Kenneth M. Sterling

Heavy metal detoxification in crustacean epithelial lysosomes: role of anions in the compartmentalization process

SUMMARY Crustacean hepatopancreatic lysosomes are organelles of heavy metal sequestration and detoxification. Previous studies have shown that zinc uptake by lysosomal membrane vesicles (LMV) occurred by a vanadate- and thapsigargin-sensitive ATPase that was stimulated by a transmembrane proton gradient established by a co-localized V-ATPase associated with this organelle. In the present study, hepatopancreatic LMV from the American lobster Homarus americanus were prepared by standard centrifugation methods and 65Zn2+, 36Cl–, 35SO42– and 14C-oxalate2– were used to characterize the interactions between the metal and anions during vesicular detoxification events. Vesicles loaded with SO42– or PO43– led to a threefold greater steady-state accumulation of Zn2+ than similar vesicles loaded with mannitol, Cl– or oxalate2–. The stimulation of 65Zn2+ uptake by intravesicular sulfate was SO42– concentration dependent with a maximal enhancement at 500 μmol l–1. Zinc uptake in the presence of ATP was proton-gradient enhanced and electrogenic, exhibiting an apparent exchange stoichiometry of 1Zn+/3H+. 35SO42– and 14C-oxalate2– uptakes were both enhanced in vesicles loaded with intravesicular Cl– compared to vesicles containing mannitol, suggesting the presence of anion countertransport. 35SO42– influx was a sigmoidal function of external [SO42–] with 25 mmol l–1 internal [Cl–], or with several intravesicular pH values (e.g. 7.0, 8.0 and 9.0). In all instances Hill coefficients of approximately 2.0 were obtained, suggesting that 2 sulfate ions exchange with single Cl– or OH– ions. 36Cl– influx was a sigmoidal function of external [Cl–] with intravesicular pH of 7.0 and 9.0. A Hill coefficient of 2.0 was also obtained, suggesting the exchange of 2 Cl– for 1 OH–. 14C-oxalate influx was a hyperbolic function of external [oxalate2–] with 25 mmol l–1 internal [Cl–], suggesting a 1:1 exchange of oxalate2– for Cl–. As a group, these experiments suggest the presence of an anion exchange mechanism exchanging monovalent for polyvalent anions. Polyvalent inorganic anions (SO42– and PO43–) are known to associate with metals inside vesicles and a detoxification model is presented that suggests how these anions may contribute to concretion formation through precipitation with metals at appropriate vesicular pH.

Transepithelial d-glucose and d-fructose transport across the American lobster, Homarus americanus, intestine

SUMMARY Transepithelial transport of dietary d-glucose and d-fructose was examined in the lobster Homarus americanus intestine using d-[3H]glucose and d-[3H]fructose. Lobster intestines were mounted in a perfusion chamber to determine transepithelial mucosal to serosal (MS) and serosal to mucosal (SM) transport mechanisms of glucose and fructose. Both MS glucose and fructose transport, as functions of luminal sugar concentration, increased in a hyperbolic manner, suggesting the presence of mucosal transport proteins. Phloridizin inhibited the MS flux of glucose, but not that of fructose, suggesting the presence of a sodium-dependent (SGLT1)-like glucose co-transporter. Immunohistochemical analysis, using a goat anti-rabbit GLUT5 polyclonal antibody, revealed the localization of a brush border GLUT5-like fructose transport protein. MS fructose transport was decreased in the presence of mucosal phloretin in warm spring/summer animals, but the same effect was not observed in cold autumn/winter animals, suggesting a seasonal regulation of sugar transporters. Mucosal phloretin had no effect on MS glucose transport. Both SM glucose and SM fructose transport were decreased in the presence of increasing concentrations of serosal phloretin, providing evidence for the presence of a shared serosal GLUT2 transport protein for the two sugars. The transport of d-glucose and d-fructose across lobster intestine is similar to sugar uptake in mammalian intestine, suggesting evolutionarily conserved absorption processes for these solutes.

Identification of a novel sodium-dependent fructose transport activity in the hepatopancreas of the Atlantic lobster Homarus americanus.

