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PubMed1978

Calcium dependence of ionophore A23187-induced lymphocyte cytotoxicity.

Concentrations of the divalent cation ionophore, A23187, optimal for the transformation of human and pig lymphocytes, were cytotoxic to lymphocytes from rats and mice. The biochemical effects associated with A23187-induced cytolysis in rat thymocytes included inhibition of [3H]uridine uptake and incorporation into macromolecules and stimulation of [14C]-alpha-aminoisobutyric acid uptake. The biochemical effects, as well as the reduction in the number of viable cells, were dose dependent and were blocked by the omission of ionic calcium from the incubation medium. At a given ionophore concentration, the magnitude of lysis of thymocytes was proportional to the concentration of Ca2+ in the extracellular medium. Sr2+ was less effective than was Ca2+ in supporting A23187-induced thymocyte lysis. A comparison of the lytic response of lymphocytes of various origins showed that extracellular Ca2+ plays a role in ionophore-induced cytolysis in thymocytes and lymph node lymphocytes but not in mouse lymphosarcoma P1798 cells.

Aminoisobutyric Acids↗
PubMed1980

The effects of dexamethasone on metabolic activity of hepatocytes in primary monolayer culture.

The effects of dexamethasone on multiple metabolic functions of adult rat hepatocytes in monolayer culture were studied. Adult rat liver parenchymal cells were isolated by collagenase perfusion and cultured as a primary monolayer in HI/WO/BA, a serum free, completely defined, synthetic culture medium. Cells inoculated into the culture medium formed a monolayer within 24 hr. Electron microscopy showed that the cells in primary culture had a fine structure identical to liver parenchymal cells in vivo, including the observation of desmosomes and bile canaliculi in intercellular space. There was significant gluconeogenesis by the cells 24 hr postinoculation but it had decreased markedly by 48 hr. There was a marked induction of tyrosine aminotransferase (TAT) by dexamethasone, which was maintained for up to 72 hr postinoculation of cells. The transport of alpha-aminoisobutyric acid into the cells in monolayer culture was stimulated by dexamethasone and was dependent on the concentration of dexamethasone. Albumin synthesis and secretion by the cells was measured by a quantitative electroimmunoassay. Albumin production was shown to increase linearly over an incubation period of 24 to 48 hr postinoculation. Dexamethasone depressed the albumin synthesis. The effects of dexamethasone are slow, and at times require more than 6 hr to show variation from the control, indicating that dexamethasone is not a single controlling hormone. Possibly it functions in a cooperative and coordinating role in the regulation of cell metabolism.

Albumins↗
PubMed1990

Arecoline, but not haloperidol, induces changes in the permeability of the blood-brain barrier in the rat.

The aim of the present study was to investigate the existence of alterations of the blood-brain barrier (BBB) permeability in rats injected with centrally acting drugs, by calculating a unidirectional blood-to-brain transfer constant (Ki) for the circulating tracer [14C]-alpha-aminoisobutyric acid. The intraperitoneal (i.p.) injection of the dopaminergic antagonist haloperidol (1 mg kg-1) did not modify the regional BBB permeability. When the cholinomimetic agent arecoline hydrobromide (6.25 mg kg-1) was injected i.p. into methylatropine-pretreated rats, it induced a significant decrease of Ki values within the frontal cortex, parietal cortex, striatum and brain-stem. Our findings emphasize two concepts: (1) centrally acting drugs, such as arecoline, can induce changes in the BBB permeability, through several mechanisms; (2) there is no predictable correlation of drug stimulation of specific brain neuronal pathways and changes in the permeability of the BBB.

Aminoisobutyric Acids↗
PubMed1993

Transmembrane signaling by an insulin receptor lacking a cytoplasmic beta-subunit domain.

