Biomedical subjects

S Ernest

Publications and source records attributed to S Ernest.

12 recordsLinked to original sources

PubMed2000

Mariner is defective in myosin VIIA: a zebrafish model for human hereditary deafness.

The zebrafish (Danio rerio) possesses two mechanosensory organs believed to be homologous to each other: the inner ear, which is responsible for the senses of audition and equilibrium, and the lateral line organ, which is involved in the detection of water movements. Eight zebrafish circler or auditory/vestibular mutants appear to have defects specific to sensory hair cell function. The circler genes may therefore encode components of the mechanotransduction apparatus and/or be the orthologous counterparts of the genes underlying human hereditary deafness. In this report, we show that the phenotype of the circler mutant, mariner, is due to mutations in the gene encoding Myosin VIIA, an unconventional myosin which is expressed in sensory hair cells and is responsible for various types of hearing disorder in humans, namely Usher 1B syndrome, DFNB2 and DFNA11. Our analysis of the fine structure of hair bundles in the mariner mutants suggests that a missense mutation within the C-terminal FERM domain of the tail of Myosin VIIA has the potential to dissociate the two different functions of the protein in hair bundle integrity and apical endocytosis. Notably, mariner sensory hair cells display morphological and functional defects that are similar to those present in mouse shaker-1 hair cells which are defective in Myosin VIIA. Thus, this study demonstrates the striking conservation of the function of Myosin VIIA throughout vertebrate evolution and establishes mariner as the first fish model for human hereditary deafness.

Alleles↗

PubMed2000

Role of multidrug resistance P-glycoprotein in the secretion of aldosterone by human adrenal NCI-H295 cells.

We determined the role of the multidrug resistance (MDR1) gene product, P-glycoprotein (PGP), in the secretion of aldosterone by the adrenal cell line NCI-H295. Aldosterone secretion is significantly decreased by the PGP inhibitors verapamil, cyclosporin A (CSA), PSC-833, and vinblastine. Aldosterone inhibits the efflux of the PGP substrate rhodamine 123 from NCI-H295 cells and from human mesangial cells (expressing PGP). CSA, verapamil, and the monoclonal antibody UIC2 significantly decreased the efflux of fluorescein-labeled (FL)-aldosterone microinjected into NCI-H295 cells. In MCF-7/VP cells, expressing multidrug resistance-associated protein (MRP) but not PGP, and in the parental cell line MCF7 (expressing no MRP and no PGP), the efflux of microinjected FL-aldosterone was slow. In BC19/3 cells (MCF7 cells transfected with MDR1), the efflux of FL-aldosterone was rapid and it was inhibited by verapamil, indicating that transfection with MDR1 cDNA confers the ability to transport FL-aldosterone. These results strongly indicate that PGP plays a role in the secretion of aldosterone by NCI-H295 cells and in other cells expressing MDR1, including normal adrenal cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Pulmonary natriuretic peptide system during rat development.

Maturational changes in the rat lung natriuretic peptide system were studied postnatally in 1-, 4-, and 22-day-old rats. Lung atrial natriuretic factor (ANF) content increased significantly from day 1 to day 4 (712+/-188 vs. 1905+/-520 pg/mg protein; p<0.01) but decreased to 532+/-41 pg/mg protein, on day 22. These changes paralleled ANF messenger RNA (mRNA) detected by reverse transcribed polymerase chain reaction (RT-PCR). Rat pulmonary development also was associated with quantitative and qualitative alterations in ANF receptors. Competitive-binding radioreceptor assays of lung membranes with 125I-ANF and increasing concentrations of unlabeled ANF revealed that the natriuretic peptide receptor-binding sites (Bmax) progressively increased with age from 112 +/-21 fmol/mg protein at day 1 to 211+/-16 (p<0.02) and 326+/-62 fmol/mg protein (p<0.04) in 4- and 22-day-old rats, respectively. Autoradiographic studies of 125I-tyr(0)CNP binding to lung sections revealed that the levels of the natriuretic peptide receptor B (NPR-B) were undetectable. On the other hand, binding of 125I-ANF increased with age, and the higher binding at 4 days was mainly due to increased density of the clearance receptor-C (NPR-C), and at 22 days due to increased natriuretic peptide receptor-A (NPR-A). The increase in natriuretic peptide binding was confirmed at the level of synthesis, where RT-PCR revealed that NPR-A mRNA significantly increased (p<0.01) in 22-day-old rats. In conclusion, these studies demonstrate that the rat pulmonary natriuretic peptide system is altered during development. The altered synthesis of lung natriuretic peptides and their receptors may play a role in the postnatal adaptation of pulmonary circulation.

