Characterization of ATP-dependent Ca2+ transport in the basolateral membrane vesicles from proximal and distal tubules of the rabbit kidney

Basolateral membrane vesicles were prepared from purified proximal and distal tubules of the rabbit kidney. The properties of the ATP-dependent Ca2+ transport were investigated. In both membranes, there was a high affinity, ATP-dependent Ca2+ transport system (Km = 0.1 microM). The optimal concentration of Mg2+ was 0.5 mM and the optimal concentration of ATP was 1 mM. The nucleotide specificity and pH dependence of the Ca2+ transport in both membranes were similar. In basolateral membrane vesicles, calmodulin had no effect on Ca2+ transport. However, in basolateral membrane vesicles depleted of calmodulin, exogenous calmodulin increased the Ca2+ transport by increasing maximal velocity. There were no major differences in the properties of the ATP-dependent Ca2+ transport system in these two membranes. These findings are discussed in relation to why parathyroid hormone differentially modulates Ca2+ transport in these two segments of the nephron.     

Characterization of the high and low affinity components of the renal Ca2(+)-Mg2+ ATPase

The purpose of this study was to characterize the interrelationship between free calcium (Ca2+) and magnesium (Mg2+) in the Ca2+ ATPase enzyme cycle of kidney membranes. Experiments were performed with basolateral membranes from rat renal cortex and microdissected proximal and distal tubules from mice. Results were similar in the three types of preparations. We first investigated the effect of ATP concentration on Ca2(+)- and Mg2(+)-dependent ATP hydrolysis. With 0.2 microM Ca2+, the enzyme activity, as a function of ATP concentration, showed two saturable components: a high affinity component with a Km of 33 microM ATP and a low affinity component with a Km of 0.63 mM ATP. These components may represent either two distinct sites of ATP binding or two forms of the same site. For the sake of simplicity, it was assumed that the two components correspond to a high affinity and a low affinity substrate site. At the high affinity site (ATP = 50 microM), the Ca2+ dependence of ATP hydrolysis followed a single Michaelis-Menten kinetics with Km for Ca2+ of 0.08 microM. The addition of 1 mM Mg2+ resulted in a relatively constant increase in ATP hydrolysis at all Ca2+ concentrations, indicating that the effects of the two cations were additive. With high ATP concentration (ATP = 3 mM), Ca2+ also induced an ATP hydrolysis according to a saturable process, with a Km for Ca2+ of 0.2 microM. In contrast with what occurred with low concentrations of ATP, addition of millimolar Mg2+ completely curtailed the sensitivity of the enzyme to Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipids and lipoprotein profile in doxorubicin treated rats: influence of alpha-tocopherol administration

The effect of doxorubicin (DXR) on the levels of heart, liver and plasma lipids and plasma lipoproteins were studied in rats. Rats were treated with DXR (2.5 mg/kg body weight weekly for 8 weeks, iv) with or without alpha-tocopherol (alpha-TPL) (400 mg/kg body wt daily for 60 days) co-administration. DXR treated rats showed increase in plasma total cholesterol, triglycerides and phospholipids. The activities of lecithin cholesterol-acyl transferase and hepatic and extrahepatic lipoprotein lipase were lowered significantly with concomitant increase in liver and heart lipid peroxide levels in DXR treatment. HDL cholesterol level was found to be decreased significantly in DXR treated rats as a result of which there was an increase of LDLc/HDLc ratio. alpha-TPL coadministration brought back the enzyme activity to near normal and reduced the level of lipid peroxides. The lipid changes were minimum in rats treated with both alpha-TPL and DXR. This study suggests that the toxicity of DXR is reflected in lipids and lipoprotein profile.     

On the mechanism of the gamma-aminobutyric acid receptor in the mammalian (mouse) cerebral cortex. Chemical kinetic investigations with a 10-ms time resolution adapted to measurements of neuronal receptor function in single cells.

The gamma-aminobutyric acidA (GABA) receptor belongs to a superfamily of proteins involved in chemical reactions that regulate signal transmission between cells of the nervous system and is the target of some of the agents most frequently used in medicine to control disorders of the central nervous system. In contrast to the nicotinic acetylcholine receptor, which initiates signal transmission and is the best characterized member of the superfamily, the GABA receptor forms anion-specific transmembrane channels and inhibits signal transmission. The chemical kinetic experiments described here, in which fast chemical reaction techniques were used, indicate that both receptor proteins may operate by the same mechanism. Also described is the use of a chemical kinetic technique with a 10-ms time resolution that we have developed for making measurements with single cells isolated from specific areas of the nervous system, in this case the cerebral cortex of embryonic mice. A flow device was used to equilibrate receptors on the cell surface with GABA, and the concentration of open transmembrane channels in the cells was then measured by recording the whole-cell currents at pH 7.2, 21-23 degrees C, and a transmembrane voltage of -70 mV. Two different receptor forms, A alpha and A beta, were detected in cerebral cortical cells. Although the ratio of A alpha to A beta varied from cell to cell, on average 35% and 65% of the receptor-controlled current was associated with receptor forms A alpha and A beta, respectively. At saturating concentrations of GABA, the rate coefficients of desensitization, alpha and beta, associated with these two forms have maximal values of 4.4 and 0.7 s-1, respectively. The constants of a mechanism that accounts for the open transmembrane channels of both receptor forms were evaluated over a 50-fold range of GABA concentration. The dissociation constant of the site controlling channel opening was 40 microM for A alpha and 320 microM for A beta. The channel-opening equilibrium constant, phi-1, was 3.5 for A alpha and 20 for A beta. The evaluated constants allow one to calculate Po, the conditional probability that at a given concentration of GABA the receptor-channel is open. Po could also be determined in the presence of 100 microM GABA by an independent method in which different assumptions are made in the interpretation of the experimental results, the single-channel current-recording technique. The value of Po obtained (0.56) was in good agreement with the Po value (0.61) calculated for receptor form A alpha from chemical kinetic measurements at 100 microM GABA.(ABSTRACT TRUNCATED AT 400 WORDS)     

