Roles of transient receptor potential canonical (TRPC) channels and reverse-mode Na/Ca exchanger on cell proliferation in human cardiac fibroblasts: Effects of transforming growth factor β1

Expression of transient receptor potential canonical channels (TRPC) and the effects of transforming growth factor-β1 (TGF-β1) on Ca²⁺ signals and fibroblast proliferation were investigated in human cardiac fibroblasts. The conventional and quantitative real-time RT-PCR, western blot, immunocytochemical analysis, and intracellular Ca²⁺ concentration [Ca²⁺]_i measurement were applied. Cell proliferation and cell cycle progression were assessed using MTT assays and fluorescence activated cell sorting. Human cardiac fibroblasts have the expression of TRPC1,3,4,6 mRNA and proteins. 1-oleoyl-2-acetyl-sn-glycerol (OAG) and thapsigargin induced extracellular Ca²⁺-mediated [Ca²⁺]_i rise. siRNA for knock down of TRPC6 reduced OAG-induced Ca²⁺ entry. Hyperforin as well as angiotensin II (Ang II) induced Ca²⁺ entry. KB-R7943, a reverse-mode Na⁺/Ca²⁺ exchanger (NCX) inhibitor, and/or replacement of Na⁺ with NMDG⁺ inhibited thapsigargin-, OAG- and Ang II-induced Ca²⁺ entry. Treatment with TGF-β1 increased thapsigargin-, OAG- and Ang II-induced Ca²⁺ entry with an enhancement of TRPC1,6 protein expression, suppressed by KB-R7943. TGF-β1 and AngII promoted cell cycle progression from G0/G1 to S/G2/M and cell proliferation. A decrease of the extracellular Ca²⁺ and KB-R7943 suppressed it. Human cardiac fibroblasts contain several TRPC-mediated Ca²⁺ influx pathways, which activate the reverse-mode NCX. TGF-β1 enhances the Ca²⁺ influx pathways requiring Ca²⁺ signals for its effect on fibroblast proliferation.     

Novel contribution of cell surface and intracellular M1‐muscarinic acetylcholine receptors to synaptic plasticity in hippocampus

Muscarinic acetylcholine receptors (mAChRs) are well known to transmit extracellular cholinergic signals into the cytoplasm from their position on the cell surface. However, we show here that M1‐mAChRs are also highly expressed on intracellular membranes in neurons of the telencephalon and activate signaling cascades distinct from those of cell surface receptors, contributing uniquely to synaptic plasticity. Radioligand‐binding experiments with cell‐permeable and ‐impermeable ligands and immunohistochemical observations revealed intracellular and surface distributions of M1‐mAChRs in the hippocampus and cortex of rats, mice, and humans, in contrast to the selective occurrence on the cell surface in other tissues. All intracellular muscarinic‐binding sites were abolished in M1‐mAChR‐gene‐knockout mice. Activation of cell surface M1‐mAChRs in rat hippocampal neurons evoked phosphatidylinositol hydrolysis and network oscillations at theta rhythm, and transiently enhanced long‐term potentiation. On the other hand, activation of intracellular M1‐mAChRs phosphorylated extracellular‐regulated kinase 1/2 and gradually enhanced long‐term potentiation. Our data thus demonstrate that M1‐mAChRs function at both surface and intracellular sites in telencephalon neurons including the hippocampus, suggesting a new mode of cholinergic transmission in the central nervous system.     

Effect of Additional Protein or Methionine and Threonine on Tyrosine Catabolism in Rats Fed Diets High in Tyrosine

The supplementation of additional protein or methionine and threonine to a high tyrosinc-low protein diet has previously been shown to prevent the tyrosine toxicity. To elucidate the mechanism, studies were performed on the effect of these supplements on the capacity to oxidize excessive tyrosine. Male weanling rats were ad libitum fed a 10% casein diet containing 5% tyrosine with and without extra casein or methionine plus threonine for 7 days, then animals were injected intraperitoneally with l-tyrosine-U-¹⁴C and the oxidation rate to ¹⁴CO₂ determined in vivo at an interval of several hours throughout a 24-hour period. The addition of extra casein or methionine and threonine to the high tyrosine diet enhanced the ability of tyrosine oxidation, and decreased the radioactivities of the TCA-soluble fractions in plasma, liver and muscle. A high level of free tyrosine in blood and tissues was also lowered by the addition of these supplements. The diurnal chenges in free tyrosine concentration of various tissues were observed. The data suggest that the beneficial effect of extra casein or methionine and threonine supplementation on tyrosine toxicity is due to an increased rate of tyrosine catabolism which results in lower tyrosine concentrations in body fluids which overcomes tyrosine toxicity.     

Effects of Alloxan Diabetes and Hypophysectomy on Growth,and Plasma and Liver Free Amino Acids of Rats Fed Excess Glycine Diets

Three chitinases (EC 3.2.1.14) were purified from yam, Dioscorea opposita THUMB, by fractionation with ammonium sulfate, chromatographies on DEAE-Cellulose and DEAE-Sephadex A-50, chromatofocusing and gel filtration on Bio-Gel P-60. The purified enzymes (E-l, E-2 and E-3) showed single bands on sodium dodecylsulfate polyacrylamide gel electrophoresis, and the molecular weights were estimated to be 33,500. The pIs were 4.05 (E-l), 4.0 (E-2) and 3.8 (E-3). All enzymes were glycoproteins and the neutral sugar contents were 3.6% (E-l), 3.6 (E-2) and 0.9% (E-3). The N-terminal amino acids of E-l and E-3 were the same and determined to be histidine. All enzymes hydrolyzed glycolchitin, but not p-nitrophenyl-2-acetamido-2-deoxy-β-d-glucopyranoside or Micrococcus lysodeikticus cell walls. E-l and E-3 were stable in the pH range of 5 ~ 11, and below 60°C. These enzymes showed two optimum pHs around 3.5 and 8.0 or 8.5 with glycolchitin as substrate.     