Animals↗

PubMed1999

Secretion of platelet-activating factor is mediated by MDR1 P-glycoprotein in cultured human mesangial cells.

MDR1 P-glycoprotein (Pgp), the product of the MDR1 gene involved in multidrug resistance in cancer cells, is also expressed in normal tissues. In the human kidney, it is localized in the mesangium, the proximal tubule, the thick ascending limb of Henle's loop, and the collecting duct. Pgp actively transports lipophilic xenobiotics, peptides, steroids, and lipids, and perhaps endogenous substrates. It has been shown previously that human mesangial cells in culture express active Pgp and that the expression of Pgp can be down-regulated by exposure to antisense oligonucleotides. Mesangial cells do not express multidrug resistance-related protein (MRP). Experiments were performed to determine whether 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (generically platelet-activating factor, PAF) is a substrate of Pgp in human mesangial cells in culture. This study found: (1) PAF C-16 and analogs inhibited Pgp-mediated efflux of rhodamine 123 by 59 to 88% in multidrug-resistant KBV-1 cells and by 85 to 97% in cultured human mesangial cells. (2) In mesangial cells stimulated with A23187, the secretion of endogenously produced PAF was inhibited by >80% by the Pgp blockers verapamil, cyclosporin A, PSC-833, vinblastine, and adriamycin. (3) Preincubation with MDR1 antisense oligonucleotides also blocked PAF secretion by human mesangial cells. PAF analogs do not modify the transport of MRP substrates in MCF-7/VP cells expressing MRP but not Pgp. These results indicate that PAF is an endogenous substrate of Pgp in human mesangial cells. Inhibition of Pgp transport may be useful in reducing glomerular damage occurring in pathologic conditions where PAF secretion is elevated.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Altered regulation of natriuretic peptides in the rat heart by prenatal exposure to morphine.

1. Both endogenous and exogenous opioids modulate blood pressure and cardiac function by stimulating cardiac synthesis of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP). Since morphine crosses the placental barrier, it could alter the ANF-BNP system in the fetal heart. The aim of this study was to characterize cardiac natriuretic peptides in normal rat development and in rats prenatally exposed to morphine. 2. Female rats received either saline or morphine (10 or 20 mg kg-1 day-1) via osmotic minipumps during gestation. The effects of this treatment were investigated in offspring at 1, 4 and 22 days of age. 3. During maturation, atrial ANF and ANF mRNA increased by 3-fold from birth to 3 weeks of age, but BNP and BNP mRNA tended to decrease. In the ventricles, both ANF and BNP content decreased at 3 weeks after birth, from 25.11 +/- 3.6 to 0.81 +/- 0.1 ng (mg protein)-1 (P < 0.001), and from 3.36 +/- 0.33 to 0.19 +/- 0.01 ng (mg protein)-1 (P < 0.001), respectively. However, whereas ventricular ANF mRNA decreased, BNP mRNA levels did not change during maturation. Prenatal exposure to morphine significantly increased ANF content in the left atria of 22-day-old rats, and in the right atria of 1-, 4- and 22-day-old rats compared with age-matched saline controls. In contrast, prenatal exposure to 20 mg kg-1 day-1 morphine significantly inhibited BNP and BNP mRNA in the ventricles at all ages studied. 4. These observations suggest that alterations in mRNA synthesis or stability and/or post-translational processing of ANF and BNP occur in the heart during maturation, and that prenatal exposure to morphine alters cardiac production, and possibly release, of both peptides.

Analgesics, Opioid↗

PubMed1998

P-glycoprotein functions and substrates: possible roles of MDR1 gene in the kidney.

