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J Lytton

Publications and source records attributed to J Lytton.

At least 37 records · Page 2Linked to original sources
PubMed1996

Apical localization of the Na-K-Cl cotransporter, rBSC1, on rat thick ascending limbs.

A bumetanide-sensitive Na-K-Cl cotransporter (rBSC1) was recently cloned from a rat renal outer medulla (OM) cDNA library and shown to be expressed predominantly in the kidney. The purpose of the present study was to examine the nephron distribution of cotransporter transcripts and protein in rat kidney. In situ hybridization showed an intense signal only in the outer medulla and extending along cortical medullary rays consistent with expression of rBSC1 transcripts in medullary (MTAL) and cortical (CTAL) thick ascending limbs. Polyclonal antibodies raised in rabbits against a unique 67 amino acid segment from the carboxyl terminus of rBSC1 identified a broad major band of 130 to 160 (midpoint of 150) kDa and at least two minor bands of 50 to 70 kD on Western blotting of homogenates from cortex (C) and outer medulla (OM), but not inner medulla (IM), of rat kidney. Thus the Na-K-Cl cotransporter protein detected by the polyclonal rBSC1 antibody in rat kidney was similar in size to the major approximately 150 kD bumetanide binding protein detected by others in mouse and dog kidneys. Immunofluorescence studies using the anti-rBSC1 polyclonal antibody on rat kidney sections showed an intense signal limited to apical surfaces of MTAL and CTAL segments. Colocalization with anti-Tamm-Horsfall antibody which is present in all TABA cells except macula densa cells confirmed the absence of anti-rBSC1 fluorescence in the macula densa cells. These results are consistent with rBSC1 encoding the, or the major isoform of the, apical Na-K-Cl cotransporter in the thick ascending limb. The Na-K-Cl cotransporter functionally detected in macula densa cells may be encoded by a different BSC isoform.

Animals↗
PubMed1996

Localization and regulation by vitamin D of calcium transport proteins in rabbit cortical collecting system.

The 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]-induced expression of Na+/Ca2+ exchanger, Ca(2+)-adenosinetriphosphatase (Ca(2+)-ATPase), and calbindin-D28k was investigated in the rabbit distal nephron. Immunocytochemical studies in rabbit kidney sections revealed colocalization of the three Ca2+ transport proteins in the majority of cells in the distal nephron, including connecting tubules and cortical collecting ducts. Subsequently, rabbit connecting and cortical collecting tubule cells were immunodissected and cultured on permeable supports. Immunocytochemical analysis of the cultured cells by confocal microscopy revealed that Na+/Ca2+ exchanger and Ca(2+)-ATPase were present at the basolateral membrane, whereas calbindin-D28k was evenly distributed throughout the cytosol. Concomitant with an increase in Ca2+ transport, 1,25(OH)2D3 increased calbindin-D28k protein and RNA content two- to threefold, as determined by Northern and Western blotting. By contrast, neither Na+/Ca2+ exchanger nor Ca(2+)-ATPase RNA or protein content was noticeably altered. Our findings suggest that 1,25(OH)2D3 stimulation of transcellular Ca2+ transport in primary cultures of rabbit cortical collecting system cells involves an increase in the gene expression of calbindin-D28k but not of Na+/Ca2+ exchanger and Ca(2+)-ATPase.

Animals↗
PubMed1995

Inhibition of sarcoplasmic/endoplasmic reticulum calcium ATPases (SERCA) by polycyclic aromatic hydrocarbons in HPB-ALL human T cells and other tissues.