SUMMARY [3H]Fructose and [3H]glucose transport were determined in brush-border membrane vesicles (BBMV), basolateral membrane vesicles (BLMV) and isolated cells (E, R, F, B) of H. americanus (Atlantic lobster) hepatopancreas. Glucose transport in BBMV was equilibrative in the absence of sodium and concentrative in the presence of sodium. Sodium-dependent glucose transport by BBMV was not inhibited by a tenfold molar excess of fructose. Glucose transport by BLMV was equilibrative and sodium independent. Fructose uptake by BBMV and BLMV was equilibrative in the absence of sodium and concentrative in the presence of sodium. This enhancement was not affected by a tenfold molar excess of glucose in the presence of sodium. E-, F- and B-cells showed sodium-dependent uptake of fructose, while R-cells did not. Sodium-dependent fructose uptake by E-cells was not inhibited by a tenfold molar excess of glucose or mannose. Western blot analysis of BBMV, BLMV and E-, R-, F- and B-cells using rabbit polyclonal antibodies directed against epitopes of mammalian GLUT2, GLUT5, SGLT1 and SGLT4 indicated the presence of cross-reacting lobster proteins. Sequence alignment of the mammalian proteins with translated, lobster expressed sequence tags also indicated significant identity between species. Comparison of fructose and glucose uptake in the absence and presence of sodium by BBMV, BLMV and isolated cells indicated the presence of a distinct sodium-dependent transport activity for each sugar in the Atlantic lobster.

Dual control of cytosolic metals by lysosomal transporters in lobster hepatopancreas

SUMMARY This study describes the membrane transport mechanisms used by lobster (Homarus americanus) hepatopancreatic epithelial lysosomes to accumulate and sequester heavy metals from the cytosol, and thereby aid in the regulation of these ions entering the animal from dietary constituents. The present investigation extends previous work describing lysosomal metal uptake by cation exchange with protons and suggests that a second, parallel, lysosomal transport process involving metal—thiol conjugates may work in conjunction with the cation antiporter to control cytoplasmic metal concentrations. Transport of 65Zn2+ by lysosomal membrane vesicles (LMV) incubated in 1 mmol l−1 glutathione (GSH) was not significantly different from metal transport in the absence of the tripeptide. However, preloading LMV with 1 mmol l−1 α-ketoglutarate (AKG), and then incubating in a medium containing 1 mmol l−1 GSH, more than doubled metal uptake, compared with vesicles equilibrated with chloride or possessing an outwardly directed chloride gradient. Kinetic analysis of lysosomal 65Zn2+ influx as a function of zinc concentration, in vesicles containing 1 mmol l−1 AKG and incubated in 1 mmol l−1 GSH, revealed the presence of a sigmoidal, low affinity, high capacity carrier process transporting the metal into the organelle. These data indicated the possible presence of an organic anion exchanger in lobster lysosomal membranes. Western blot analysis of LMV with a rabbit anti-rat OAT1 antibody showed the presence of an orthologous OAT1-like protein (approximate molecular mass of 80 kDa) signal from these membranes. These results, and those published previously, suggest the occurrence of two metal transporters on hepatopancreatic membranes, a high affinity, low capacity cation antiporter and a low affinity, high capacity organic anion exchanger. Together these two systems have the potential to regulate cytoplasmic metals over a wide concentration range.

H(+) V-ATPase-energized transporters in brush border membrane vesicles from whole larvae of Aedes aegypti.

Brush border membrane vesicles (BBMVs) from Whole larvae of Aedes aegypti (AeBBMVWs) contain an H(+) V-ATPase (V), a Na(+)/H(+) antiporter, NHA1 (A) and a Na(+)-coupled, nutrient amino acid transporter, NAT8 (N), VAN for short. All V-ATPase subunits are present in the Ae. aegypti genome and in the vesicles. AgNAT8 was cloned from Anopheles gambiae, localized in BBMs and characterized in Xenopus laevis oocytes. AgNHA1 was cloned and localized in BBMs but characterization in oocytes was compromised by an endogenous cation conductance. AeBBMVWs complement Xenopus oocytes for characterizing membrane proteins, can be energized by voltage from the V-ATPase and are in their natural lipid environment. BBMVs from caterpillars were used in radio-labeled solute uptake experiments but approximately 10,000 mosquito larvae are needed to equal 10 caterpillars. By contrast, functional AeBBMVWs can be prepared from 10,000 whole larvae in 4h. Na(+)-coupled (3H)phenylalanine uptake mediated by AeNAT8 in AeBBMVs can be compared to the Phe-induced inward Na(+) currents mediated by AgNAT8 in oocytes. Western blots and light micrographs of samples taken during AeBBMVW isolation are labeled with antibodies against all of the VAN components. The use of AeBBMVWs to study coupling between electrogenic V-ATPases and the electrophoretic transporters is discussed.