There is a renewed attention on the multidrug resistance genes and their products, P-glycoproteins, since recent molecular and functional studies revealed unexpected functions in normal tissues. There are two types of human P-glycoprotein: Type I, encoded by the MDR1 gene, present in excretory organs and in non-polarized cells; and Type II, encoded by MDR2, present in the canalicular membrane of hepatocytes. MDR1 Pgp transports xenobiotics, peptides, steroids, and phospholipids, and is also a regulator of swelling-activated chloride channels. MDR2 Pgp is exclusively a phosphatidylcholine translocase. In the kidney, the MDR1 gene and protein are expressed in mesangial, proximal tubule, thick loop of Henle, and collecting duct cells. In mesangial and proximal tubule cells Pgp transports xenobiotics. Concomitant exposure of kidney cells to two Pgp substrates results in increased cell toxicity. Extracts from supernatants of mesangial cell cultures inhibit Pgp-mediated transport, suggesting that a mesangial-cell metabolite could be a substrate of Pgp. Active vitamin D3 and platelet activating factor inhibit Pgp transport and are possible endogenous substrates in proximal tubule and mesangial cells, respectively. Pgp could be also a regulator of swelling-activated chloride channels present in the kidney.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

Expression of MDR1 (multidrug resistance) gene and its protein in normal human kidney.

P-glycoprotein (Pgp), the product of the multidrug resistance (MDR) gene overexpressed in cancer cells, is present also in normal tissues. In the kidney, MDR1 Pgp has been found in the proximal tubule and in cultured mesangial cells. In situ hybridization and immunohistochemistry were used to determine the complete nephronal localization of MDR mRNA and its product, Pgp, in the human kidney. MDR mRNA expression was studied with the use of nonradioactive in situ MDR RNA probes. MDR1 Pgp was immunolocalized using the specific monoclonal antibody MRK16. The presence of MDR mRNA was confirmed in proximal tubules and mesangium, and demonstrated as well in thick limb of Henle's loops and in collecting ducts. MDR1 Pgp colocalized in the same nephronal segments. This suggests that, in addition to secreting xenobiotics, Pgp may play a role in the transport of endogenous substrates or in the regulation of Cl- channels.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1996

Xenobiotic transport differences in mouse mesangial cell clones expressing mdr1 and mdr3.

P-glycoprotein (PGP), which confers multidrug resistance to cancer cells, is expressed in mouse kidney proximal tubule and mesangium. We report on the expression of PGP and its xenobiotic transport function in mesangial cells. Studies were performed in a mouse mesangial cell line (TKGM) and two cell clones. Ribonuclease protection assay and Western blot analysis demonstrated that TKGM cells expressed mdr1 and mdr3, the isoforms responsible for multidrug resistance. TKGM-F12 cells coexpressed mdr1 and mdr3 whereas TKGM-G2 cells expressed only mdr1. The drug transport function, measured by rhodamine 123 (R-123) efflux, was smaller in TKGM-F12 than in TKGM-G2 cells. The PGP substrates adriamycin, cyclosporin A, vinblastine, and verapamil inhibited R-123 transport in TKGM and TKGM-G2 cells. In the cells studied, PGP conferred some resistance to adriamycin; concomitant exposure to adriamycin with another PGP substrate impaired cell growth. The differential expression of mdr1 and mdr3 in mouse mesangial cell clones, the ability of mdr1 PGP to transport R-123, and the impairment of PGP-mediated transport in TKGM-F12 cells, coexpressing mdr1 and mdr3 products, are demonstrated. PGP may play a physiological role in mesangial cells.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed1995

Expression and function of P-glycoprotein in a mouse kidney cell line.