To assess the function of the cytoplasmic domain of the insulin receptor (IR) beta subunit, we have studied a mutant IR truncated by 365 aa (HIR delta 978), thereby deleting > 90% of the cytoplasmic domain. HIR delta 978 receptors were processed normally to homodimers that were expressed at the cell surface where they bind insulin with normal affinity. Although these truncated IRs were inactive with respect to ligand-induced internalization and autophosphorylation, insulin stimulated endogenous substrate (pp185) phosphorylation significantly more in HIR delta 978 cells than in untransfected Rat1 cells. Importantly, despite absence of the beta-subunit cytoplasmic domain, fibroblasts expressing HIR delta 978 receptors displayed enhanced sensitivity to insulin for stimulation of glucose incorporation into glycogen, alpha-aminoisobutyric acid uptake, thymidine incorporation, and S6 kinase activity compared with parental fibroblasts. Insulin also induced the expression of the protooncogene c-fos and the early growth response gene Egr-1 in HIR delta 978 cells far greater than in parental Rat1 fibroblasts. Furthermore, an agonistic monoclonal antibody specific for the human IR stimulated insulin action in fibroblasts expressing wild-type human IR but had no effect on HIR delta 978 cells. In conclusion, the HIR delta 978 truncated IRs appear to confer enhanced insulin sensitivity by augmenting the signaling properties of the endogenous rodent IRs.

Aminoisobutyric Acids↗
PubMed1970

Transport and retention of K+ and other metabolites in a marine pseudomonad and their relation to the mechanism of optical effects.

Suspensions of cells of a marine pseudomonad washed with 0.05 m MgSO(4) showed an immediate increase in optical density (first-phase optical change) when the salt concentration of the suspending medium was increased; a subsequent slow decrease in optical density (second-phase optical change) occurred if K(+) was present. The rate of the second-phase change was similar to the rate of uptake of (42)K(+) by the cells. Glutamate increased the rate and extent of the second-phase change and produced a parallel increase in the rate and extent of uptake of (42)K(+). Citrate increased the extent of the second-phase change in cells adapted to oxidize citrate but not in unadapted cells. Adapted, but not unadapted, cells accumulated (14)C-citrate. The nonmetabolizable alpha-aminoisobutyric acid (AIB) also increased the extent of the second-phase change under conditions leading to the uptake of (14)C-AIB by the cells. Cells maintained in a salt solution optimal for the retention of intracellular solutes were found to contain 0.184 m K(+). In the same salt solution, cells preloaded with (42)K(+) retained the isotope, but they lost it rapidly when suspended in 0.05 m MgSO(4). The second-phase changes can be accounted for by the energy-dependent accumulation in an osmotically active form of K(+) and other metabolites by cells depleted of intracellular solutes.

Aminoisobutyric Acids↗
PubMed1977

The mitogenic effect of A23187 in human peripheral lymphocytes.

The mitogenic action of the divalent ionophore A23187 was confirmed and shown to be very sensitive to changes in extracellular calcium ion concentration. At optimal calcium and ionophore concentrations, an increase in [3H]-thymidine incorporation was seen that was similar to that seen after phytohemagglutinin addition. A calcium-dependent stimulation of alpha-aminoisobutyric acid transport was also seen after A23187 addition. Studies with three inhibitors demonstrate a similarity between proliferation induced by phytohemagglutinin and by A23187. Isoproterenol (10(-4) M) and ouabain (10(-7) M) blocked the effects of phytohemagglutinin and A23187. A drug, D-600 that has been shown to block calcium channels in cardiac muscle, inhibited proliferation induced by either phytohemagglutinin or A23187. This concentration of D600 had no effect on either phytohemagglutinin- or A23187-induced 45Ca2+ uptake. Furthermore, the (+) and (-) isomers separated from racemic D600, which have been shown to block sodium and calcium channels respectively in smooth muscle, had equal potency in blocking lymphocyte proliferation.

Aminoisobutyric Acids↗
PubMedDate not supplied

Rapid shape change and release of ninhydrin-positive substances by Leishmania major promastigotes in response to hypo-osmotic stress.

