Search PubMed⌕ Search

Biomedical subjects

A S Pollock

Publications and source records attributed to A S Pollock.

31 records · Page 2Linked to original sources
PubMed1991

Molecular cloning of gp42, a cell-surface molecule that is selectively induced on rat natural killer cells by interleukin 2: glycolipid membrane anchoring and capacity for transmembrane signaling.

We have previously shown that in vitro culture of rat natural killer (NK) cells in high concentrations of recombinant interleukin 2 (rIL-2) leads to the expression of a surface glycoprotein with a molecular mass of approximately 42 kD. This glycoprotein, gp42, is not induced on other lymphocytes and thus provides a lineage-specific marker for rIL-2-activated NK cells. We here present the nucleotide sequence for gp42 cDNA. The open reading frame encodes 233 amino acids with three potential sites for N-linked glycosylation. The deduced amino acid sequence lacks an apparent transmembrane domain and instead contains a hydrophobic COOH terminus that is characteristic of glycosylphosphatidylinositol (GPI)-anchored surface proteins. Consistent with this, gp42 is cleaved from the NK-like cell line, RNK-16, by phosphatidylinositol-specific phospholipase C (PI-PLC), as is gp42 expressed on CHO cells that have been transformed with gp42 cDNA. On rIL-2-activated NK cells, gp42 is resistant to PI-PLC, though our studies suggest that gp42 on these cells is still expressed as a GPI-anchored molecule. Antibody to gp42 stimulates in RNK-16 cells an increase in inositol phosphates and in intracellular calciu, signals that are associated with the activation of lymphocytes, including NK cells. rIL-2-activated NK cells, however, lack this response to gp42 as well as to other stimuli. Thus, gp42, the only NK-specific activation antigen, is a GPI-anchored surface molecule with the capacity to stimulate transmembrane signaling.

Amino Acid Sequence↗
PubMed1990

Transfection-mediated expression of a dominant cAMP-resistant phenotype in the opossum kidney (OK) cell line prevents parathyroid hormone-induced inhibition of Na-phosphate cotransport. A protein kinase-A-mediated event.

Sodium-phosphate cotransport in the PTH-responsive opossum kidney (OK) cell line is inhibited by PTH, cAMP, and activators of protein kinase C. In order to probe the role of cAMP, we stably transfected OK cells with an expression vector for a cAMP-binding mutation of the murine protein kinase A regulatory subunit. Two-dimensional electrophoresis of cAMP-binding proteins from transfected cells indicated a 20-fold overexpression of the mutant regulatory unit. Protein kinase A from these cells had a 20-fold increase in the concentration of cAMP required for half-maximal activation, 2.8 microM vs. 0.15 microM for wild type cells. In the transfected cells, Na-phosphate cotransport was insensitive to up to 1 mM 8-Br-cAMP and 1 microM PTH, while these same agonists caused a significant inhibition of transport in the wild type cells. The effects on Na-phosphate cotransport of the protein kinase C activators oleoyl-acetyl glycerol and tetradecanoyl-phorbol acetate, which were marked in the wild type cells, were still present, although attenuated, in the transfected mutants. With prolonged passage, the cAMP-insensitive phenotype reverted to wild type cAMP sensitivity despite continued selection for the cotransfected neo marker. The revertant cells had a normal cAMP requirement for half-maximal activation of protein kinase A, 0.13 microM, and the PTH and cAMP-sensitive inhibition of Na-phosphate cotransport was restored. We suggest that an intact and normally cAMP-sensitive protein kinase A pathway is an absolute requirement for PTH inhibition of Na-phosphate cotransport in the OK cell.

Animals↗
PubMed1989

The 5'region of the rat phosphoenolpyruvate carboxykinase gene confers pH sensitivity to chimeric genes expressed in renal and liver cell lines capable of expressing PEPCK.