Polycyclic aromatic hydrocarbons (PAHs) are immunosuppressive chemicals found in the environment that have been shown to disrupt intracellular Ca2+ homeostasis and Ca(2+)-dependent signaling in human and murine lymphocytes. Many PAHs produce a rapid and sustained increase in intracellular free Ca2+ in lymphocytes. The mechanism of persistent Ca2+ perturbation remains undefined. In the present studies, ATP-dependent 44Ca2+ uptake into vesicles prepared from a 15,000g supernatant of HPB-ALL human T cell lysates was significantly inhibited by 0.1, 1, and 10 microM concentrations of the immunotoxic PAHs 7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (BAP), benz[a]anthracene, and 9,10-dimethylanthracene, but not by the less immunotoxic compounds anthracene (ANT) and benzo[e]pyrene (BEP). Ca(2+)-ATPase catalytic activity was determined by quantitating hydrolysis of ATP in the presence or absence of PAHs, with known ATPase inhibitors included as controls. Formation of inorganic phosphate was significantly decreased (> 65% of control at 10 microM) by DMBA and BAP, whereas ANT and BEP caused only a slight reduction in activity (10% of control at 10 microM). Anthracene partially reversed the inhibitory effect of DMBA and BAP on ATP hydrolysis when agents were coincubated. Both DMBA and BAP, but not ANT and BEP, inhibited the activity of all known SERCA-type Ca(2+)-ATPases, while not affecting either Na+, K(+)-ATPase activity or plasma membrane Ca(2+)-ATPase activities. These results demonstrate that immunotoxic and carcinogenic polycyclic aromatic hydrocarbons have a thapsigargin-like effect in human lymphocytes and SERCA-containing tissues from various species. Inhibition of SERCA activity may play an important role in altered Ca2+ homeostasis in lymphocytes and other tissues.

Adenosine Triphosphate↗
PubMed1995

Localization and quantification of endoplasmic reticulum Ca(2+)-ATPase isoform transcripts.

The Ca(2+)-adenosinetriphosphatase pump of the sarcoplasmic or endoplasmic reticulum (SERCA) plays a critical role in Ca2+ signaling and homeostasis in all cells and is encoded by a family of homologous and alternatively spliced genes. To understand more clearly the role the different isoforms play in cell physiology, we have undertaken a quantitative and qualitative assessment of the tissue distribution of transcripts encoding each SERCA isoform. SERCA1 expression is restricted to fast-twitch striated muscles, SERCA2a to cardiac and slow-twitch striated muscles, whereas SERCA2b is ubiquitously expressed. SERCA3 is expressed most abundantly in large and small intestine, thymus, and cerebellum and at lower levels in spleen, lymph node, and lung. In situ hybridization analyses revealed SERCA3 transcripts in cells of the intestinal crypt, the thymic cortex, and Purkinje cells in cerebellum. In addition, SERCA3 was expressed abundantly in isolated rat spleen lymphocytes, in various murine lymphoid cell lines, and in primary cultured microvascular endothelial cells. This analysis demonstrates that SERCA3 is expressed selectively in cells in which Ca2+ signaling plays a critical and sensitive role in regulating physiological processes.

Animals↗
PubMed1995

Molecular characterization of renal calcium channel beta-subunit transcripts.

An apical, hormone-regulated, calcium entry channel in the distal convoluted tubule and/or connecting tubule (DCT/CNT) is thought to play an important role in controlling renal calcium excretion. We previously identified a gene transcript encoding the pore-forming alpha 1-subunit of a calcium channel (alpha 1A, or CaCh4) which may be a candidate for such a molecule. The properties of voltage-dependent calcium channels are known to be modulated by their beta-subunits. To identify the accessory beta-subunit of DCT/CNT calcium channels, degenerate primers based on published beta-subunit sequences were used to amplify rat kidney cDNA by the polymerase chain reaction (PCR), and the products were subcloned and sequenced. Alternatively spliced transcripts of three beta-subunit genes (beta 2, beta 3, and beta 4) were identified. Northern blot analysis indicated that beta 4-subunit is preferentially expressed in kidney cortex. Transcripts of all three beta-subunit genes were detected by PCR in microdissected nephron segments, but only beta 4-subunit was found in DCT/CNT. As the beta 4- and alpha 1A-subunits colocalize to the DCT/CNT, we hypothesize that they may be constituent subunits of a renal calcium channel regulated by a hormone(s).

Alternative Splicing↗
PubMed1994

Molecular cloning, primary structure, and characterization of two members of the mammalian electroneutral sodium-(potassium)-chloride cotransporter family expressed in kidney.

Electrically silent Na(+)-(K+)-Cl- transporter systems are present in a wide variety of cells and serve diverse physiological functions. In chloride secretory and absorbing epithelia, these cotransporters provide the chloride entry mechanism crucial for transcellular chloride transport. We have isolated cDNAs encoding the two major electroneutral sodium-chloride transporters present in the mammalian kidney, the bumetanide-sensitive Na(+)-K(+)-Cl- symporter and thiazide-sensitive Na(+)-Cl- cotransporter, and have characterized their functional activity in Xenopus laevis oocytes. Despite their differing sensitivities to bumetanide and thiazides and their different requirements for potassium, these approximately 115-kDa proteins share significant sequence similarity (approximately 60%) and exhibit a topology featuring 12 potential membrane-spanning helices flanked by long non-hydrophobic domains at the NH2 and COOH termini. Northern blot analysis and in situ hybridization indicate that these transporters are expressed predominantly in kidney with an intrarenal distribution consistent with their recognized functional localization. These proteins establish a new family of Na(+)-(K+)-Cl- cotransporters.

