Biomedical subjects

S R Gullans

Publications and source records attributed to S R Gullans.

At least 55 records · Page 3Linked to original sources

PubMed1994

Molecular characterization of ROSIT, a renal osmotic stress-induced Na(+)-Cl(-)-organic solute cotransporter.

A gene family encoding many Na(+)- and Cl(-)-dependent organic solute cotransporters has recently been recognized. Among the cotransporters that have been characterized are those for neurotransmitters, amino acids, and organic osmolytes. Using polymerase chain reaction and mRNA derived from renal cortex, we isolated a new member of this gene family, ROSIT, a renal osmotic stress-induced transporter. The cDNA is 2,354 bp long with an open reading frame of 1,845 bp. The 615 deduced amino sequence shows ROSIT to be most clearly related to two orphan cDNAs of this family isolated from brain. Northern analysis showed the mRNA is normally expressed in renal cortex but not in brain, heart, colon, liver, stomach, or skeletal muscle. Moreover, hypernatremic rats displayed a marked increase in mRNA levels in renal cortex, renal outer medulla, and perhaps intestine. Heterologous expression of the cRNA in Xenopus laevis oocytes failed to reveal the function of this gene product when analyzed with isotope fluxes or electrophysiological measurements using a wide variety of organic solutes. Although its function remains unknown, ROSIT is likely to be involved in kidney reclamation of an organic osmolyte or osmolyte precursor required for adaptation to hypertonic stress.

Amino Acid Sequence↗

PubMed1993

Rapid correction of hyponatremia produces differential effects on brain osmolyte and electrolyte reaccumulation in rats.

Studies from these and other laboratories have shown that hyponatremia causes marked depletion of both electrolytes and organic osmolytes from the brain. The present studies evaluated brain reaccumulation of both classes of solute after correction of chronic hyponatremia. Hyponatremia was induced by subcutaneous infusions of 1-deamino-[8-D-arginine]-vasopressin (dDAVP) in rats fed a balanced liquid diet. After 14 days of sustained hyponatremia the dDAVP minipumps were removed causing rapid correction of plasma sodium concentrations from 104 +/- 1 mmol/l to 139 +/- 1 mmol/l in 24 h. Water and solute contents were measured in brain extracts both before and for 5 days after correction of the hyponatremia, and compared to values in normonatremic rats maintained on the same diet for 14 days. Our results demonstrate that electrolytes, particularly Na+ and Cl-, reaccumulate rapidly in the brain, resulting in a significant overshoot above normal control brain Na+ and Cl- contents as early as 24 h after correction. In contrast, organic osmolyte reaccumulation occurs more slowly, requiring 5 or more days for a return to normal control brain contents in most cases. A prominent exception to this pattern was glutamate, which also returned rapidly to normal brain contents within 24 h similar to the electrolytes. Quantitative analysis of brain water and solute contents after correction of hyponatremia indicated that the reaccumulation of electrolytes and organic osmolytes was sufficient to account for the changes in brain volume that occurred.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Control of brain volume during hyperosmolar and hypoosmolar conditions.

The brain is particularly vulnerable to disturbances of body fluid osmolality. Studies in animals indicate that brain adaptation to osmotic stresses is a very complex process involving transient changes in water content and sustained changes in electrolyte and organic osmolyte contents. Appreciation of the nature of the adaptation process enables a better understanding of the marked variations in neurological sequelae that characterize hyper- and hypoosmolar states and provides a basis for more rational therapy.

Animals↗

PubMed1993

Urea induces Egr-1 and c-fos expression in renal epithelial cells.

