, NH4+ excretion, extracellular acid–base balance were monitored during exposure to control (pH 8.1) and acidified conditions of pH 7.7 and 7.3 along a period of 168 h. Metabolic rates were significantly depressed by 40% after exposure to pH 7.3 conditions for 168 h. Animals fully restored extracellular pH accompanied by an increase in blood HCO3− levels within 20 hours. This compensation reaction was accompanied by increased transcript abundance of branchial acid–base transporters including V-type H+-ATPase (VHA), Rhesus protein (RhP), Na+/HCO3− cotransporter (NBC) and cytosolic carbonic anhydrase (CAc). Immunocytochemistry demonstrated the sub-cellular localization of Na+/K+-ATPase (NKA), VHA in basolateral and Na+/H+-exchanger 3 (NHE3) and RhP in apical membranes of the ion-transporting branchial epithelium. Branchial VHA and RhP responded with increased mRNA and protein levels in response to acidified conditions indicating the importance of active NH4+ transport to mediate acid–base balance in cephalopods. The present work demonstrated that cephalopods have a well developed branchial acid–base regulatory machinery. However, pelagic squids that evolved a lifestyle at the edge of energetic limits are probably more sensitive to prolonged exposure to acidified conditions compared to their more sluggish relatives including cuttlefish and octopods.

A1A2BO and Rh gene frequencies among six populations of Jammu and Kashmir, India

A study was undertaken to record gene frequencies of ABO blood groups, their subtypes and Rh antigen for six different endogamous groups including a tribal population. The ABO phenotypic frequency varies among six different populations showing significant difference (p<0.0005). Gujjar and Bakarwal (a tribal population) shows highest (42.29%) of B blood phenotypes. A1 is the highest among Syeds (39.31%), O blood group frequency highest among Mughals (43.23%) and A1B and A2B are rare phenotypes showing very low frequency among all populations. The pattern of allele frequencies (p<0.025) is in order of I(O)>I(B)>I(A1)>I(A2), except Syeds (I(O)>I(A1)>I(B)>I(A2)). The rhesus protein (Rh) phenotypic frequency (p<0.01) shows significant increase in Rh(D) positive (87.86% in Syed to 96.03% in Khan) among all populations. The Rh allele (p<0.05) and genotype (p<0.02) frequencies shows a significant difference. Heterozygosity for Rh protein is less than homozygosity among six populations. The result from this study provides information on the genetic variation in blood antigens and rhesus protein among human populations inhabiting Jammu and Kashmir.

Two Rhesus protein ammonia transporters team up to eliminate ammonium into urine

renal ammoniagenesis serves the de novo synthesis of bicarbonate consumed by metabolism and the excretion of acid. Ammonium is eventually excreted into urine after accumulation in the medullary interstitium. The process of ammonium excretion was long thought to be a mostly passive process mediated

Branchial NH

BackgroundCephalopods have evolved strong acid–base regulatory abilities to cope with CO2 induced pH fluctuations in their extracellular compartments to protect gas transport via highly pH sensitive hemocyanins. To date, the mechanistic basis of branchial acid–base regulation in cephalopods is still poorly understood, and associated energetic limitations may represent a critical factor in high power squids during prolonged exposure to seawater acidification.ResultsThe present work used adult squid Sepioteuthis lessoniana to investigate the effects of short-term (few hours) to medium-term (up to 168 h) seawater acidification on pelagic squids. Routine metabolic rates, NH4+ excretion, extracellular acid–base balance were monitored during exposure to control (pH 8.1) and acidified conditions of pH 7.7 and 7.3 along a period of 168 h. Metabolic rates were significantly depressed by 40% after exposure to pH 7.3 conditions for 168 h. Animals fully restored extracellular pH accompanied by an increase in blood HCO3− levels within 20 hours. This compensation reaction was accompanied by increased transcript abundance of branchial acid–base transporters including V-type H+-ATPase (VHA), Rhesus protein (RhP), Na+/HCO3− cotransporter (NBC) and cytosolic carbonic anhydrase (CAc). Immunocytochemistry demonstrated the sub-cellular localization of Na+/K+-ATPase (NKA), VHA in basolateral and Na+/H+-exchanger 3 (NHE3) and RhP in apical membranes of the ion-transporting branchial epithelium. Branchial VHA and RhP responded with increased mRNA and protein levels in response to acidified conditions indicating the importance of active NH4+ transport to mediate acid–base balance in cephalopods.ConclusionThe present work demonstrated that cephalopods have a well developed branchial acid–base regulatory machinery. However, pelagic squids that evolved a lifestyle at the edge of energetic limits are probably more sensitive to prolonged exposure to acidified conditions compared to their more sluggish relatives including cuttlefish and octopods.

