Roberta Giunti

Fatty acyl-CoA esters induce calcium release from terminal cisternae of skeletal muscle

The effect of palmitoyl-CoA (PCoA) on Ca2+ fluxes in unfractionated SR, longitudinal tubules (LSR) and terminal cisternae (TC) subfractions, obtained from rabbit fast-twitch skeletal muscles, was investigated. After MgATP-dependent Ca2+ preloading, PCoA released Ca2+ from unfractionated SR and TC, but not from LSR. Both the extent and the rate of PCoA-induced Ca2+ release from TC were increased in a dose-dependent manner, the half-maximal effect being attained at [PCoA] of approximately 6 microM. Ruthenium red, a Ca2+ release channel blocker, completely inhibited PCoA-induced Ca2+ release, whereas caffeine, a Ca2+ release channel agonist, depleted TC of Ca2+ and prevented the PCoA action. Scatchard plot analysis of [3H]-ryanodine binding showed that PCoA increased the affinity without affecting Bmax. The action of PCoA was mimicked by a nonhydrolysable analog. The present results indicate that PCoA interacts and opens the Ca2+ release channel (ryanodine receptor) of TC and that the mechanism of action involves binding rather than hydrolysis.

Contribution of Fructose-6-Phosphate to Glucocorticoid Activation in the Endoplasmic Reticulum: Possible Implication in the Metabolic Syndrome

Both fructose consumption and increased intracellular glucocorticoid activation have been implicated in the pathogenesis of the metabolic syndrome. Glucocorticoid activation by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) depends on hexose-6-phosphate dehydrogenase (H6PD), which physically interacts with 11β-HSD1 at the luminal surface of the endoplasmic reticulum (ER) membrane and generates reduced nicotinamide adenine dinucleotide phosphate for the reduction of glucocorticoids. The reducing equivalents for the reaction are provided by glucose-6-phosphate (G6P) that is transported by G6P translocase into the ER. Here, we show that fructose-6-phosphate (F6P) can substitute for G6P and is sufficient to maintain reductase activity of 11β-HSD1 in isolated microsomes. Our findings indicate that the mechanisms of F6P and G6P transport across the ER membrane are distinct and provide evidence that F6P is converted to G6P in the ER lumen, thus yielding substrate for H6PD-dependent reduced nicotinamide adenine dinucleotide phosphate generation. Using the purified enzyme, we show that F6P cannot be directly dehydrogenated by H6PD, and we also excluded H6PD as a phosphohexose isomerase. Therefore, we postulate the existence of an ER luminal hexose-phosphate isomerase different from the cytosolic enzyme. The results suggest that cytosolic F6P promotes prereceptor glucocorticoid activation in white adipose tissue, which might have a role in the pathophysiology of the metabolic syndrome.

Low levels of glucose-6-phosphate hydrolysis in the sarcoplasmic reticulum of skeletal muscle: involvement of glucose-6-phosphatase

Glucose-6-phosphate hydrolysis was measured in a fraction obtained from rabbit fast-twitch skeletal muscle and corresponding to total sarcoplasmic reticulum, as well as in three subfractions containing longitudinal tubules, terminal cisternae or both structures. In all cases the levels of hydrolysis measured both in native and disrupted membranes were approximately 60-100 times lower than the microsomal glucose-6-phosphatase activity of the corresponding livers. In contrast to liver microsomes, most (up to 80%) of the glucose-6-phosphate hydrolysing activity in muscle sarcoplasmic reticulum membranes was not inactivated by pH 5.0 pre-incubation indicating that it was not catalysed by the specific glucose-6-phosphatase enzyme. Osmotically induced changes in light-scattering intensity of sarcoplasmic reticulum vesicles revealed that, in contrast to liver microsomes, sarcoplasmic reticulum vesicles were not selectively permeable to glucose-6-phosphate as mannose-6-phosphate was also permeable and in addition they were poorly permeable to glucose. Immunoblot experiments using antibodies raised against the glucose-6-phosphatase enzyme, and liver endoplasmic reticulum glucose and Pi translocases, failed to detect the presence of these protein components in sarcoplasmic reticulum membranes. Southern blot analysis of reverse transcriptase-polymerase chain reaction products from rat muscle revealed that glucose-6-phosphatase mRNA is present in muscle. Quantification of Northern blot analysis of liver and muscle mRNA indicated that muscle contains less than 2% of the amount of glucose-6-phosphate mRNA found in corresponding livers. We conclude that very low levels of specific glucose-6-phosphatase (e.g. as in liver; E.C. 3.1.3.9) are present in muscle sarcoplasmic reticulum and that the muscle and liver glucose-6-phosphatase systems have several different properties.