The membrane-permeant solute urea, in concentrations present in the mammalian renal medulla, increased expression at the mRNA level of two immediate-early gene (IEG) transcription factors, Egr-1 and c-fos, in a time- and dose-dependent fashion in confluent growth-suppressed Madin-Darby canine kidney (MDCK) cells. This upregulation occurred in the absence of both cytotoxicity and an inhibition of protein synthesis, two potential nonspecific inducers of IEG expression. These findings were of interest because we have previously shown that hyperosmotic stress induced by the functionally membrane-impermeant solute NaCl increased expression of these IEG, whereas hyperosmotic stress induced by the membrane-permeant solute glycerol failed to do so. The urea-induced increase in Egr-1 mRNA expression was not secondary to enhanced message stability as determined by actinomycin D experiments and is therefore likely a consequence of urea-induced transcriptional activation. Augmented Egr-1 expression in response to urea treatment was also observed in another renal epithelial cell line, LLC-PK1, but not in other cell types examined. Therefore cells of renal epithelial origin may be uniquely capable of responding to hyperosmotic urea with increased expression of IEG transcription factors, and this increase is likely transcriptionally mediated.

Animals↗

PubMed1993

Urea selectively induces DNA synthesis in renal epithelial cells.

Hyperosmotic stress with the functionally impermeant solute NaCl has been shown by us and others to inhibit cell growth and DNA synthesis. Several lines of evidence suggest that urea, the other principal renal medullary solute, may exert a growth-promoting effect on renal epithelial cells. Among these is the finding that urea upregulates expression at the mRNA level of two growth-associated immediate-early genes, Egr-1 and c-fos. In the present study, urea, in concentrations characteristic of the renal medulla, increased [3H]thymidine incorporation approximately threefold in confluent, growth-suppressed Madin-Darby canine kidney (MDCK) cells, whereas another readily membrane-permeant solute, glycerol, did not. Urea also overcame the inhibitory effect of hyperosmotic NaCl on DNA synthesis. The urea-induced increase in [3H]thymidine incorporation was also evident in the renal epithelial LLC-PK1 cell line, but not in renal nonepithelial and epithelial nonrenal cell types examined. In addition, it was associated with a 15% increase in total DNA content measured fluorometrically at 24 h of treatment. There was, however, no associated increase in cell proliferation as measured by cell number, total protein content, or cell cycle distribution. Urea also failed to induce polyploidy or aneuploidy. Therefore cells of renal epithelial origin may be uniquely capable of responding to hyperosmotic urea with increased DNA synthesis through an undefined and potentially novel mechanism.

Animals↗

PubMed1993

An osmotically tolerant inner medullary collecting duct cell line from an SV40 transgenic mouse.

The terminal inner medullary collecting duct (IMCD) plays an important role in determining the final urinary composition. Currently, there is no continuous cell line derived from this nephron segment. We have developed a cell line derived from the terminal IMCD of mice transgenic for the early region of simian virus SV40 (large T antigen). This cell line, mIMCD-3, retains many differentiated characteristics of this nephron segment including high transepithelial resistance (1,368 +/- 172 omega.cm2), inhibition of apical-to-basal sodium flux by amiloride (41 +/- 7%) and by atrial natriuretic peptide (ANP) (40 +/- 9%), the presence of the amiloride-sensitive sodium channel as determined by Western blot analysis, and accumulation of the major organic osmolytes in response to hypertonic stress. Significantly, mIMCD-3 cells adapted readily and were able to grow in hypertonic medium supplemented with NaCl and urea up to 910 mosmol/kgH2O. These extreme osmotic conditions exist in the renal medulla in vivo but are known to be lethal to most other cells. This cell line should be highly useful for the study of the cellular adaptation to osmotic stress and the cell biology and transport physiology of this nephron segment.

Adaptation, Physiological↗

PubMed1993

The role of organic osmolytes in the cerebral cell volume regulatory response to acute and chronic renal failure.