Dysregulation of the Glutamine Transporter Slc38a3 (SNAT3) and Ammoniagenic Enzymes in Obese, Glucose-Intolerant Mice

Background/Aims: Uric acid nephrolithiasis is prevalent among patients with type 2 diabetes and metabolic syndrome; it is correlated with an acidic urine and lower urinary ammonium excretion and is likely associated with insulin resistance. Insulin stimulates ammoniagenesis in renal cell lines via increased phosphate-dependent glutaminase (PDG) activity and glutamine metabolism. Ammonium excretion into the proximal tubule is mediated at least in part by the Na⁺/H⁺-exchanger NHE3 and in the collecting duct involving the Rhesus protein RhCG. Here we tested, whether obesity and insulin resistance in a diet-induced mouse model could contribute to deranged ammonium excretion. Methods: Obesity was induced by diet in mice and the impact on key molecules of proximal tubular ammoniagenesis and urinary acid excretion tested. Results: Diet-induced obesity was confirmed by pathological intraperitoneal glucose tolerance tests (IPGTT). Three groups of mice were compared: control mice; obese, glucose-intolerant with abnormal IPGTT (O-GI); or moderate weight with normal IPGTT (Non-Responders, NR). Basal urinary ammonium excretion did not differ among groups. However, acid loading increased urinary ammonium excretion in all groups, but to a lesser extent in the O-GI group. SNAT3 mRNA expression was enhanced in both obese groups. PDG expression was elevated only in acid-loaded O-GI mice, whereas PEPCK was enhanced in both O-GI and NR groups given NH₄CI. NHE activity in the brush border membrane of the proximal tubule was strongly reduced in the O-GI group whereas RhCG expression was similar. Conclusion: In sum, obesity and glucose intolerance impairs renal ammonium excretion in response to NH₄CI feeding most likely through reduced NHE activity. The stimulation of SNAT3 and ammoniagenic enzyme expression may be compensatory but futile.

Development in a naturally acidified environment: Na+/H+-exchanger 3-based proton secretion leads to CO2 tolerance in cephalopod embryos.

BackgroundRegulation of pH homeostasis is a central feature of all animals to cope with acid–base disturbances caused by respiratory CO2. Although a large body of knowledge is available for vertebrate and mammalian pH regulatory systems, the mechanisms of pH regulation in marine invertebrates remain largely unexplored.ResultsWe used squid (Sepioteuthis lessoniana), which are known as powerful acid–base regulators to investigate the pH regulatory machinery with a special focus on proton secretion pathways during environmental hypercapnia. We cloned a Rhesus protein (slRhP), V-type H+-ATPase (slVHA) and the Na+/H+ exchanger 3 (slNHE3) from S. lessoniana, which are hypothesized to represent key players in proton secretion pathways among different animal taxa. Specifically designed antibodies for S. lessoniana demonstrated the sub-cellular localization of NKA, VHA (basolateral) and NHE3 (apical) in epidermal ionocytes of early life stages. Gene expression analyses demonstrated that slNHE3, slVHA and slRhP are up regulated in response to environmental hypercapnia (pH 7.31; 0.46 kPa pCO2) in body and yolk tissues compared to control conditions (pH 8.1; 0.045 kPa pCO2). This observation is supported by H+ selective electrode measurements, which detected increased proton gradients in CO2 treated embryos. This compensatory proton secretion is EIPA sensitive and thus confirms the central role of NHE based proton secretion in cephalopods.ConclusionThe present work shows that in convergence to teleosts and mammalian pH regulatory systems, cephalopod early life stages have evolved a unique acid–base regulatory machinery located in epidermal ionocytes. Using cephalopod molluscs as an invertebrate model this work provides important insights regarding the unifying evolutionary principles of pH regulation in different animal taxa that enables them to cope with CO2 induced acid–base disturbances.

Exploring acid secretion machinery in embryos of marine mollusks cephalopod under hypercapnic environment

The present studies investigated effects of elevated seawater pCO2 on acid secretion mechanism in early life stages of squid Sepioteuthis lessoniana. Although cephalopods have already proved as powerful acid‐base regulators, which tightly regulate their pH homeostasis; however the acid‐base regulatory machinery is virtually unexplored. Thus, a major focus of this work has been dedicated to the mechanistic understanding of proton secretion pathways in epithelia of squid embryos during environmental hypercapnia. We cloned a Na+/H+ exchanger 3 (slNHE3), V‐type H+‐ATPase (slVHA) and Rhesus protein (slRhP) from squid, S. lessoniana, which are hypothesized to represent key players in proton secretion pathways of marine organisms. In situ hybridization and immunohistochemical analyses demonstrate the expression of slNHE3 and slVHA in epidermal ionocytes, which are rich in mitochondria, and thus, can be referred to as mitochondria‐rich cells (MRCs). Gene expression analysis demonstrates that putative acid‐base transporters, including slNHE3, slVHA and slRhP are up‐regulated in response to environmental hypercapnia (pH 7.4; 0.46 kPa pCO2) in body and yolk tissues. This observation is further supported by selective ion electrode measurements, which demonstrate increased proton gradients on the yolk epithelium of CO2 treated embryos. The present work suggests that in convergence to teleosts, cephalopod embryos have evolved sophisticated physiological mechanisms to cope with acid‐base disturbances during their oviparous development.