The 5' flanking regions of the rat phosphoenolpyruvate carboxykinase gene were used to form chimeric gene constructs with the human growth hormone gene. These constructs were transfected into several renal and one liver cell line and the production of growth hormone (HGH) measured by immunoassay. Cyclic-AMP and glucocorticoid responsiveness of HGH production was observed in all cell lines. In two lines, the rat NRK52E renal epithelial line and the rat H4IIE hepatoma cell line, both capable of expressing PEPCK, lowering extracellular pH increased HGH production several fold. Comparison of hormone and pH effect on cells transfected with a thymidine kinase promoter-HGH chimera indicated that the PEPCK 5' flanking region effects were specific. Thus, part of the pH responsiveness of the PEPCK gene in vivo may be attributed to properties of the 5' flanking regions.

Animals↗
PubMed1989

Induction of renal phosphoenolpyruvate carboxykinase mRNA: suppressive effect of glucose.

The mRNA for the important gluconeogenic enzyme phosphoenolpyruvate carboxykinase (GTP) (PEPCK; EC 4.1.1.32) is expressed in liver and kidney. In the kidney, acidosis is a unique and potent stimulus, whereas insulin, the major counterregulatory hormone of gluconeogenesis, has no effect. In this study, we find that oral glucose administration to rats rapidly decreases the abundance of renal PEPCK mRNA by 50-72%. This reduction takes place in normal euglycemic, in insulin-induced hypoglycemic, and in streptozotocin-induced hyperglycemic diabetic animals. The effect of glucose is not seen in the presence of metabolic acidosis, whether induced by NH4Cl or by prolonged fasting. Therefore, it appears that oral glucose loading is a physiological suppressor of renal PEPCK message abundance, although not in acidosis.

Acidosis↗
PubMed1987

Modification of the internal pH sensitivity of the Na+/H+ antiporter by parathyroid hormone in a cultured renal cell line.

Sodium-proton antiporter activity can be modulated through changes Vmax and/or intracellular proton sensitivity of the antiporter. To characterize a parathyroid hormone (PTH)-induced decrease in antiporter activity in a continuous renal cell line (opossum kidney cells), the extracellular sodium and intracellular proton dependence of amiloride-inhibitable 22Na uptake was studied. The Km for extracellular sodium at intracellular pH 6.32 was 28 mM and was unaltered by PTH, whereas the Vmax was decreased by 26%. When intracellular pH was set over the range 5.87-7.57 by the potassium-nigericin method, antiporter activity increased as intracellular pH decreased. Hill analysis revealed Hill coefficients of 1.25 and 1.01 and half-maximal antiporter activity at intracellular pH values of 6.90 and 6.35 for control and PTH-treated cells, respectively. PTH decreased the apparent Vmax at low pH by 15% and the intracellular pH at which Na+/H+ exchange is half-maximal by 0.55 pH units.

Animals↗
PubMed1987

The effects of cycloheximide on Na+/H+ antiporter activity in cultured opossum kidney cells.

These studies examined the effects of cycloheximide on the Na+/H+ antiporter in cultured opossum kidney cells. The effects of cycloheximide on antiporter activity depended on the basal level of activity. These data suggest that the Na+/H+ antiporter may be regulated by several processes which are sensitive to protein synthesis inhibition.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1986

Parathyroid hormone inhibition of Na+-H+ antiporter activity in a cultured renal cell line.

The renal effects of parathyroid hormone (PTH) include a decreased rate of acidification by the proximal tubule. To determine whether this effect represented a PTH action on the Na+-H+ antiporter, we investigated the effect of PTH on the established opossum kidney (OK) cell line. This cell line retains several features characteristic of proximal tubule cells, including an amiloride-sensitive Na+-H+ antiporter and high-affinity PTH receptors with a coupled cAMP response. We measured steady-state intracellular pH and amiloride-sensitive 22Na+ uptake as a reflection of the activity of the Na+-H+ antiporter. Under bicarbonate and CO2-free conditions, the steady-state intracellular pH of OK cell cultures was modified by altering the rate of Na+-H+ exchange. When Na+-H+ exchange was inhibited by amiloride, intracellular pH fell. Conversely, augmenting antiporter activity by addition of monensin, a Na+-H+ exchange ionophore, raised intracellular pH. PTH (2.5 X 10(-8) M) lowered intracellular pH by up to 0.17 pH units, and half of the maximum PTH effect was present at a concentration of 10(-12) M. This effect was not seen in the presence of amiloride or in the absence of sodium, suggesting that a functional Na+-H+ antiporter is necessary for its expression. The decrease in intracellular pH was reproduced by forskolin and 8-bromo-cAMP, suggesting that this is a cAMP-mediated effect. PTH, forskolin, and 8-bromo-cAMP also decreased the amiloride-sensitive component of 22Na+ uptake in OK cells by up to 64%, whereas the amiloride-insensitive component was unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1984

Intracellular pH of hepatocytes in primary monolayer culture.