Amino Acid Sequence↗
PubMed1994

Tissue-specific expression of Na(+)-Ca2+ exchanger isoforms.

The sodium-calcium exchanger (NCE) plays a critical role in diverse processes in many different tissues including heart, nerve, and kidney. Surprisingly, the NCE is encoded by a single gene. We have isolated and sequenced a rat renal NCE clone, denoted F1, that was identical to previous rat NCEs, except for two unique sequences: one in the 5'-untranslated region and the other at a site of alternative splicing in the coding sequence. To explore these regions further, we examined NCE transcripts in several tissues using "rapid amplification of cDNA 5'-ends" and polymerase chain reaction amplification. Three species were identified each with a different 5'-end exon spliced to a common NCE core at nucleotide -34 in the 5'-untranslated region. Based on Northern analysis, each of these species had a unique tissue distribution. Whereas the F1 5'-end variant was abundantly expressed only in kidney, a second variant was expressed mostly in heart, and the third variant was expressed ubiquitously elsewhere. Investigation of the region of alternative splicing in the coding sequence also revealed tissue-specific expression of five major species. These findings indicate that the NCE expression is controlled and regulated under the influence of different promoters in a tissue-specific fashion. Therefore, we propose that the structural complexity of the single NCE gene allows it to respond independently to the unique demands of different environments.

Alternative Splicing↗
PubMed1993

Cloning and characterization of an extracellular Ca(2+)-sensing receptor from bovine parathyroid.

Maintenance of a stable internal environment within complex organisms requires specialized cells that sense changes in the extracellular concentration of specific ions (such as Ca2+). Although the molecular nature of such ion sensors is unknown, parathyroid cells possess a cell surface Ca(2+)-sensing mechanism that also recognizes trivalent and polyvalent cations (such as neomycin) and couples by changes in phosphoinositide turnover and cytosolic Ca2+ to regulation of parathyroid hormone secretion. The latter restores normocalcaemia by acting on kidney and bone. We now report the cloning of complementary DNA encoding an extracellular Ca(2+)-sensing receptor from bovine parathyroid with pharmacological and functional properties nearly identical to those of the native receptor. The novel approximately 120K receptor shares limited similarity with the metabotropic glutamate receptors and features a large extracellular domain, containing clusters of acidic amino-acid residues possibly involved in calcium binding, coupled to a seven-membrane-spanning domain like those in the G-protein-coupled receptor superfamily.

Amino Acid Sequence↗
PubMed1993

Primary structure and functional expression of a cDNA encoding the thiazide-sensitive, electroneutral sodium-chloride cotransporter.

Electroneutral Na+:Cl- cotransport systems are involved in a number of important physiological processes including salt absorption and secretion by epithelia and cell volume regulation. One group of Na+:Cl- cotransporters is specifically inhibited by the benzothiadiazine (thiazide) class of diuretic agents and can be distinguished from Na+:K+:2Cl- cotransporters based on a lack of K+ requirement and insensitivity to sulfamoylbenzoic acid diruetics like bumetanide. We report here the isolation of a cDNA encoding a thiazide-sensitive, electroneutral sodium-chloride cotransporter from the winter flounder urinary bladder using an expression cloning strategy. The pharmacological and kinetic characteristics of the cloned cotransporter are consistent with the properties of native thiazide-sensitive sodium-chloride cotransporters in teleost urinary bladder and mammalian renal distal tubule epithelia. The nucleotide sequence predicts a protein of 1023 amino acids (112 kDa) with 12 putative membrane-spanning regions, which is not related to other previously cloned sodium or chloride transporters. Northern hybridization shows two different gene products: a 3.7-kb mRNA localized only to the urinary bladder and a 3.0-kb mRNA present in several non-bladder/kidney tissues.

Amino Acid Sequence↗
PubMed1993

Cloning and expression of an inwardly rectifying ATP-regulated potassium channel.

