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S C Hebert

Publications and source records attributed to S C Hebert.

At least 109 records · Page 6Linked to original sources
PubMed1995

The cloning of extracellular Ca(2+)-sensing receptors from parathyroid and kidney: molecular mechanisms of extracellular Ca(2+)-sensing.

The parathyroid cell detects changes in the extracellular ionized calcium concentration (Ca2 + o) with exquisite sensitivity, but the mechanisms through which it senses Ca2 + o have remained obscure. Recently, we isolated a cDNA encoding a Ca2 + o-sensing receptor from bovine parathyroid using expression cloning in Xenopus laevis oocytes. The expressed receptor stimulates phospholipase C and has a pharmacological profile almost identical to that of the native receptor. Furthermore, its deduced amino acid sequence confirms that it belongs to the superfamily of G-protein-coupled receptors. Receptor transcripts are present in parathyroid and other tissues sensing Ca2 + o (e.g., kidney and thyroidal C-cells) as well as those not known to be involved in Ca2+ homeostasis (viz., in the brain). We have also shown that mutations in the receptor cause three inherited disorders of calcium metabolism: Familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT) result from inactivating mutations, when present in the heterozygous and homozygous states, respectively, whereas an autosomal dominant form of hypocalcemia is due to an activating mutation. Thus this Ca2 + o-sensing receptor permits Ca2+o to act as an extracellular, first messenger in addition to its better known role as an intracellular second messenger.

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↗
PubMed1995

ROMK inwardly rectifying ATP-sensitive K+ channel. I. Expression in rat distal nephron segments.

The inwardly rectifying, ATP-sensitive K+ channel (ROMK) was localized by in situ hybridization in the rat kidney. Tissue in situ hybridization revealed that transcripts encoding the ROMK channel were expressed predominantly in cortical and outer medullary nephron segments. The localization of ROMK mRNA to specific nephron segments was assessed by hybridization of isolated nephron segments with an ROMK-specific probe (single segment in situ hybridization). ROMK mRNA was present in cortical and medullary thick ascending limb, distal tubule, and cortical and outer medullary collecting ducts, but not in proximal tubule. A weak hybridization was observed with inner medullary collecting ducts. To confirm these results, serial cryosections were alternatively stained by hybridization histochemistry for ROMK mRNA or by immunocytochemistry using antibodies specific for S1, S2, or S3 proximal tubular segments. Tubular cells that displayed immunoreactivity with the proximal tubular segment-specific antibodies showed little, if any, ROMK message. In addition, using an in situ hybridization and immunocytochemistry double-labeling technique, ROMK transcripts and vitamin D-dependent calcium-binding protein were shown to colocalize to the distal tubule (distal convoluted tubule and connecting tubule). The overall nephron localization of ROMK mRNA shown in these studies is consistent with the possibility that this novel channel may represent the low-conductance ATP-sensitive K+ channel that has been identified in apical membranes of thick limb and collecting duct segments and is believed to participate in K+ secretion.

Animals↗
PubMed1995

ROMK inwardly rectifying ATP-sensitive K+ channel. II. Cloning and distribution of alternative forms.

The rat ROMK gene encodes inwardly rectifying, ATP-regulated K+ channels [K. Ho, C. G. Nichols, W. J. Lederer, J. Lytton, P. M. Vassilev, M. V. Kanazirska, and S. C. Hebert. Nature Lond. 362: 31-38, 1993; H. Zhou, S. S. Tate, and L. G. Palmer. Am. J. Physiol. 266 (Cell Physiol. 35): C809-C824, 1994], and mRNA encoding these channels is widely expressed in distal cortical and outer medullary nephron segments [see companion study; W.-S. Lee and S. C. Hebert. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F1124-F1131, 1995]. Using approaches based on homology to ROMK1, we have identified two additional ROMK isoforms, ROMK2b and ROMK3. Analysis of the nucleotide sequences of the ROMK isoforms indicates that molecular diversity of ROMK transcripts is due to alternative splicing at both the 5'-coding and 3'-noncoding regions. The splicing at the 5' end of ROMK gives rise to channel proteins with variable-length NH2 termini containing different initial amino acid sequences. Functional expression of these isoforms in Xenopus oocytes showed that they form functional Ba(2+)-sensitive K+ channels. The nephron distribution of mRNAs encoding alternatively spliced isoforms of ROMK (ROMK1-ROMK3) was investigated by reverse transcription-polymerase chain reaction (RT-PCR) of nephron segments dissected from rat kidney. Nondegenerate PCR primer pairs were designed to span at least one intron and to amplify specific alternatively spliced forms of ROMK.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Calcium receptor messenger ribonucleic acid levels in the parathyroid glands and kidney of vitamin D-deficient rats are not regulated by plasma calcium or 1,25-dihydroxyvitamin D3.