Leishmania major promastigotes were grown to late-log phase and washed and resuspended in an isosmotic buffer. When osmolality was suddenly decreased by 50%, the cells rapidly became shorter and increased in width. Cell volume, calculated assuming a prolate-ellipsoidal shape, increased 1.4 times after 1 min. Over the next several minutes, the average length and width returned to control values while the volume returned to baseline, indicating the ability to regulate volume. Concomitantly with the swelling, large amounts of alanine and other ninhydrin-positive substances were released. All of the alanine pool was released within 1 min after reduction of the osmolality by 66%. Cells pre-loaded with [14C]-aminoisobutyric acid also released it very rapidly upon hypo-osmotic stress. Release of ninhydrin-positive substances resulted from decreased osmolality rather than changes in ionic composition. The same results were obtained if osmolality was decreased by reducing only the NaCl content of the buffer instead of diluting it with water, and mannitol could substitute for the NaCl. Promastigotes were able to grow well over several days in media as low as 154 mOsm/kg. The nature of the signalling mechanisms(s) that initiates the rapid shape change and efflux of ninhydrin-positive substances in response to hypo-osmotic stress is at present unknown.

Alanine↗
PubMed1979

Unlinked control of multiple glucocorticoid-induced processes in HTC cells.

HTC cell variants chosen for their lack of tyrosine aminotransferase (EC 2.6.1.5) (TAT) induction by glucocorticoids were tested for interrelated effects on other glucocorticoid responses: TAT induction by dibutyryl cyclic AMP (dBcAMP) +/- dexamethasone, glutamine synthetase (GS) induction, cyclic nucleotide phosphodieterase (PDE) suppression, inhibition of alpha-aminoisobutyric acid (AIB) uptake, inhibition of plasminogen activator (PA), and induction of mouse mammary tumor virus (MTV). Loss of TAT induction by steroid was accompanied by loss of TAT induction by dBcAMP and of PDE suppression by steroid. In addition, subclones of MTV-infected cells were examined for the effect of the virus on glutamine synthetase (GS) and TAT induction. The virus had no effect on their induction in wild-type cells and no effect on GS induction in the variants. One MTV-infected subclone from a TAT variant, however, showed significant return of TAT induction.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1985

Insulin receptors on cultured hypothalamic cells: functional and structural differences from receptors on peripheral target cells.

We have studied the functional and structural characteristics of insulin receptors on cultured rat hypothalamic cells. The receptors on these cells are specific for insulin, but have a lower binding affinity than that measured in nonneuronal tissues. Neither acute (2-h) nor long term (24-h) exposure of the hypothalamic cells to high insulin concentrations resulted in receptor down-regulation. However, insulin is internalized in these cells and accumulated in the presence of the lysomotropic agent chloroquine. Acute exposure to insulin does not alter initial rate of 2-deoxyglucose transport in hypothalamic cells, but does cause a stimulation of aminoisobutyric acid uptake. Photoaffinity labeling of the receptors of the hypothalamic cells with a biologically active photosensitive insulin revealed a major specifically labeled band of 115K mol wt and a minor band of 40K mol wt under disulfide-reducing conditions compared to bands of 125K and 90K mol wt seen after labeling of the insulin receptors of adipocytes. The receptor proteins in hypothalamic cells under nonreducing conditions (420K, 370K, and 310K mol wt) were also smaller than those in adipocytes. Thus, the insulin receptors of cultured hypothalamic cells differ from insulin receptors on peripheral target tissues in both functional and structural aspects.

Adipose Tissue↗
PubMed2000

Exogenous and endogenous corticosteroids modulate blood-brain barrier development in the ovine fetus.