Nonproliferating rat hepatocytes in primary monolayer culture were used for determining liver cell intracellular pH and the degree of intracellular pH homeostasis. The dimethyloxazolidinedione weak acid distribution method was adapted for use in monolayer culture. Intracellular pH of cultured hepatocytes in bicarbonate:CO2 medium was relatively constant at 6.85-7.05 over the external pH range of 7.0-8.0. Below an external pH of 7.0, intracellular pH fell below 6.8. Varying PCO2 between 15 and 40 mmHg did not alter the extracellular versus intracellular pH curve. In N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid medium, in the absence of bicarbonate, intracellular pH homeostasis was less well defended. In this setting, the intracellular versus extracellular pH relationship curve could be described by a straight line with slope of 0.59 +/- 0.04. The system responded to the addition of the protonophore carbonyl cyanide p-trifluoromethoxyphenyl hydrazone with an increase in the transmembrane pH gradient. Addition of nigericin in 5 mM K+ medium resulted in intracellular acidification to pH 5.5 +/- 0.2. Metabolism of 20 mM added fructose resulted in intracellular acidification. Incubation in sodium-free media at extracellular pH of 7.6 reduced intracellular pH to 6.67 +/- 0.02 compared with an intracellular pH of 6.99 +/- 0.04 in cultures exposed to medium sodium concentrations of 20-80 meq/liter.

Animals↗
PubMed1980

Abnormalities of cell volume regulation and their functional consequences.

Disturbances of body fluid osmolality are common as clinical entities. The primary clinical manifestations of both hyper- and hyposmolal states are central nervous system dysfunction. With hyperosmolal perturbations in plasma osmolality, the brain, like other tissues, initially acts as a "perfect osmometer," passively shrinking as a result of secondary substantial cellular water loss. In hours to days, depending on the extracellular solute, restoration of brain volume may be achieved if the solute is endogenous (Na+, urea, glucose). This occurs largely by the generation of new, nonelectrolyte intracellular solute in brain. This de novo solute appears only when hyperosmolality is caused by endogenous substances and not with mannitol, glycerol, or radiographic contrast media. Under the latter circumstances, the brain remains dehydrated and idiogenic osmoles are not observed. In hyposmolal states, the brain initially acts as an "imperfect osmometer," expanding its volume less than expected on the basis of passive water movement. Other tissues (red cell, muscle, and liver) behave more as perfect osmometers. In time, restoration of cell volume is achieved largely through loss of intracellular electrolytes (Na+ and K+) and other solutes such as amino acids. Teleologically, these mechanisms appear to protect brain volume at the expense of the intracellular milieu. The resultant alteration of intracellular composition may be largely responsible for the diffuse alterations in brain function observable in patients and experimental animals with such afflictions.

Alcoholism↗
PubMed1978

Zygomycosis of the maxillary sinus and palate caused by Basidiobolus haptosporus.

Basidiobolus haptosporus is known to cause subcutaneous zygomycosis in tropical Africa and Asia. We cared for a 49-year-old hyperglycemic, asplenic man who had never traveled outside the United States and who was seen initially for a painless palatal ulcer with cutaneous hypesthesia of the right cheek and upper lip. An invasive process involved the right middle nasal turbinate, maxillary antrum, maxillary division of the trigeminal nerve, and bony palate. Histological examination of biopsy tissue showed necrotizing granulomata with broad, nonseptate hyphae. Basidiobolus haptosporus was cultured from this tissue. Various laboratory studies revealed no immunologic defect and his lesions responded to therapy with amphotericin B. To our knowledge, this represents the first case of zygomycosis caused by B haptosporus in the Americas and the first culture-documented case of invasive mycosis caused by this mold.

Fungi↗