The level of extracellular ionized calcium ([Ca2+]o) is the primary physiological regulator of PTH secretion. Complementary DNAs encoding the calcium receptor (CaR) protein that mediates this response have been cloned from bovine and human parathyroid glands. This protein is a seven-transmembrane, G-protein-coupled receptor linked to the mobilization of intracellular Ca2+ in response to increases in [Ca2+]o. More recently, a rat kidney CaR has been cloned and shown to be 92% identical at the amino acid level to the bovine parathyroid CaR. Homologous or heterologous regulation of the expression and/or function of a variety of G-protein-coupled receptors has been documented in numerous cell types. Therefore, we determined whether [Ca2+]o and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], major regulators of PTH synthesis and secretion, affect CaR gene expression in parathyroid gland and kidney in rats. CaR messenger RNA (mRNA) levels were quantified in pairs of parathyroid glands and single kidneys from individual animals using a solution hybridization assay. The effects of Ca2+ and 1,25-(OH)2D3 on CaR gene expression were assessed independently in vitamin D-deficient (-D) rats. A wide range of plasma Ca2+ levels (0.7-1.9 mM) was produced by supplementing -D diets with varying amounts of calcium and by infusing CaCl2 i.v. for 7 days using osmotic minipumps. There was no correlation between plasma Ca2+ levels and steady state CaR mRNA levels in parathyroid gland (r = -0.18) or kidney (r = 0.25). In another group of -D rats, 1,25-(OH)2D3 was infused sc at 25 and 275 ng/kg.day for 10-12 days. Dietary calcium was adjusted to maintain normocalcemia in some of the groups. No effect of 1,25-(OH)2D3 administration on CaR mRNA levels occurred in parathyroid glands or kidney regardless of the resultant plasma Ca2+ or 1,25-(OH)2D3 levels. In conclusion, neither parathyroid gland nor kidney CaR mRNA levels are regulated by plasma Ca2+ and 1,25-(OH)2D3 levels in the experimental models examined here.

Animals↗
PubMed1995

The reduced responsiveness of cultured bovine parathyroid cells to extracellular Ca2+ is associated with marked reduction in the expression of extracellular Ca(2+)-sensing receptor messenger ribonucleic acid and protein.

PTH secretion from dispersed bovine parathyroid cells maintained in culture becomes progressively less responsive to changes in the extracellular Ca2+ concentration (Ca2+o) over several days. We have now investigated whether this change in secretory control is associated with alterations in the expression of the Ca2+o-sensing receptor (BoPCaR) recently cloned from bovine parathyroid, which plays a central role in Ca2+o-regulated PTH secretion. BoPCaR messenger RNA levels dropped rapidly in cultured bovine parathyroid cells, as assessed by Northern analysis, decreasing by 78% within 18 h and remaining low for at least 4 days. The level of receptor protein decreased to a comparable extent (approximately 72-82%) after 3-4 days in culture, as determined by immunocytochemistry with specific antibodies directed at the extracellular domain of the receptor. The half-time for the reduction in receptor protein (approximately 2 days) was considerably longer, however, than that for BoPCaR messenger RNA, but was comparable to that for the loss of sensitivity of PTH secretion to Ca2+o. Indeed, there was a close linear correlation between maximal suppressibility of PTH secretion and the intensity of staining for the receptor protein (r = 0.88; P = 0.004). We conclude that alterations in the expression of BoPCaR could explain much of the reduced responsiveness of cultured bovine parathyroid cells to Ca2+o.

Animals↗
PubMed1995

Molecular mechanisms underlying the sensing of extracellular Ca2+ by parathyroid and kidney cells.

Mineral ion homeostasis in mammalian species is maintained by a complex mechanism comprising sensors of the extracellular calcium concentration (Ca2+0) (i.e. parathyroid cells) as well as effectors that modify their translocation of mineral ions into and out of the extracellular fluid (e.g. kidney) in response to calciotropic hormones. Indirect evidence accumulated over the past decade suggested that parathyroid cells sense Ca2+0 through a cell surface receptor coupled to intracellular second messenger systems via one or more guanine nucleotide regulatory (G) proteins. More recently, Brown et al. employed expression cloning in Xenopus laevis oocytes to isolate a cDNA encoding a Ca2+0-sensing receptor from bovine parathyroid. The expressed receptor activates phospholipase C in a G-protein dependent manner and shows pharmacological properties almost identical to those of the native parathyroid receptor. Agonists for the receptor include not only divalent cations (e.g. Ca2+ and Mg2+) but also trivalent cations and even organic polycations such as neomycin. The deduced amino acid sequence of the cloned receptor confirms that it is a member of the superfamily of G-protein-coupled receptors. Receptor transcripts are present in parathyroid as well as in kidney, thyroid and brain. Therefore, this receptor may mediate the sensing of Ca2+0 not only by parathyroid cells but also by other tissues directly regulated by Ca2+0 (e.g. the thyroidal C cells and certain kidney cells) as well as those not currently known to be involved in calcium homeostasis (viz. in the brain).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

A cloned Ca(2+)-sensing receptor: a mediator of direct effects of extracellular Ca2+ on renal function?