We previously reported decreases in blood-brain barrier permeability in the ovine fetus at 80% of gestation after antenatal corticosteroids and shown that permeability is not reduced in newborn lambs after postnatal corticosteroids. We now test the hypotheses that exogenous antenatal corticosteroids decrease blood-brain barrier permeability at 60% but not 90% of gestation in ovine fetuses and that endogenous increases in plasma cortisol concentrations are associated with ontogenic decreases in barrier permeability during gestation. Chronically instrumented ovine fetuses were studied 12 h after the last of four 6-mg dexamethasone or placebo injections were given 12 h apart over 48 h to ewes. Fetuses at 80% of gestation from placebo-treated ewes studied under the same protocol were also included. Blood-brain barrier function was quantified with the blood-to-brain transfer constant (K(i)) to alpha-aminoisobutyric acid. K(i) values were lower in cerebral cortex, caudate nucleus, hippocampus, superior colliculus, thalamus, medulla, and cervical spinal cord in fetuses of dexamethasone- than placebo-treated ewes at 60% but not 90% of gestation. Regional brain K(i) values demonstrated inverse correlations with increases in gestation and plasma cortisol concentrations in most brain regions. We conclude that maternal treatment with exogenous corticosteroids was associated with decreases in blood-brain barrier permeability at 60% but not 90% of gestation and that increases in gestation and endogenous cortisol concentrations were associated with ontogenic decreases in barrier permeability during fetal development.

Adrenal Cortex Hormones↗
PubMed1976

Pleiotypic response of rat heart cells in culture to serum stimulation.

The pleiotypic effects of medium replacement were studied in rat heart cell cultures. After each medium change alpha-aminoisobutyric acid and glucose transport are increased, RNA and protein syntheses are activated. DNA synthesis did not begin before 12 hours and was followed by a wave of mitoses. This sequence of events suggests that the stimulated cells were in early G 1 phase. DNA synthesis, following the shift to a fresh medium, is linearly related to the amount of serum used as is protein synthesis. However when serum concentrations higher than 20 percent were used no increased protein synthesis could be observed suggesting the existence of another limiting factor, which was probably the isoleucine content of the medium. The serum stimulating factor is heat stable, dialysable and was found in both human and fetal calf sera.

Aminoisobutyric Acids↗
PubMed1979

Greatly decreased susceptibility of nonmetabolizing cells towards detergents.

The addition of different detergents to Chlorella cells that had previously accumulated 6-deoxyglucose causes a rapid release of the hexose analogue into the medium. This effect is independent of the nature of the detergent and is observed only when the cells are in an energized state. Thus, in the presence of the uncoupler p-trifluoromethoxycarbonylcyanide phenylhydrazone or of inhibitors such as N-ethylmaleimide, the cells show a greatly reduced susceptibility towards detergents. Similarly, the detergent-induced loss of accumulated alpha-aminoisobutyric acid from Saccharomyces cerevisiae and of potassium from Escherichia coli is also strongly affected by the energy state of the cells. The differential susceptibility of energized and nonenergized cells was observed at all detergent concentrations tested. Measurements of substrate efflux at different concentrations of Triton indicated that only Triton monomers are responsible for the increase in permeability. The absorption of [14C]Triton X-100 by Chlorella and the binding of detergent to the cells were measured in the presence of metabolic inhibitors. Again, nonenergized cells bound a significantly lower amount of Triton X-100. The amphiphilic antibiotic nystatin produced effects on cell permeability similar to those of detergents, whereas toluene, which is apolar, gave opposite results.

Aminoisobutyric Acids↗
PubMed1980

Effects of carbachol on extracellular Na-dependent AIB uptake in rat parotid gland.

In rat parotid glands the uptake of 2-[1-14C]aminoisobutyric acid (AIB), in vitro, depends on a Na concentration gradient between the intra- and extracellular medium. Ouabain (1 mM) which inhibits the Na+-K+-ATPase and a Na+ ionophore, monensin (which dissipates the Na+ gradient), both suppress this amino acid uptake. Carbachol (5 microM) (through muscarinic receptors) evokes a decrease in AIB uptake, and in the presence of 0.1 mM ouabain the cholinergic effect is enhanced. Ouabain alone (0.1 mM) very slightly depresses the [14C]AIB uptake. Neither 1 microM isoproterenol, nor 1 microM Ca2+ ionophore A23187, which affect the membrane potential in rat parotid acinar cells, modifies the AIB uptake. When the Ca is removed from the incubation medium, carbachol still evokes a small decreasing effect on AIB uptake. From these data we can suggest that the reduced AIB uptake (induced by the cholinergic agonist) appears to be related to a process dependent on variation of intracellular Na concentration that may be triggered by the cholinergic agonist.

Absorption↗