Brain cells respond to increased osmolality of the extracellular fluid by accumulating inorganic electrolytes and nonperturbing organic osmolytes to limit the extent of brain cell shrinkage. It is unclear whether urea is an effective osmole that triggers this adaptive response. Therefore, the amount of brain water and the cerebral content of organic osmolytes in rats with acute renal failure induced by bilateral ureteral ligation and in animals with chronic renal failure for 6 weeks created by a 75% reduction in renal mass were measured. Eight hours after the onset of acute renal failure, the BUN concentration and serum osmolality were 61 +/- 4 mg/dL and 314 +/- 2 mosmol/kg, respectively, compared with 13 +/- 1 mg/dL and 288 +/- 1 mosmol/kg, respectively, in sham-operated rats. This hyperosmolal state reduced brain water from 79.7 +/- 0.1% in controls to 79.2 +/- 0.1% in uremic animals (P < 0.01). During acute renal failure lasting 48 h, the BUN concentration and serum osmolality rose to 251 +/- 10 mg/dL and 370 +/- 4 mosmol/kg versus 18 +/- 1 mg/dL and 286 +/- 4 mosmol/kg, respectively, in sham-operated control rats. The percentage of brain water content was unchanged in rats with azotemia for 48 h--79.6 +/- 0.1 compared with 79.5 +/- 0.2% in controls. Stabilization of brain volume was associated with a 19% increment in total organic osmolyte content in brain cells from 123 +/- 6 to 146 +/- 2 mmol/kg dry wt (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Kidney Injury↗

PubMed1993

Differential actions of cisplatin on renal proximal tubule and inner medullary collecting duct cells.

Cisplatin (CP) nephrotoxicity in vivo is characterized by proximal tubule (PT) and collecting duct dysfunction. We reported previously that mitochondrial injury is an important early event in CP toxicity to PT cells and precedes inhibition of Na+,K(+)-ATPase activity and loss of cell K+. In the present study, we monitored oxygen consumption (QO2) and net K+ fluxes in intact inner medullary collecting duct (IMCD) and PT cells in vitro, using O2- and K(+)-sensitive electrodes, to determine if CP has similar effects on IMCD cells. Short-term exposure of IMCD cells to CP resulted in inhibition of spontaneous, ouabain-sensitive and ouabain-oversensitive QO2, but to a lesser degree than in PT. Ouabain-sensitive K+ transport and cell K+ content were also reduced in intact IMCD cells in this setting, confirming inhibition of Na+,K(+)-ATPase activity. In contrast, Na+,K(+)-ATPase activity measured in IMCD cell lysates was not altered. These results suggested that CP inhibited Na+,K(+)-ATPase activity in intact IMCD cells indirectly either by blocking Na+ entry or by inhibiting mitochondrial oxidative phosphorylation. Nystatin (Na+ ionophore) and carbonyl cyanide m-chlorophenylhydrazone (CCCP, uncoupler of oxidative phosphorylation) were used to distinguish between these possibilities. Nystatin-stimulated and CCCP-uncoupled QO2 were reduced in CP-treated IMCD cells by 34 +/- 10% and 25 +/- 5%, respectively, indicating mitochondrial injury. Again, the effects of CP on nystatin-stimulated and CCCP-uncoupled QO2 in IMCD cells were significantly less dramatic than in PT cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Expression of GLUT-2 cDNA in human B lymphocytes: analysis of glucose transport using flow cytometry.

The molecular characterization of transport proteins is often limited by transient functional expression or the need for a simple method to select functional cDNA clones. We used a mammalian expression system to obtain long-term expression of GLUT-2, an isoform of glucose permease. Rat GLUT-2 cDNA was ligated into an EBV vector (pLPP) and transfected into B lymphocytes which lack GLUT-2. Northern and Western analyses confirmed expression of GLUT-2 protein in membranes of transfected cells. Two functional assays using flow cytometry were developed to distinguish GLUT-2 transfectants from control/pLPP transfectants. Uptake of NBD-glucosamine, a fluorescent analogue of glucose, was increased in GLUT-2 transfectants. In addition, when exposed to hypertonic glucose medium, GLUT-2 transfectants and control/pLPP transfectants exhibited a difference in forward-angle light scatter (FALS), an index of cell volume, indicating a difference in glucose permeability. Independent measurements of glucose uptake (isotopic) and cell volume (video microscopy) confirmed the flow cytometry observations. This expression system used in combination with flow cytometry is useful for studying the functional properties of glucose and other solute transporters.

4-Chloro-7-nitrobenzofurazan↗

PubMed1991

Hyponatremia causes large sustained reductions in brain content of multiple organic osmolytes in rats.