Changes in the extracellular calcium concentration [Ca2+]o modulate several aspects of renal function through unknown mechanism(s). cDNA encoding a Ca2+o-sensing receptor from bovine parathyroid and rat kidney that appears to mediate several of the known effects of Ca2+o on parathyroid and renal function were recently isolated. The expressed receptor activates phospholipase C, showing a pharmacologic profile very similar to that of the native receptor. Its deduced amino acid sequence identifies it as a member of the superfamily of G protein-coupled receptors. The physiologic relevance of the receptor has been established by the demonstration that mutations in it cause three inherited diseases of calcium metabolism. Two hypercalcemic disorders, familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism, result from inactivating mutations when present in the heterozygous and homozygous states, respectively. An activating mutation, in contrast, causes an autosomal dominant form of hypocalcemia. In the kidney, the receptor is expressed most abundantly in the thick ascending limb, where it likely modulates sodium chloride, calcium, and magnesium reabsorption and, perhaps, urinary concentrating ability. Studies are currently underway to determine whether it also mediates the effects of Ca2+o on other parameters of kidney function, such as RBF, glomerular filtration, renin secretion, and vitamin D metabolism. Thus, this Ca2+o-sensing receptor permits extracellular calcium ions to act not only as an intracellular second messenger but also in a "hormone-like" role as an extracellular first messenger.

Animals↗
PubMed1994

Molecular cloning and chromosome localization of a putative basolateral Na(+)-K(+)-2Cl- cotransporter from mouse inner medullary collecting duct (mIMCD-3) cells.

Electroneutral Na(+)-K(+)-2Cl- cotransporters represent one of the major routes for Cl- movement in epithelia. A secretory form of the cotransporter has been described in the basolateral membrane of a variety of epithelia from fish to mammals. We isolated a putative bumetanide-sensitive Na(+)-K(+)-2Cl- cotransporter cDNA, BSC2, from mIMCD-3 cells. Northern analysis indicates that in contrast to BSC1, the recently cloned renal-specific apical isoform of the cotransporter, BSC2 is expressed in secretory epithelia and thus appears to represent the basolateral isoform. Furthermore, BSC2 is also expressed in non-polarized cells, such as red cells and myocytes. Sequence comparison and chromosome localization demonstrate that BSC2 and BSC1 are different genes that diverged before the evolution of vertebrates.

Amino Acid Sequence↗
PubMed1994

Regulation of ROMK1 K+ channel activity involves phosphorylation processes.

An inwardly rectifying, ATP-regulated K+ channel with a distinctive molecular architecture, ROMK1, was recently cloned from rat kidney. Using patch clamp techniques, we have investigated the regulation of ROMK1 with particular emphasis on phosphorylation/dephosphorylation processes. Spontaneous channel rundown occurred after excision of membrane patches into ATP-free bath solutions in the presence of Mg2+. Channel rundown was almost completely abolished after excision of patches into either Mg(2+)-free bathing solutions or after preincubation with the broad-spectrum phosphatase inhibitor, orthovanadate, in the presence of Mg2+. MgATP preincubation also inhibited channel rundown in a dose-dependent manner. In addition, the effect of the specific phosphatase inhibitors okadaic acid (1 microM) and calyculin A (1 microM) was also investigated. The presence of either okadaic acid or calyculin A failed to inhibit channel rundown. Taken together, these data suggest that rundown of ROMK1 involves a Mg(2+)-dependent dephosphorylation process. Channel activity was also partially restored after the addition of MgATP to the bath solution. Addition of exogenous cAMP-dependent protein kinase A (PKA) catalytic subunit led to a further increase in channel open probability. Addition of Na2ATP, in the absence of Mg2+, was ineffective, suggesting that restoration of channel activity is a Mg(2+)-dependent process. Addition of the specific PKA inhibitor, PKI, to the bath solution led to a partial, reversible inhibition in channel activity. Thus, PKA-dependent phosphorylation processes are involved in the modulation of channel activity. This observation is consistent with the presence of potential PKA phosphorylation sites on ROMK1.

Adenosine Triphosphate↗
PubMed1994

The neuronal and epithelial human high affinity glutamate transporter. Insights into structure and mechanism of transport.