A complementary DNA encoding an ATP-regulated potassium channel has been isolated by expression cloning from rat kidney. The predicted 45K protein, which features two potential membrane-spanning helices and a proposed ATP-binding domain, represents a major departure from the basic structural design characteristic of voltage-gated and second messenger-gated ion channels. But the presence of an H5 region, which is likely to form the ion conduction pathway, indicates that the protein may share a common origin with voltage-gated potassium channel proteins.

Adenosine Triphosphate↗
PubMed1993

Differences in the subcellular localization of calreticulin and organellar Ca(2+)-ATPase in neurons.

It has become clear that calcium is an important mediator in the transduction of signals due to ligand binding to cell surface receptors. Cytosolic calcium is typically maintained at low levels in both muscle and non-muscle cells and intracellular sequestering of calcium appears to be important in this process. The identification of intracellular calcium pools has been the subject of much recent study, and it has been proposed that such pools would contain three components: a calcium-activated pump or Ca(2+)-ATPase, a calcium channel such as the inositol trisphosphate receptor or ryanodine receptor, and a high-capacity calcium-binding protein such as calsequestrin or calreticulin. We report here on the localization of two components, the organellar Ca(2+)-ATPase (SERCA) and calreticulin, in neuronal tissues. Using immunofluorescence and subcellular fractionation, we have found that for the most part, these two proteins do not co-localize in neuron cell bodies, dendrites, or axons; but may co-localize at the axon terminal.

Animals↗
PubMed1993

Molecular cloning and quantification of sarcoplasmic reticulum Ca(2+)-ATPase isoforms in rat muscles.

A cDNA encoding the full-length adult rat fast-twitch muscle Ca(2+)-adenosinetriphosphatase (ATPase) was cloned. The deduced amino acid sequence of this molecule has 97 and 90% identity with those of rabbit fast-twitch muscle and chicken skeletal muscle Ca(2+)-ATPases, respectively. Specific probes from the 3'-untranslated region of each sarcoplasmic or endoplasmic reticulum Ca(2+)-ATPase (SERCA) gene product and full-length cRNA transcript standards were used to determine the quantity of mRNA encoding each isoform in various rat muscles. Quantitative immunoblotting was also used to determine the protein content of each SERCA isoform. Fast-twitch fibers expressed both SERCA1 mRNA and protein at a level two- to fivefold higher than SERCA2 was expressed in slow-twitch fibers. We observed a protein-to-mRNA ratio that varied from approximately 500,000 molecules per molecule in the fast-twitch muscles to approximately 200,000 in cardiac and smooth muscles. There was no difference, however, between the ratio for different isoforms in the same muscle. The content of Ca2+ pump in a given muscle therefore depends on at least three factors: 1) the efficiency of gene transcription and message stability (fiber type dependent), 2) the efficiency of translation and protein stability (muscle identity dependent), and 3) fiber composition of the muscle.

Amino Acid Sequence↗
PubMed1992

Molecular characterization and nephron distribution of a family of transcripts encoding the pore-forming subunit of Ca2+ channels in the kidney.

Active, transepithelial, Ca2+ reabsorption in kidney occurs primarily in the distal convoluted tubule. Recent evidence suggests that entry of Ca2+ at the apical membrane through channels bearing resemblance to those of the voltage-dependent L type may be the rate-determining step in Ca2+ reabsorption. To determine the molecular identity of the pore-forming subunit of voltage-dependent Ca2+ channel(s) in the kidney, a homology-based PCR cloning strategy was employed. Nondegenerate primers, based on conserved regions of the published cDNA sequences of voltage-dependent Ca2+ channel alpha 1 subunits, were used to amplify cDNA from rat kidney, and the products were subcloned and sequenced. A family of molecular species was identified, representing alternatively spliced transcripts of four known genes encoding these channel subunits. Northern blot analysis indicated that the expression of each of the genes exhibits a distinct spatial distribution within the kidney. One gene, CaCh4, is expressed primarily in the cortex, and by microdissected-tubule PCR was found predominantly in the distal convoluted tubule, consistent with a role in transepithelial Ca2+ reabsorption at this site.

Amino Acid Sequence↗
PubMed1992

Functional comparisons between isoforms of the sarcoplasmic or endoplasmic reticulum family of calcium pumps.

