Cadmium inhibits glucose uptake in primary cultures of mouse cortical tubule cells

1990 ◽  
Vol 258 (6) ◽  
pp. F1625-F1633 ◽  
Author(s):  
S. S. Blumenthal ◽  
D. L. Lewand ◽  
M. A. Buday ◽  
J. G. Kleinman ◽  
S. K. Krezoski ◽  
...  
Keyword(s):  
Amino Acid ◽  
Glucose Uptake ◽  
Cultured Cells ◽  
Primary Cultures ◽  
Defined Medium ◽  
Isobutyric Acid ◽  
Cell Permeability ◽  
Dependent Manner ◽  
Lactate Dehydrogenase Activity ◽  
Tubule Cells

We studied the effect of cadmium (Cd2+) on transport of alpha-methylglucoside in primary cultures of mouse kidney cortical tubule cells grown in defined medium. When cultured cells were exposed to Cd2+ concentrations from 0 to 6 microM for 24 h, uptake of alpha-methylglucoside was inhibited in a dose-dependent manner by up to 50%. By contrast, acute exposure of the cells to 7 microM Cd2+ for 60 min did not inhibit alpha-methylglucoside uptake. Increasing Cd2+ concentrations progressively decreased the Vmax of Na(+)-dependent glucose cotransport but not the Km for glucose. Cell ATP/ADP ratios of unexposed monolayers and of cells exposed to 4.5 microM Cd2+ for 24 h were 5.0 and 4.9, respectively (n = 3). Intracellular volume, lactate dehydrogenase activity, and cell Na+ and K+ concentrations were unaltered even after 24 h of exposure to 7 microM Cd2+. Untreated and Cd2+-treated monolayers preloaded with alpha-methylglucoside released the sugar analogue into the medium at nearly identical rates, indicating that Cd2+ did not alter cell permeability to glucose. Uptake of the amino acid analogue alpha-(methylamino)isobutyric acid was not affected by prior Cd2+ exposure. Whereas cell DNA content declined in Cd2(+)-exposed plates, both Na(+)-glucose and Na(+)-amino acid cotransport were enhanced at lower cell densities. Protein and DNA synthesis, estimated, respectively, by incorporation of [3H]leucine and [3H]thymidine into acid-insoluble material, were not significantly affected at 6 microM Cd2+. We conclude that after a lag time Cd2+ selectively inhibits renal Na(+)-dependent glucose transport despite an unchanged gradient for Na+ across the cell membrane.

scholarly journals Effect of serum proteins on sodium-linked transport processes in cultured kidney cells.

1995 ◽  
Vol 5 (11) ◽  
pp. 1964-1970
Author(s):  
S S Blumenthal ◽  
D L Lewand ◽  
P A Tipnis ◽  
J G Kleinman
Keyword(s):  
Amino Acid ◽  
Dextran Sulfate ◽  
Cultured Cells ◽  
Serum Proteins ◽  
Fluid Phase ◽  
Defined Medium ◽  
Transport Processes ◽  
Transport Systems ◽  
Heat Treated ◽  
Fluid Phase Endocytosis

The mechanism for increased Na+ retention in the nephrotic syndrome is unknown. To determine if Na+ transport systems in the proximal tubule might be affected by filtered proteins, mouse cortical tubule cells grown in defined medium were exposed to concentrations of bovine serum albumin (BSA) ranging from 0.01 to 0.5%. Activity of the Na(+)-glucose cotransporter, measured as Na(+)-dependent uptake of alpha-methylglucoside, increased progressively to a maximum of 2.3-fold above baseline (P < 0.001; N = 10). The increase in transporter activity was due to an increased Vmax, and the magnitude of the increase was inversely related to the basal cotransporter activity of the cultures. Increased cotransporter activity was detectable 6 h after exposure, was sustained for 24 h after cells were removed from an albumin-free medium, and was prevented by cycloheximide. Heat-treated BSA, fatty-acid and globulin-free BSA, and gamma-globulins were as effective at increasing Na(+)-glucose cotransporter activity as untreated Fraction V BSA. Dextran, dextran-sulfate, and amino acid supplements were ineffective. Neither protease inhibitors nor chloroquine added to an albumin-containing medium prevented increased alpha-methylglucoside uptake. Albumin did not change the rate of fluid-phase endocytosis in the cultured cells. Na(+)-amino acid cotransport and Na(+)-H+ exchange were either decreased or unchanged after BSA exposure. Exposing apical surfaces of cells grown on permeable membranes to BSA led to a greater increase in activity of the Na(+)-glucose cotransporter relative to controls than did exposing the basolateral surface (145 versus 89%; P < 0.05; N = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of pH on growth of mouse renal cortical tubule cells in primary culture