Brain adaptation to hypoosmolality is known to involve volume regulatory losses of both extracellular and intracellular electrolytes. We studied the effects of acute and chronic hypoosmolality on brain content of organic osmolytes as well as electrolytes in rats to ascertain the relative contributions of different brain solutes to the brain volume regulation that occurs under these conditions. Brains were dissected from rats after 2, 7 and 14 d of sustained hyponatremia induced by continuous infusion of 1-deamino-[8-D-arginine]-vasopressin (DDVAP) in combination with a liquid formula, along with control rats fed the same formula in the absence of DDAVP infusions. One half of each brain was analyzed for organic osmolyte contents and the other half for water and electrolyte contents. Brain Na+, K+ and Cl- and multiple organic osmolytes (glutamate, creatine, taurine, myo-inositol, glutamine and glycerophosphoryl-choline) decreased markedly by 2 d of hyponatremia, and brain electrolyte and most organic osmolyte contents then remained at these reduced levels throughout the duration of the hyponatremia. Although the absolute magnitude of the brain electrolyte losses was greater than the magnitude of the brain organic osmolyte losses, the organic osmolyte losses accounted for approximately 35% of the total measured brain solute losses during sustained hyponatremia. These results demonstrate that organic osmolytes constitute a significant proportion of the brain solute losses that take place during hyponatremia, and indicate that reductions in both organic osmolyte and electrolyte contents are necessary to accomplish brain volume regulation during adaptation to sustained hypoosmolality.

Animals↗

PubMed1991

Application of missing pulse steady state free precession to the study of renal microcirculation.

Missing pulse steady state free precession (MP-SSFP), an extension of steady state free precession (SSFP), was evaluated for its ability to measure slow fluid flows. In experiments using flow phantoms, the MP-SSFP signal was sensitive to fluid velocities in the millimeters per second range. Isolated perfused rabbit kidneys were then used to determine if MP-SSFP could measure perfusion in a biological tissue. The signal intensities in the different anatomical regions of the kidney were observed to be related to the total flow to the organ. Furthermore, increasing the flow sensitivity of the pulse sequence by increasing the gradient strength resulted in decreases in the image signal intensity. The MP-SSFP signal was more sensitive to flow in the medulla than in the cortex. This can be related to slow flow sensitivity of MP-SSFP and the known differences in velocity profiles between these two regions. These results suggest that MP-SSFP may be a powerful tool for the noninvasive measurement of slow fluid flows in different regions of the kidney.

Animals↗

PubMed1991

Regional 1H transverse magnetization studies in perfused rabbit kidney.

A Carr-Purcell-Meiboom-Gill imaging sequence consisting of 128 echoes is used to extract transverse magnetization decay curves (TDCs) at 1.9 T from 1.7 x 1.7 x 5-mm3 voxels within the cortex, outer medulla, and inner medulla of perfused rabbit kidneys. The spatially localized TDCs within each tissue type are found to be better approximated by biexponential, as opposed to monoexponential, functions. The biexponential parameters characterizing the TDCs demonstrate an improved degree of tissue specificity over that available from monoexponential analyses. The fraction of the quickly relaxing TDC component and the relaxation rate of this component are observed to decrease from cortex to inner medulla. A two-site exchange analysis is used to convert biexponential TDC parameters into water volume fractions and exchange rates. The exchange rates between the fast and slowly relaxing pools increased from cortex to inner medulla. All exchange rates were less than 1.5 Hz, indicating a relatively slow water exchange process. The imaging methods and subsequent analyses offer the potential to generate unconventional MR images with tissue contrast dependent upon water compartmentation and exchange.

Animals↗

PubMed1991

Objective verification of dietary intake by measurement of urine osmolality.

A new method for assessing the accuracy of dietary nutrient intakes in metabolic studies is described. The theoretical basis of the method is the comparison of measurements of the urine osmole excretion rate (OER, the product of urine osmolality and urine weight) with values for OER predicted from dietary nitrogen, sodium, and potassium. The method was tested in 34 healthy male and female volunteers aged 18-78 y who made complete 24-h urine collections and consumed a diet over 6 d in metabolic-balance studies involving either overfeeding, weight maintenance, or weight loss. The coefficients of variation for equations relating measured OER to dietary nitrogen, sodium, and potassium intakes ranged from 14.1% for 1-d measurements to 6.94% for 6-d means. These results indicate that it should be possible to use measurements of the urine OER to identify dietary noncompliance in metabolic studies.