ATP-dependent calcium pumps that reside in intracellular organelles are encoded by a family of structurally related enzymes, termed the sarcoplasmic or endoplasmic reticulum Ca(2+)-ATPases (SERCA), which each have a distinct pattern of tissue-specific and developmentally regulated expression. A COS-1 cell expression system was used to examine the biochemical properties of the isoforms: SERCA1 (fast-twitch skeletal muscle). SERCA2a (cardiac/slow-twitch skeletal muscle), SERCA2b (ubiquitous smooth- and non-muscle), and SERCA3 (non-muscle). Each isoform was expressed efficiently and appeared to be targeted to the endoplasmic reticulum. All isoforms displayed qualitatively similar enzymatic properties and were activated by calcium in a cooperative manner with a Hill coefficient of 2. The quantitative properties of SERCA1 and SERCA2a (the muscle isoforms) were identical in all respects. SERCA2b, however, appeared to have a lower turnover rate for both calcium transport and ATP hydrolysis. SERCA3 displayed a reduced apparent affinity for calcium, an increased apparent affinity for vanadate, and an altered pH dependence when compared with the other isoforms. These properties are consistent with an enzyme in which the equilibrium between the E1 and E2 conformations is shifted toward the E2 state.

Adenosine Triphosphate↗
PubMed1992

The nucleotide binding/hinge domain plays a crucial role in determining isoform-specific Ca2+ dependence of organellar Ca(2+)-ATPases.

Several isoforms of organellar Ca(2+)-ATPases have been identified, each of which is expressed in a tissue-specific manner. In order to examine the functional properties of fast-twitch (SERCA 1a), cardiac/slow-twitch (SERCA 2a), and non-muscle (SERCA 3) isoforms of the Ca(2+)-ATPase, cDNAs of each type were expressed transiently in COS-1 cells. A study of the Ca2+ dependence of Ca2+ uptake showed that SERCA 1 and SERCA 2 have identical Ca2+ dependences (K0.5 = pCa 6.87 +/- 0.03 and pCa 6.87 +/- 0.02, respectively), but SERCA 3 has a lower Ca2+ dependence (K0.5 = pCa 6.32 +/- 0.03). A study of the ATP dependence of Ca2+ uptake showed that SERCA 1, 2, and 3 have almost identical ATP dependences. Average Hill coefficients derived from Ca2+ uptake curves ranged from 1.7 to 1.8 for the three isoforms. In order to identify which regions of the linear sequence determine this difference in Ca2+ dependence, chimeric Ca(2+)-ATPases between SERCA 2 and SERCA 3 were constructed. Chimeric Ca(2+)-ATPases containing the nucleotide binding/hinge domain of SERCA 2 had SERCA 2 type Ca2+ dependence, but both nucleotide binding/hinge and COOH-terminal transmembrane domains of SERCA 3 were required for SERCA 3 type Ca2+ dependence. Accordingly, structural interactions between the nucleotide binding/hinge and COOH-terminal transmembrane domains appear to determine isoform-specific Ca2+ dependences.

Animals↗
PubMed1992

Intracellular calcium: molecules and pools.

The complex nature of intracellular calcium storage pools has been examined at many levels in the past year. Additional molecules associated with calcium stores have been identified and their localization examined. The convergence of molecular biology, cell biology and biochemistry has now allowed the details of calcium signalling to be meaningfully explored.

Animals↗
PubMed1992

Identification and localization of renal Na(+)-Ca2+ exchanger by polymerase chain reaction.

The molecular identity of the renal Na(+)-Ca2+ exchanger was determined by a homology-based polymerase chain reaction (PCR) cloning strategy. Rat kidney RNA was amplified by PCR, using oligonucleotide primers based on regions of low degeneracy in the published canine cardiac Na(+)-Ca2+ exchanger cDNA sequence, and the products were subcloned and sequenced. A 452-bp clone (NCX1) was identified, which shares 89% nucleotide and 98% amino acid sequence identity with the canine cardiac exchanger, suggesting that they are products of the same gene. NCX1 was shown, by Northern analysis, to hybridize to an abundant major transcript of 7 kb and a minor one of approximately 14 kb both localized predominantly to kidney cortex. Microdissected tubule PCR analysis revealed that NCX1 was enriched in distal convoluted tubule compared with other cortical nephron segments. Such a location is consistent with a Na(+)-Ca2+ exchanger corresponding to NCX1 playing a major role in active Ca2+ reabsorption at this site.

Animals↗