1989 ◽  
Vol 257 (3) ◽  
pp. C419-C426 ◽  
Author(s):  
S. S. Blumenthal ◽  
D. L. Lewand ◽  
M. A. Buday ◽  
N. S. Mandel ◽  
G. S. Mandel ◽  
...  
Keyword(s):  
Water Content ◽  
Prostaglandin E1 ◽  
Primary Cultures ◽  
Defined Medium ◽  
Thymidine Uptake ◽  
Medium Ph ◽  
Medium Acidification ◽  
Cell Ph ◽  
Glass Slides ◽  
Tubule Cells

We examined the effect of the medium pH on growth of primary cultures of mouse cortical tubule cells grown in defined medium. A significantly higher DNA content was observed within 24 h of lowering medium pH from 7.4 to 6.8 or 7.1 and persisted for the duration of the study. Further studies revealed that either medium acidification or insulin plus prostaglandin E1 nearly doubled uptake of [3H]thymidine in cells deprived of other growth factors for the previous 72-110 h. Moreover, the effects of insulin, prostaglandin E1, and medium acidification on [3H]thymidine uptake of quiescent cells were additive. An alkaline medium pH appeared to have a small but significant effect on cell hypertrophy, since cells exposed to pH 7.4 and 7.7 had a higher protein-to-DNA ratio than cells incubated at a lower pH. Cell pH of monolayers grown on glass slides determined from fluorescence of the carboxyfluorescein analogue 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) was linearly correlated with medium pH, and changes in medium pH resulted in changes in steady-state cell pH of a similar magnitude. Four hours after medium acidification, relative increases in cell Na+ and water content occurred, whereas medium alkalinization led to decreases in cell Na+ and water content. The increases in cell Na+ and cell water content at pH 6.8 could be inhibited by amiloride. We conclude that decreasing the cell pH can be a mitogenic stimulus for renal tubule cells. Medium acidification is accompanied by changes in cell Na+ transport, which may be mediated in part by altered Na+-H+ antiporter activity.

ASC system activity is altered by development of cell polarity in trophoblast from human placenta

1993 ◽  
Vol 265 (1) ◽  
pp. C212-C217 ◽  
Author(s):  
T. C. Furesz ◽  
C. H. Smith ◽  
A. J. Moe
Keyword(s):  
Amino Acid ◽  
Primary Cultures ◽  
Amino Acid Uptake ◽  
Selective Inhibition ◽  
Isobutyric Acid ◽  
Neutral Amino Acid ◽  
Transport Systems ◽  
Bewo Cell ◽  
Acid Uptake

Pathways of neutral amino acid uptake were investigated in vitro during differentiation of primary cultures of trophoblast isolated from full-term human placentas and a clone (b30) of the BeWo cell line. Inhibition of initial alanine (0.1 microM) uptake by 2-(methylamino)isobutyric acid and unlabeled alanine revealed two Na(+)-dependent systems and one Na(+)-independent transporter. Characterization of these transporters, by selective inhibition, suggested system A, ASC, and L-like transporters. Concomitant with formation of microvillous membrane and syncytium, system ASC activity decreased from 16.1 +/- 2.8 pmol.mg DNA-1.min-1 at 24 h to 2.4 +/- 1.1 pmol.mg DNA-1.min-1 at 72 h. Na(+)-independent alanine uptake increased from 6.0 +/- 2.0 to 12.9 +/- 0.9 pmol.mg DNA-1.min-1 at 24 and 72 h, respectively. Similarly, alpha-(methylamino)isobutyric acid-insensitive, Na(+)-dependent activity in b30 cells (100 microM alanine) decreased from 6.5 +/- 1.6 to 1.2 +/- 1.2 nmol.mg DNA-1.min-1 for control and forskolin-treated cells, respectively. We conclude that membrane specialization accompanying fusion and differentiation of the cytotrophoblast to form syncytiotrophoblast results in a polarization of neutral amino acid transport systems.