High affinity transport of glutamate across plasma membranes of brain neurons and epithelial is mediated by a Na(+)- and K(+)-coupled electrogenic transporter. Here we report the primary structure and functional characterization of the human high affinity glutamate transporter (HEAAC1). A unique characteristic of HEAAC1-mediated transport is that the affinity for glutamate and the maximal transport rate are strongly dependent on membrane potential. Our data provide new insights into individual steps of high affinity glutamate transport and show that the transport mechanism is distinct from that of the gamma-aminobutyric acid transporter GAT-1 and the Na+/glucose transporter SGLT1. Under voltage clamp condition, HEAAC1 mediated large substrate-evoked inward currents (up to 1 microA). The substrate specificity, stereospecificity, the Km value (30 +/- 3 microM at -60 mV) of the L-glutamate-evoked current, and Northern analysis all agree with previously reported characteristics of high affinity glutamate transport in brain. In contrast to SGLT1 and GAT-1, voltage jump studies of HEAAC1 yielded only minor relaxation currents. Classic inhibitors of brain glutamate uptake such as DL-threo-beta-hydroxyaspartate, L-trans-pyrrolidine 2,4,-dicarboxylic acid (PDC), and dihydrokainate were found to be either transport substrates or to have no significant effect on glutamate transport. We also found that the maximal transport rate for PDC was markedly reduced compared to that for L-glutamate. We propose that PDC most likely reduces the turnover rate of the transporter. A search of the sequence data bases revealed weak homology of HEAAC1 to the H(+)-coupled vesicular monoamine transporter, suggesting an evolutionary link between plasma membrane and vesicular transporters.

Adult↗
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

Critical role for intracellular calcium in tight junction biogenesis.

Using the Madin Darby canine kidney (MDCK) cell "calcium switch," we have previously demonstrated that, as MDCK cells establish contact and ultimately form tight junctions, there are marked global and localized changes in intracellular calcium at the sites of cell-cell contact (Nigam et al., 1992, Proc. Natl. Acad. Sci. USA, 89:6162-6166). We have now examined whether intracellular Ca++ is critical to the biogenesis of tight junctions by chelating this ion and monitoring the formation of junctions by electrical, immunocytochemical, and biochemical criteria. Intracellular Ca++ was chelated with the cell-permeant chelators, dimethyl-BAPTA-AM and BAPTA-AM. By digital imaging of fura-2 loaded cells, it was demonstrated that both agents efficiently chelated Ca++ during the "switch" in a dose-dependent manner which paralleled their respective in vitro affinities for Ca++. Chelation of Ca++ during the switch markedly attenuated the development of transepithelial electrical resistance (TER), a measure of tight junction assembly. Immunofluorescent staining of the tight junctional protein, zonula occludens-1 (ZO-1), revealed that chelation of intracellular Ca++ retarded the movement of ZO-1 from intracellular sites to the plasma membrane during the switch. During the development of tight junctions, a fraction of ZO-1 redistributed from the Triton X-100 soluble to the Triton X-100 insoluble pool; chelation of Ca++ during the induction of cell-cell contact prevented this stabilization into the Triton X-100 insoluble fraction. Taken together, these data indicate an important role for intracellular Ca++ in tight junction biogenesis and suggest a specific role for calcium in the early sorting and possible cytoskeletal association of tight junction components.

Animals↗
PubMed1994

Molecular cloning and characterization of the renal diuretic-sensitive electroneutral sodium-(potassium)-chloride cotransporters.

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, were isolated and their functional activity characterized in Xenopus laevis oocytes [2]. Although they differ in sensitivities to bumetanide and thiazides and have different requirements for potassium, these approximately 115-kDa proteins share about 60% sequence similarity and exhibit a topology featuring 12 potential membrane-spanning helices flanked by large hydrophilic domains at the NH2- and COOH-termini. These molecules, together with the Na-Cl cotransporter from the flounder urinary bladder, which exhibits a significant homology suggestive of common ancestry, define a new family of electroneutral Na(+)-(K+)-Cl- cotransporters. 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. The kidney-specific distribution of transcripts encoding these cotransporters suggest that other, probably related, genes encode non-renal Na(+)-(K+)-Cl- cotransporters.

Animals↗
PubMed1994

Autosomal dominant hypocalcaemia caused by a Ca(2+)-sensing receptor gene mutation.

Defects in the human Ca(2+)-sensing receptor gene have recently been shown to cause familial hypocalciuric hypercalcaemia and neonatal severe hyperparathyroidism. We now demonstrate that a missense mutation (Glu128Ala) in this gene causes familial hypocalcaemia in affected members of one family. Xenopus oocytes expressing the mutant receptor exhibit a larger increase in inositol 1,4,5-triphosphate in response to Ca2+ than oocytes expressing the wild-type receptor. We conclude that this extracellular domain mutation increases the receptor's activity at low Ca2+ concentrations, causing hypocalcaemia in patients heterozygous for such a mutation.

Amino Acid Sequence↗