Adolescent↗

PubMed1991

Upregulation of inositol transport mediates inositol accumulation in hyperosmolar brain cells.

Attempts to understand brain volume regulation have been greatly hampered by the structural complexity of the mammalian central nervous system, indicating a need for the investigation of cultured brain cell lines whose behavior reflects that observed in situ. We demonstrate here that rat C6 glioma cells exhibit a pattern of hyperosmolar volume regulation qualitatively similar to that of the intact brain. Chronic (2-6 days) acclimation of C6 cells to high NaCl media (440 or 590 mosM) resulted in a 46-133 mM increase in cellular inositol, a known major brain osmolyte. C6 cells exposed acutely to 440 mosM medium shrank abruptly and then underwent a complete regulatory volume increase (RVI) within 4 h. Inositol levels began to increase after 10 h of hyperosmolar stress and reached maximal values by 24 h, suggesting that RVI is initially mediated by inorganic ion uptake. [3H]inositol uptake measurements revealed a sevenfold stimulation of phlorizin-inhibitable inositol transport in hyperosmotic cells. The enhancement of inositol transport paralleled the rise in cellular inositol content. Phlorizin reduced inositol accumulation in hyperosmolar cells by 44%. Our studies provide the first demonstration of RVI and organic osmolyte accumulation in a cultured brain cell line.

Animals↗

PubMed1991

Immediate early gene and HSP70 expression in hyperosmotic stress in MDCK cells.

The early genetic response to hyperosmotic stress remains to be elucidated in eukaryotes. We observed that hyperosmotic NaCl in Madin-Darby canine kidney cells increased levels of mRNA encoding the immediate early gene (IEG) transcription factors Egr-1 and c-fos at 2 h of treatment by two- and threefold, respectively. Sham treatment and hyperosmotic glycerol, and ineffective osmole, had no effect. Hyperosmotic NaCl, but not glycerol, also increased the mRNA level of the stress protein HSP70 by four- to fivefold at 2, 6, and 24 h. These changes occurred despite inhibition of total RNA transcription rate and DNA synthesis rate by NaCl. Neither NaCl nor glycerol treatment manifested significant cytotoxicity. NaCl, and to a lesser extent glycerol, suppressed protein synthesis, a phenomenon previously correlated with IEG superinduction. Therefore, hyperosmotic stimuli with different physiological effects result in differential expression of IEGs and the stress protein HSP70; induction of the former may govern the ensuing program of gene expression that culminates in the osmolyte response, while the latter may serve as a temporizing protective measure.

Animals↗

PubMed1990

Sulfhydryl-reactive heavy metals increase cell membrane K+ and Ca2+ transport in renal proximal tubule.

The cellular mechanisms by which nephrotoxic heavy metals injure the proximal tubule are incompletely defined. We used extracellular electrodes to measure the early effects of heavy metals and other sulfhydryl reagents on net K+ and Ca2+ transport and respiration (QO2) of proximal tubule suspensions. Hg2+, Cu2+, and Au3+ (10(-4)M) each caused a rapid net K+ efflux and a delayed inhibition of QO2. The Hg2(+)-induced net K+ release represented passive K+ transport and was not inhibited by barium, tetraethylammonium, or furosemide. Both Hg2+ and Ag+ promoted a net Ca2+ uptake that was nearly coincident with the onset of the net K+ efflux. A delayed inhibition of ouabain-sensitive QO2 and nystatin-stimulated QO2, indicative of Na+, K(+)-ATPase inhibition, was observed after 30 sec of exposure to Hg2+. More prolonged treatment (2 min) of the tubules with Hg2+ resulted in a 40% reduction in the CCCP-uncoupled QO2, indicating delayed injury to the mitochondria. The net K+ efflux was mimicked by the sulfhydryl reagents pCMBS and N-ethylmale-imide (10(-4) M) and prevented by dithiothreitol (DTT) or reduced glutathione (GSH) (10(-4) M). In addition, both DTT and GSH immediately reversed the Ag(+)-induced net Ca2+ uptake. Thus, sulfhydryl-reactive heavy metals cause rapid, dramatic changes in the membrane ionic permeability of the proximal tubule before disrupting Na+, K(+)-ATPase activity or mitochondrial function. These alterations appear to be the result of an interaction of the metal ions with sulfhydryl groups of cell membrane proteins responsible for the modulation of cation permeability.