Insulin-mimetic agents vanadate and pervanadate stimulate glucose but inhibit amino acid uptake

1997 ◽  
Vol 272 (1) ◽  
pp. C156-C162 ◽  
Author(s):  
E. Tsiani ◽  
N. Abdullah ◽  
I. G. Fantus
Keyword(s):  
Amino Acid ◽  
Glucose Uptake ◽  
Tyrosine Phosphatase ◽  
Amino Acid Uptake ◽  
Inhibitory Effect ◽  
Acid Uptake ◽  
Dependent Manner ◽  
Transport Capacity ◽  
Insulin Mimetic ◽  
Biologic Effects

The protein tyrosine phosphatase (PTP) inhibitors vanadate and pervanadate (pV) exert insulin-like biologic effects. In cultured differentiated rat L6 skeletal muscle cells, vanadate and pV stimulated 2-deoxy-D-[3H]glucose uptake in a dose- and time-dependent manner. There was no increase in maximum stimulation by additional insulin. In contrast, whereas insulin stimulated [14C]methylaminoisobutyric acid (MeAIB) uptake, basal uptake was inhibited by vanadate and pV. Insulin-stimulated MeAIB uptake was also inhibited in a dose-dependent manner and completely abolished by 5 mM vanadate or 0.1 mM pV. The inhibitory effect on basal MeAIB uptake was associated with a decrease in transporter affinity and a small decrease in maximum transport capacity, whereas the insulin-stimulated increase in maximum transport capacity was completely inhibited. Inhibition of MeAIB uptake by vanadate and pV was not blocked by cycloheximide, and oubain did not inhibit uptake. Vanadate also inhibited amino acid deprivation-stimulated MeAIB uptake. Insulin-stimulated MeAIB uptake was also inhibited in rat hepatoma cells. Thus vanadate and pV mimic insulin to stimulate glucose uptake but inhibit system A amino acid uptake. The relative inhibitory concentrations of vanadate and pV suggest that the mechanism may involve PTP inhibition.

Effects on the ATP Content of Cultured Cells after Radiographic Contrast Media Exposure

1989 ◽  
Vol 30 (5) ◽  
pp. 541-547 ◽  
Author(s):  
A. Nordby ◽  
K. Thorstensen ◽  
J. Halgunset ◽  
O. A. Haugen ◽  
S. Solberg
Keyword(s):  
Contrast Media ◽  
Cultured Cells ◽  
Primary Cultures ◽  
Established Cell Line ◽  
Dependent Manner ◽  
Atp Content ◽  
Concentration Dependent Manner ◽  
Short Period ◽  
Radiographic Contrast Media ◽  
Established Cell

The ATP content of cultured cells after exposure to meglumine-calcium metrizoate, sodium metrizoate, iohexol, iopamidol and saline was studied. Initially, the ATP content diminished rapidly for a short period and thereafter slowly during the incubation. After incubation with contrast media or saline, the ATP content slowly increased to normal when the cells were reincubated with fresh nutrient medium. Different contrast media and saline with the same final osmolality produced a similar effect on the ATP content of the cultured cells. Cellular association of meglumine-sodium diatrizoate, sodium metrizoate, sodium-iothalamate, iohexol and iopamidol was also examined. The established cell line NHIK 3025 as well as primary cultures of human umbilical endothelium were found to accumulate contrast media in a time-and concentration-dependent manner. When the incubation was carried out at 4°C, the cellular accumulation of contrast medium was less than 35 per cent of that seen at 37°C. It therefore seems that energy-dependent processes are involved to some degree.