A new Conus peptide ligand for mammalian presynaptic Ca2+ channels.

Voltage-sensitive Ca2+ channels that control neurotransmitter release are blocked by omega-conotoxin (omega-CgTx) GVIA from the marine snail Conus geographus, the most widely used inhibitor of neurotransmitter release. However, many mammalian synapses are omega-CgTx-GVIA insensitive. We describe a new Conus peptide, omega-CgTx-MVIIC, that is an effective inhibitor of omega-CgTx-GVIA-resistant synaptic transmission. Ca2+ channel targets that are inhibited by omega-CgTx-MVIIC but not by omega-CgTx-GVIA include those mediating depolarization-induced 45Ca2+ uptake in rat synaptosome preparations, "P" currents in cerebellar Purkinje cells, and a subset of omega-CgTx-GVIA-resistant currents in CA1 hippocampal pyramidal cells. The characterization of omega-CgTx-MVIIC by a combination of molecular genetics and chemical synthesis defines a general approach for obtaining ligands with novel receptor subtype specificity from Conus.     

STUDIES ON MEVALONATE KINASE, PHOSPHOMEVALONATE KINASE AND PYROPHOSPHOMEVALONATE DECARBOXYLASE IN DEVELOPING RAT BRAIN

Abstract— We have in the present study investigated the properties of mevalonate kinase, phosphomevalonate kinase and pyrophosphomevalonate decarboxylase in the 105,000 g supernatant fractions from rat brain, and determined whether the activities of these enzymes change during brain development. All three enzymes in brain showed a specific requirement for ATP for optimal activity. The presence of Mg²⁺ as divalent cation was also required for optimal activity of mevalonate kinase and phosphomevalonate kinase. Both Mg²⁺ and Mn²⁺ were equally effective divalent metal ions for pyrophosphomevalonate decarboxylase in brain. Mevalonate kinase as well as phosphomevalonate kinase were active in a broad pH range of 6.5–8 while the pH curve for pyrophosphomevalonate decarboxylase showed a peak activity at approx 6. No age-dependent change occurred in the activities of mevalonate kinase and phosphomevalonate kinase in developing brain, whereas pyrophosphomevalonate decarboxylase activity in brain increased during the 1st week after birth, reached a peak value at about the 8th day of age and declined slowly thereafter. The K_m for brain mevalonate kinase in 2, 13 and 52 day old rats were 312, 400 and 434 μM, respectively. The V _max for the kinase in 2, 13 and 52 day old rats were in the range of 45–52 nmol/h/mg protein, respectively. This suggests that, like in liver (R amachandran & S hah , 1976), pyrophosphomevalonate decarboxylase in brain may also be one regulatory step for cholesterol synthesis.     

IL-1 induces p62/SQSTM1 and autophagy in ERα+/PR+ BCa cell lines concomitant with ERα and PR repression,conferring an ERα−/PR− BCa-like phenotype

Estrogen receptor α (ERα)^low/− tumors are associated with breast cancer (BCa) endocrine resistance, where ERα low tumors show a poor prognosis and a molecular profile similar to triple negative BCa tumors. Interleukin-1 (IL-1) downregulates ERα accumulation in BCa cell lines, yet the cells can remain viable. In kind, IL-1 and ERα show inverse accumulation in BCa patient tumors and IL-1 is implicated in BCa progression. IL-1 represses the androgen receptor hormone receptor in prostate cancer cells concomitant with the upregulation of the prosurvival, autophagy-related protein, Sequestome-1 (p62/SQSTM1; hereinafter, p62); and given their similar etiology, we hypothesized that IL-1 also upregulates p62 in BCa cells concomitant with hormone receptor repression. To test our hypothesis, BCa cell lines were exposed to conditioned medium from IL-1-secreting bone marrow stromal cells (BMSCs), IL-1, or IL-1 receptor antagonist. Cells were analyzed for the accumulation of ERα, progesterone receptor (PR), p62, or the autophagosome membrane protein, microtubule-associated protein 1 light chain 3 (LC3), and for p62-LC3 interaction. We found that IL-1 is sufficient to mediate BMSC-induced ERα and PR repression, p62 and autophagy upregulation, and p62-LC3 interaction in ERα⁺/PR⁺ BCa cell lines. However, IL-1 does not significantly elevate the high basal p62 accumulation or high basal autophagy in the ERα⁻/PR⁻ BCa cell lines. Thus, our observations imply that IL-1 confers a prosurvival ERα⁻/PR⁻ molecular phenotype in ERα⁺/PR⁺ BCa cells that may be dependent on p62 function and autophagy and may underlie endocrine resistance.