Effect of nifedipine on capacitive calcium entry in Jurkat T lymphocytes

The effect of nifedipine-an antagonist of L-type calcium (Ca(2+)) channels-on capacitative Ca(2+) entry (CCE) was studied in Jurkat T lymphocytes. CCE was induced by a variety of treatments each of which depleted intracellular Ca(2+) stores. Cells were treated with thapsigargin, ionomycin, anti-CD3 antibodies, and phytohaemagglutinin, or pre-incubated in a Ca(2+)-free medium. Activity of CCE was evaluated with a Ca(2+)-free/Ca(2+)-readmission protocol, in Fluo-3 pre-loaded cells. Nifedipine inhibited CCE in a dose-dependent manner. CCE inhibition was not due to non-specific effects on K(+) channels. Nifedipine, did not induce any membrane depolarization, as revealed by measurements of the plasma membrane potential with the fluorescent probe bis-oxonol. Moreover, experiments done under depolarizing conditions (i.e. by substituting Na(+) with K(+) ions in the medium) revealed that nifedipine could inhibit capacitative Ca(2+) entry independently of plasma membrane depolarization. We also demonstrated the presence in our Jurkat T-cells of transcripts for Ca(V)1.3 (alpha(1D)) and Ca(V)1.4 (alpha(1F)) L-type Ca(2+) channels. Verapamil and diltiazem, two unrelated blockers of L-type Ca(2+) channels, were less inhibitory on CCE. Possible mechanisms by which nifedipine interferes with Ca(2+) entry in these cells are discussed.

Inhibition of glucuronidation by an acyl-CoA-mediated indirect mechanism

The mechanism of the inhibition of glucuronidation by long-chain fatty acyl-CoAs was studied in rat liver microsomal membranes and in isolated hepatocytes. Palmitoyl- and oleoyl-CoA did not affect p-nitrophenol UDP-glucuronosyltransferase activity in native microsomes but were inhibitory in permeabilised vesicles. The extent of inhibition was dependent on the effectiveness of permeabilisation and was constant in time in fully permeabilised microsomes. Fatty acyl-CoAs mobilised calcium from calcium-loaded microsomes. Elevation of the intracellular acyl-CoA level by the addition of palmitate or oleate inhibited the glucuronidation of p-nitrophenol in isolated hepatocytes. This effect could be abolished by emptying the intracellular calcium stores. Therefore, it is concluded that fatty acyl-CoAs inhibit glucuronidation indirectly, presumably via calcium mobilisation.

Expression of hexose-6-phosphate dehydrogenase in rat tissues

Hexose-6-phosphate dehydrogenase (H6PD) is the main NADPH generating enzyme in the lumen of the endoplasmic reticulum. H6PD is regarded as an ancillary enzyme in prereceptorial glucocorticoid activation and probably acts as a nutrient sensor and as a prosurvival factor. H6PD expression was determined in a variety of rat and human tissues by detecting mRNA and protein levels, and by measuring its dehydrogenase and lactonase activities. It was found that H6PD was present in all investigated tissues; both expression and activity remained within an order of magnitude. Correlation was found between the dehydrogenase activity and protein or mRNA levels. The results confirmed the supposed housekeeping feature of the enzyme.

The glucose-6-phosphate transport is not mediated by a glucose-6-phosphate/phosphate exchange in liver microsomes

A phosphate‐linked antiporter activity of the glucose‐6‐phosphate transporter (G6PT) has been recently described in liposomes including the reconstituded transporter protein. We directly investigated the mechanism of glucose‐6‐phosphate (G6P) transport in rat liver microsomal vesicles. Pre‐loading with inorganic phosphate (Pi) did not stimulate G6P or Pi microsomal inward transport. Pi efflux from pre‐loaded microsomes could not be enhanced by G6P or Pi addition. Rapid G6P or Pi influx was registered by light‐scattering in microsomes not containing G6P or Pi. The G6PT inhibitor, S3483, blocked G6P transport irrespectively of experimental conditions. We conclude that hepatic G6PT functions as an uniporter.