Comparison of peptide release from fetal rat hypothalamic neurones cultured in defined media and serum-containing media

1988 ◽  
Vol 116 (3) ◽  
pp. 349-356 ◽  
Author(s):  
M. J. O. Clarke ◽  
G. E. Gillies
Keyword(s):  
Primary Cultures ◽  
Defined Medium ◽  
Maximal Response ◽  
Dependent Manner ◽  
Corticotrophin Releasing Factor ◽  
Fetal Rat ◽  
Concentration Step ◽  
And Control

ABSTRACT Primary cultures of rat hypothalamic neurones were maintained either in a serum-supplemented medium or in a serum-free chemically defined medium for up to 6 weeks. The release of the 41 amino acid-containing peptide, corticotrophin-releasing factor (CRF-41), vasopressin (AVP) and somatostatin (SRIF) were followed using immunoassays. In response to K+ (56 mmol/l) depolarization both the quantities of peptides released and the magnitude of responses were significantly greater from cultures maintained in the fully supplemented defined medium. As a consequence, release of CRF-41 and AVP could be measured directly, without requiring the concentration step necessary for cultures grown in serum. The response to K+ depolarization increased with the age of the culture, suggesting neuronal maturation. Responses to K+ depolarization were Ca2+-dependent, and the addition of corticosterone (100 nmol/l) to the defined medium caused a significant reduction in the response of neurones secreting CRF-41 and AVP, but not those secreting SRIF, to depolarization. This suggests the retention in vitro of the responsiveness of stress-associated neuropeptides to the negative feedback effects of corticosterone. Neurones producing CRF-41 and AVP responded significantly in a dose-dependent manner to acetylcholine stimulation, whereas those producing SRIF did not. As cultures matured, the CRF-41- and AVP-producing neurones became more sensitive to acetylcholine with the maximal response at 1 nmol acetylcholine/1. In conclusion, the culture of rat hypothalamic neurones is improved in terms of peptide output when the cultures are maintained in a defined medium. Differential responses of the peptidergic neurones may be seen in the presence of corticosterone and neurotransmitters, illustrating the retention in vitro of specific receptor-mediated responses which have been observed in vivo. This model should prove useful in the further study of the physiological, pharmacological and biochemical maturation and control of peptidergic neurones. J. Endocr. (1988) 116, 349–356

Regulation of albumin synthesis by hormones and amino acids in primary cultures of rat hepatocytes

1987 ◽  
Vol 252 (3) ◽  
pp. E291-E298 ◽  
Author(s):  
S. M. Hutson ◽  
C. Stinson-Fisher ◽  
R. Shiman ◽  
L. S. Jefferson
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Cell Number ◽  
Dependent Manner ◽  
Albumin Synthesis ◽  
Albumin Secretion ◽  
Culture Dish ◽  
Experimental Conditions

Culture conditions necessary for optimizing albumin secretion were studied in rat hepatocytes maintained in a chemically defined, serum-free medium. Amino acid analysis of the culture medium, which was based on a 1:1 mixture of Ham's F12:Dulbecco's modified Eagle's medium (unsupplemented medium), revealed that certain essential amino acids were depleted from this medium over a 24-h incubation. Rates of albumin secretion were significantly higher and better maintained when the medium was supplemented with additional amino acids (supplemented medium). Moreover, selective removal of an essential amino acid resulted in an immediate decrease in total protein and albumin synthesis and after 48 h a further selective decrease in albumin synthesis. Linear rates of albumin secretion were observed over a wide variety of experimental conditions, but secretion was not strictly proportional to cell number. Maximal rates of secretion were obtained at plating densities of 2–3 X 10(6) cells/60 mm culture dish. Albumin secretion also increased with time in culture reaching a maximum on days 3 and 4. When added singly, either insulin or dexamethasone increased rates of albumin secretion in a dose-dependent manner, but both hormones and an adequate supply of amino acids were necessary for maximal rates of secretion as well as long-term maintenance of the hepatocytes (greater than 3–4 days). In the presence of dexamethasone the dose-response curve for insulin was shifted toward physiological insulin concentrations. Changes in rates of albumin secretion in response to added hormones in supplemented media were found to parallel changes in albumin synthesis and relative amounts of albumin mRNA. Changes in gene transcription were probably involved.