Influence of Forced-Feeding of Individual Essential Amino Acids on the Activities of All Urea Cycle Enzymes in Rat Liver

The response of all urea cycle enzymes, i.e. carbamyl phosphate synthetase, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinase and arginase, has been determined in the liver of protein-depleted young rats which were forcibly fed individual essential l-amino acids along with or without caloric sources. The feeding of individual amino acids produced different effects on the level of each of the enzymes, and generally the response of carbamyl phosphate synthetase, argininosuccinate synthetase, argininosuccinase and arginase was greater than that of ornithine transcarbamylase. Of all the essential amino acids tested tryptophan was most effective on the elevation of these enzymes. Several amino acids, phenylalanine, leucine, threonine and methionine had also somewhat effect on the increase of some enzyme activities, but other amino acids had little or no effect on the response of these enzymes. On the contrary, histidine and lysine caused appreciable decrease of arginase activity. These enzyme activities in rats fed tryptophan alone were extremely higher than those of animals fed it along with caloric sources. The response level of the enzymes was essentially dependent on the tryptophan content in diets under the proper conditions. Tryptophan feeding did not produce any increase in both levels of urine and plasma urea despite the elevation of all urea cycle enzyme activities occured.     

Supplemental Effects of Arginine and Methionine on Growth,and on Formations of Urea and Creatine of Adrenalectomized Rats Fed High Glycine Diets

The effects of adrenalectomy on growth, some enzyme activities in the liver and kidney, and urinary excretion of urea, creatinine and creatine were investigated in rats fed the 10% casein diets containing 7% glycine with or without l-arginine and l-methionine (10C, 10C7G and 10C7ArgMet).

Body weight gains of the intact 10C and 10C7GArgMet groups were almost same as the corresponding adrenalectomized groups. The body weight of the adrenalectomized 10C7G group was extremely decreased though that of the intact 10C7G group was maintained almost constant; but the decrease was recovered by the administration of hydrocortisone. The activities of liver arginase and carbamylphosphate synthetase were not affected by those diets. Liver serine dehydratase and ornithine δ-aminotransferase activities were increased in the intact 10C7G and 10C7GArgMet groups, but these increases were depressed by adrenalectomy. Glutamate-pyruvate transminase activities in the liver of intact 10C7G and 10C7GArgMet groups were also enhanced, but were extremely decreased in the corresponding adrenalectomized groups. Kidney transamidinase activity was not affected by adrenalectomy. The amount of urinary excreted urea was almost unchanged by adrenalectomy, but was increased by hydrocortisone administration. The amounts of excreted creatine of the adrenalectomized groups were generally larger than the corresponding intact groups, but slightly decreased by the administration of hydrocortisone. The amount of excreted creatinine was not generally affected by adrenalectomy.     

Glycine-U-14C Metabolism in Young Rats Fed the 10% Casein Diets Containing Excess Glycine

Nine hours after rats fed ad libitum for 14 days a 10% caein diet (10C), a 10% casein diet containing 7% glycine (10C7G) and a 10% casein diet containing 7% glycine with 1.4% l-arginine HCI and 0.9% l-methionine (10C7GArgMet) were force-fed 10 ml of each diet suspension containing 5 μCi of glycine-U-¹⁴C per 100 g of body weight, the radioactivity recoveries of ¹⁴C in expired CO₂, tissue components and urine were determined.

The radioactivity recovery of ¹⁴C in the expired C0₂ of the 10C7G group was generally higher than that of the 10C7GArgMet group, and those of both groups would have been much higher than that of the 10C group unless the isotope had been diluted. The amount of expiratory ¹⁴C of rats fed a 25 % casein diet containing 7% glycine was not different from that of the 10C7G group. The recovery of ¹⁴C in the trichloroacetic acid (TCA) soluble fraction of muscle of the 10C7G and the 10C7GArgMet groups were greater than that of the 10C group, but there was no difference between the 10C7G and the 10C7GArgMet groups. The recoveries of ¹⁴C in the TCA soluble fraction and protein of plasma and liver, and the muscle protein were negligible in all the groups. The amount of glycine-¹⁴C incorporated into the carcass lipids of the 10C7GArgMet group was larger than that of other groups. Those in the carcass lipids of the 10C7G and the 10C7GArgMet groups would have been much higher than that of the 10C group unless the dilution of the isotope had taken place. The recoveries of ¹⁴C in the liver and muscle glycogen, and liver lipids were remarkably small in all the groups. From the above results, it was suggested that the degradation of glycine-¹⁴C to expiratory CO₂ was not accelerated, but the rate of incorporation of the isotope into carcass lipids was increased by the supplementation of l-arginine and l-methionine to the 10C7G diet as compared with that of rats fed the 10C7G diet.