P-glycoprotein (PGP), a transporter conferring multidrug resistance to cancer cells, is expressed in the kidney. C219 monoclonal antibody binding revealed PGP in proximal tubules and mesangium of mouse kidneys. A cell line (TKPTS) expressing PGP was developed from proximal tubules of the 8Tg(SV40E)Bri7 mouse. Northern blot analysis demonstrated a 5.0-kb message identified as mdr1 by ribonuclease protection assay. Cyclosporin A (CSA) at 0.15 and 10 microM increased cellular accumulation of verapamil (VRP) by 32 and 121%, respectively (P < 0.001). VRP at 5 microM increased steady-state cellular accumulation of CSA by 46% (P = 0.02). Basal-to-apical transport of the PGP substrate vinblastine was inhibited by VRP. Rhodamine-123 (R-123) influx was rapid and independent of PGP. R-123 efflux was inhibited by VRP and CSA. Inhibition of PGP transport by VRP, CSA, and PSC-833 decreased the 50% effective dose of adriamycin. The concomitant administration of VRP and CSA was not deleterious and coincided with preferential accumulation of VRP over CSA. Inhibition of PGP-mediated transport is demonstrated as a mechanism of renal cell toxicity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Deprivation of phosphate increases IGF-II mRNA in MDCK cells but IGFs are not involved in phosphate transport adaptation to phosphate deprivation.

Phosphate (Pi) deprivation and IGFs stimulate renal Pi reabsorption. We studied the involvement of IGFs in the adaptation of Pi transport to Pi deprivation in MDCK cells. Deprivation of Pi for 15 h increased the steady-state content of IGF-II mRNA (77 +/- 12%) whereas IGF-I mRNA was not detectable in MDCK cells in either control or Pi-deprived cells. IGF-II (10(-7) M) and IGF-I (10(-8) M) stimulated the Na-dependent Pi uptake (1.23- and 1.3-fold increase at 15 h respectively). The effect of IGF-I appeared after 15 h and increased up to 40 h of treatment (2.15-fold increase). In contrast, Pi uptake was increased by Pi deprivation as early as 8 h (1.5-fold) and up to 40 h of Pi deprivation (1.9-fold increase). IGF-II mRNA was not increased before 15 h of Pi deprivation and returned to control at 40 h. The combination of IGF-I and Pi deprivation had a more than additive effect on Pi transport (fivefold increase) (P < 0.001). At variance with Pi deprivation, high concentrations of insulin stimulated Na-coupled alanine transport (6 +/- 2% and 16 +/- 4% in Pi-treated and Pi-depleted cells respectively). Pi deprivation and high concentrations of insulin decreased Na,K-ATPase activity (-48 and -64% respectively) and these effects were not additive.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Expression and function of P-glycoprotein in human mesangial cells.

P-glycoprotein (PGP), responsible for multidrug resistance (MDR) in cancer cells, is normally expressed in kidney proximal tubules and mesangium. PGP expression and function were studied in human mesangial cell cultures. MDR1 gene expression was demonstrated by reverse transcription-polymerase chain reaction. PGP expression was determined using MRK16 monoclonal antibody and its function was assessed by the efflux of rhodamine-123 (R123). R123 efflux had a half time of 25 +/- 5 s. Efflux was inhibited by cyclosporin A (10 microM), verapamil (10 microM), and vinblastine (100 microM) with half times of 380, 535, and 312 s, respectively. Incubation with MDR1-antisense oligonucleotide decreased R123 efflux (half time = 304 s). Verapamil, cyclosporin A, and PSC-833 augmented the cytotoxicity of Adriamycin by reducing the 50% maximal growth-inhibitory dose from 730 nM to 130, 110, and 90 nM, respectively. We conclude that human mesangial cells express MDR1 and demonstrate xenobiotic transport inhibitable by several known PGP substrates. Concomitant exposure of mesangial cells to PGP-transported drugs causes intracellular accumulation of toxic PGP substrates and ultimately damages the mesangial cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1989

Model of gentamicin-induced nephrotoxicity and its amelioration by calcium and thyroxine.

The exact mechanism of gentamicin-induced acute renal failure is presently unknown; various mechanisms have been proposed but there is no proposed commonality between them. In animals, dietary calcium loading and L-thyroxine administration have been shown to ameliorate toxicity, with again no common process. A mechanism of competitive displacement of calcium and other cations from anionic phospholipids at the plasma and organelle membrane level, resulting in a decrease in Na+ -K+ ATPase, adenylate cyclase, mitochondrial function and ATP production, protein synthesis, solute reabsorption and overall cellular function is proposed. A further proposal is dietary calcium loading and thyroxine (which increases intracellular calcium) reverse gentamicin-induced acute renal failure by increasing the calcium and solute flux, thereby competitively inhibiting the primary lesion: anionic phospholipid binding.

Acute Kidney Injury↗