Effects of parathyroid hormone on cytosolic calcium in renal proximal tubular primary cultures

1986 ◽  
Vol 251 (2) ◽  
pp. F188-F198 ◽  
Author(s):  
K. A. Hruska ◽  
M. Goligorsky ◽  
J. Scoble ◽  
M. Tsutsumi ◽  
S. Westbrook ◽  
...  
Keyword(s):  
Parathyroid Hormone ◽  
Cultured Cells ◽  
Primary Cultures ◽  
Cytosolic Calcium ◽  
Proximal Tubule Cells ◽  
Proximal Tubular ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells ◽  
Renal Proximal Tubular ◽  
Mitochondrial Uncouplers

The effects of parathyroid hormone on the cytoplasmic Ca2+ concentration of canine renal proximal tubule cells grown in primary culture were determined using the fluorescent Ca2+ indicator quin 2. The cultured cells exhibited responses to hormones, enzyme activities, transport functions, and morphology characteristic of the proximal convoluted tubule. Parathyroid hormone stimulated an immediate rise in cytoplasmic Ca2+, both in suspended cells and cells studied as a monolayer on Nuclepore filters. The rise in cytoplasmic Ca2+ induced by the hormone was sustained for 15-30 min, was dose dependent, and was not mimicked by cyclic AMP. Removing Ca2+ from the extracellular media markedly decreased cytoplasmic Ca2+ and abolished the effects of parathyroid hormone on cytosolic Ca2+. 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate blocked the effects of the hormone on cytosolic Ca2+, but mitochondrial uncouplers failed to inhibit the effects of the hormone to increase cytoplasmic Ca2+. These studies support a role of Ca2+ in the activation of proximal renal tubule cells by parathyroid hormone.

Investigation of the ontogenetic patterns of rat hypothalamic dopaminergic neurone morphology and function in vitro

1993 ◽  
Vol 139 (3) ◽  
pp. 403-NP ◽  
Author(s):  
H. E. Murray ◽  
G. E. Gillies
Keyword(s):  
Primary Culture ◽  
High Performance ◽  
Cultured Cells ◽  
Defined Medium ◽  
Dependent Manner ◽  
Uptake Mechanism ◽  
Functional Maturation ◽  
Functional Development

ABSTRACT Using fetal rat hypothalamic cells in primary culture maintained in a serum-free defined medium we have investigated the morphological and functional development of the dopamine (DA)-containing neurones intrinsic to the hypothalamus. Immunocytochemical studies demonstrated the presence of three morphologically distinct subtypes of tyrosine hydroxylase-immunopositive neurones. On day 3 in vitro unipolar, bipolar and multipolar cell types were apparent. The latter two subtypes persisted to later days in culture and increased both in perikarya size and neurite length. All subtypes have been shown to have correlates in vivo. Biochemical studies employing [3H]DA demonstrated a time- and temperature-dependent uptake mechanism within the cultures which was significantly attenuated by the uptake inhibitors benztropine and nomifensine in a dose-dependent manner. [3H]DA was also released under both basal and 56 mmol K+/l-stimulated conditions and the magnitude of the response was reduced by exclusion of calcium from the release medium. The amount of [3H]DA accumulated and released by the cultured cells increased with the age of the culture, suggesting functional maturation of the DA-containing neurones within this preparation. The role of oestradiol-17β in regulating hypothalamic dopaminergic function was also investigated both indirectly with the use of [3H]DA and by direct measurement of endogenously synthesized DA using high-performance liquid chromatography coupled with electrochemical detection. Both uptake and release of [3H] and release of endogenous DA were significantly modulated by the concentration of steroid in the defined medium. These results demonstrate that hypothalamic dopaminergic neurones, when maintained in primary culture, undergo morphological and functional maturation which have several correlates in vivo. In addition, we have demonstrated that at least one sub-population of dopaminergic neurones within this preparation is responsive to oestradiol-17β. As DA is considered to be a vital component in the regulation of neuroendocrine activity we suggest that this model is valuable for the investigation of the functional development of the DA systems of the hypothalamus and the relationship existing between neurotransmitters, neuropeptides and neuroactive steroids. Journal of Endocrinology (1993) 139, 403–414

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