Animals↗

PubMed1990

Influence of Na+ intake on dopamine-induced inhibition of renal cortical Na(+)-K(+)-ATPase.

The enzyme L-amino acid decarboxylase (L-AADC), found in abundance in rat proximal tubule cell cytosol, converts L-dopa to dopamine. Dopamine, in turn, suppresses proximal tubule sodium transport by inhibiting Na(+)-K(+)-ATPase activity. We sought to determine whether changes in dietary sodium intake in rats lead to adaptation of dopamine formation and dopamine-induced Na(+)-K(+)-ATPase inhibition. In rats on a high-salt (HS) diet, the maximal velocity (Vmax) of renal cortical L-AADC was 78 +/- 19% higher than that in rats on a low-salt (LS) diet. The Michaelis constant (Km) of the enzyme remained unchanged. In renal cortical tubule cell suspensions the L-dopa-induced inhibition of ouabain-sensitive oxygen consumption (QO2) was significantly greater in rats on HS diet than in rats on LS diet. Furthermore, L-dopa completely inhibited the nystatin-induced rise in QO2 in the HS but not in the LS group. Carbidopa, an inhibitor of L-AADC, abolished the L-dopa-induced inhibition of nystatin-stimulated QO2 in cells from HS rats and was without significant effect in cells from LS rats. L-Dopa-stimulated K+ efflux was greater in cells from HS rats at 28 +/- 1 nmol.min-1.mg protein-1, compared with 7 +/- 6 nmol.min-1.ng protein-1 in cells from LS rats. By contrast, ouabain-stimulated K+ efflux did not differ between the groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Mitochondrial injury: an early event in cisplatin toxicity to renal proximal tubules.

Oxygen consumption (QO2) and net K+ transport were studied in rabbit proximal tubule suspensions to define the early effects of cisplatin on proximal tubule function. Cisplatin caused dose-dependent inhibition of QO2, which was delayed in onset. The concentration of cisplatin required for inhibition decreased as the duration of exposure was increased [40-min exposure, threshold concentration of 10(-4) M, inhibitor constant (Ki) of 10(-3) M; 4-h exposure, threshold concentration of 3 X 10(-5) M, Ki of 10(-4) M]. Both ouabain-sensitive and ouabain-insensitive QO2 were reduced, indicating inhibition of all adenosinetriphosphatases, including Na(+)- K(+)-ATPase activity. There was a parallel fall in ouabain-sensitive net K+ transport and cytosolic K+ content, confirming the latter observation. Na(+)-K(+)-ATPase activity was unchanged in cell membranes prepared by hypotonic lysis from cisplatin-treated tubules, indicating an indirect cytosol-dependent mechanism of enzyme inhibition. Nystatin-stimulated QO2 was reduced in cisplatin-treated tubules, excluding inhibition of Na+ entry as the mechanism of injury and suggesting mitochondrial injury. The latter was confirmed by measurement of carbonylcyanide-m-chlorophenylhydrazone (CCCP)-uncoupled QO2 in intact cells and ADP-stimulated (state 3) QO2 in digitonin-permeabilized tubules. Furthermore, by maximally stimulating mitochondrial respiration with CCCP and nystatin, it was possible to demonstrate mitochondrial injury at a time when basal QO2 and K+ transport were apparently normal. These data suggest that mitochondrial injury is a central event in cisplatin toxicity to the proximal tubule.

Adenosine Diphosphate↗