Glucose and fructose uptake by Limulus polyphemus hepatopancreatic brush border and basolateral membrane vesicles: evidence for Na+-dependent sugar transport activity

Abstract[3H]-fructose and [3H]-glucose transport activities were determined in brush border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) from Limulus polyphemus (horseshoe crab) hepatopancreas. Glucose transport was equilibrative in the absence of sodium and sodium dependent in the presence of sodium in BBMV, suggesting GLUT-like and SGLT-like transport activity. Glucose transport by BLMV was equilibrative and sodium independent. Fructose uptake by BBMV and BLMV was equilibrative in the absence of sodium and sodium dependent in the presence of sodium. Western blot analysis using a rabbit anti-mouse SGLT-1 polyclonal antibody indicated the presence of a cross-reacting horseshoe crab BBMV protein of similar molecular weight to the mammalian SGLT1. Sequence alignment of the mouse SGLT-4 and SGLT1 with a translated, horseshoe crab-expressed sequence tag also indicated significant identity between species. Fructose and glucose uptake in the absence and presence of sodium by hepatopancreas BBMV and BLMV indicated the presence of sodium-dependent transport activity for each sugar that may result from the presence of transporters similar to those described for other species.

The procollagen type III, alpha 1 (COL3A1) gene first intron expresses poly‐A+ RNA corresponding to multiple ESTs and putative miRNAs

The mouse COL3A1 first intron is 9684 bp. RNAs of approximately 1.6 and 3.0 kb were detected by Northern hybridization analysis of poly‐A RNA from fetal mice and total RNA from suckling and adult mouse intestine using 32P‐labeled, anti‐sense RNA synthesized from a mouse COL3A1 first intron, 5 prime region, 5.4 kb Xba I fragment (1655–7030 bp), recombinant plasmid (pPI5.4x). Expression of the 1.6 and 3.0 kb RNAs was significantly reduced in adult mouse intestine, indicating that these RNAs are developmentally regulated. “BLAST” analysis indicated that the mouse first intron 5 prime sequence has 94–100% identity to 13 mouse ESTs. These mouse first intron ESTs lie within the 5.4 Xba I fragment of the mouse COL3A1 first intron. Two of the mouse first intron ESTs have significant identity to known miRNA, mature sequences, mmu‐miR‐466f‐3P, mmu‐miR‐1187, and mmu‐miR‐574‐5P as well as others. Predicted targets for mmu‐miR‐466f‐3P include COL1A1, COL19A1, COL11A2, COL4A1, and COL4A5 indicating that COL3A1 intronic miRNAs may regulate the expression of other collagen genes in development. J. Cell. Biochem. 112: 541–547, 2011.

Heavy Metal Transport and Detoxification by Crustacean Epithelial Lysosomes

Lysosomes are multi-functional organelles that aid in the disassembly of large organic molecules and store a variety of xenobiotics. Lysosomes, and vacuolar components of the endo-membrane system, play apparently ubiquitous sequestration and detoxification roles for heavy metals in cells of many organisms. X-ray microprobe analysis of metal-containing granules (concretions) in these organelles from many animal phyla suggest that monovalent, divalent, and trivalent metal cations can be stored in these compartments in conjunction with anionic elements such as phosphorus and sulfur. There is also evidence that thiol-containing compounds such as glutathione and metallothionein, which bind metals in the cytoplasm with high affinity, may also be translocated across lysosomal membranes for metal storage. Few studies have examined the nature of the sequestration and detoxification processes for heavy metals displayed by invertebrate lysosomes or other endo-membrane components. This review summarizes recent investigations focused on lysosomal function in crustacean hepatopancreatic absorptive epithelia. It describes the carrier-mediated transport processes that occur on lysosomal membranes for accumulating metals from the cytoplasm and how these metal transporters are linked with the uptake of multivalent anions that may precipitate concretions within the organelle at appropriate ion concentrations and pH conditions. In addition, preliminary data describing the potential role of the Organic Anion Transporter (OAT) in transporting glutathione with its associated metal load from cytoplasm to lysosomal interior are described. A model summarizing proposed coupling between cationic metal and polyvalent anion transports and how they might be linked with concretion formation and metal detoxification is presented.