Stimulatory effect of menaquinone-7 (vitamim K2) on osteoblastic bone formation in vitro

Menaquinone-7, which is vitamin K₂ (menatetrenone) with seven isoprene units, is highly contained in the fermented soybean. The effect of menaquinone-7 (MK-7) on osteoblastic bone formation was investigated. Femoral-diaphyseal and metaphyseal tissues of young male rats (4 weeks old) were cultured for 48 h in a medium containing either vehicle or MK-7 (10^–7–10^–5 M). Calcium content, alkaline phosphatase activity, and deoxyribonuclic acid (DNA) content in the diaphyseal and metaphyseal tissues was significantly increased in the presence of MK-7 (10^–6 and 10^–5 M). The effect of MK-7 in increasing the diaphyseal and metaphyseal calcium content and alkaline phosphatase activity was completely prevented in the presence of cycloheximide (10^–6 M), an inhibitor of protein synthesis. Moreover, osteoblastic MC3T3-E1 cells after subculture were cultured for 24 h in a serum-free medium containing MK-7 (10^–7–10^–5 M). Protein content, alkaline phophatase activity, osteocalcin and DNA content in the cells was significantly increased in the presence of MK-7 (10^–6 and 10^–5 M). The effect of MK-7 in increasing protein content, alkaline phosphatase activity, and osteocalcin production in the cells was completely blocked by cycloheximide. This study demonstrates that MK-7 has an anabolic effect on bone tissue and osteoblastic MC3T3-E1 cells in vitro, suggesting that the compound can stimulate osteoblastic bone formation.     

Adriamycin-induced leakage of lysosomal enzymes in vitro

Chronic treatment of rats with adriamycin has been shown to affect myocardial lysosomes as well as enzyme activities in the serum fraction. In this study, we examined in vitro effects of adriamycin (10^–6 to 10^–3 M) on the lysosomal fraction isolated from rat ventricular tissue. Morphological examination revealed that the isolated fraction was mainly vesicular in nature. Higher concentrations of adriamycin (10^–3 M) caused a significant loss of acid phosphatase and N-acetyl-B-d-glucosaminidase activity from the lyosomal vesicles. The enzyme leakage was not accompanied by any intravesicular localization of lanthanum, an extravesicular electron dense tracer. Preincubation of lysosomal vesicles with 10 g/ml superoxide dismutase did not protect against adriamycin-induced loss of lysosomal enzymes. The study shows that adriamycin induces loss of lysosomal enzymes in vitro and the superoxide radical may not be involved in this change.     

Stimulatory effect of genistein and daidzein on protein synthesis in osteoblastic MC3T3-E1 cells: Activation of aminoacyl-tRNA synthetase

The effect of genistein and daidzein on protein synthesis in osteoblastic MC3T3-E1 cells in vitro was investigated to determine a cellular mechanism by which the isoflavones stimulate bone formation. Cells were cultured for 48 h in -minimal essential medium containing either vehicle, genistein (10^–7–10^–5 M) or daidzein (10^–7–10^–5 M). The 5,500 g supernatant of cell homogenate was used for assay of protein synthesis with [³H]leucine incorporation in vitro. The culture with genistein or daidzein caused a significant elevation of protein synthesis in the cell homogenate. The effect of genistein (10^–5 M) or daidzein (10^–5 M) in elevating protein synthesis was significantly prevented, when cells were cultured for 48 h in a medium containing either actinomycin D (10^–7 M) or cycloheximide (10^–6 M) in the absence or presence of isoflavones. Moreover, when genistein (10^–7–10^–5 M) or daidzein (10^–6 and 10^–5 M) was added to the reaction mixture containing the cell homogenate obtained from osteoblastic cells cultured without isoflavone, protein synthesis was significantly raised. This increase was markedly blocked by the addition of cycloheximide (10^–7 M). In addition, [³H]leucyl-tRNA synthetase activity in the cytosol of osteoblastic cells was significantly increased by the addition of genistein (10^–6 and 10^–5 M) or daidzein (10^–5 M) into the enzyme reaction mixture. The present study demonstrates that genistein or daidzein can stimulate protein synthesis in osteoblastic MC3T3-E1 cells. The isoflavones may have a stimulatory effect on osteoblastic bone formation due to increasing protein synthesis.     

Zinc compounds inhibit osteoclast-like cell formation at the earlier stage of rat marrow culture but not osteoclast function

The effect of zinc compounds on osteoclast-like cell formation in rat marrow culture in vitro was investigated. The bone marrow cells were cultured for 7 days in -minimal essential medium containing a well-known bone resorbing hormone (1, 25-dihydroxyvitamin D₃ and parathyroid hormone [1–34]). Osteoclast-like cell formation was estimated by staining for tartrateresistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of 1, 25-dihydroxyvitamin D₃ (10^–8 M) or parathyroid hormone (PTH; 10^–8 M) induced a remarkable increase in osteoclast-like multinucleated cells (MNC). These increases were clearly inhibited by the presence of zinc sulfate or zinc-chelating dipeptide (-alanyl-L-histidinato zinc; AHZ) in the concentration range of 10^–7 to 10^–5 M. The inhibitory effect was seen at the earlier stage of osteoclast-like MNC formation. However, zinc compounds (10^–6 M) did not have an effect on PTH (10^–8 M)-induced osteoclast-like cell formation in the presence of EGTA (5 × 10^–4 M), dibucaine (10^–5 M) or staurosporine (10^–9 M). Moreover, when osteoclasts isolated from rat femoraldiaphyseal tissues were cultured for 24 h in the presence of zinc compounds (10^–7 to 10^–5 M), the compounds did not have an effect on cell numbers or lysosomal enzymes activity (acid phosphatase and -glucuronidase) in the cells. The present study clearly demonstrates that zinc compounds inhibit osteoclast-like cell formation at the earlier stage with differentiation of marrow cells.     

Effect of Insulin and Insulin-Like Growth Factor 1 (IGF-1) on Activity of Glycogen Synthase in Skeletal Muscles of the Lamprey Lampetra fluviatilis

Activity of glycogen synthase (GS) in the muscle tissue of the lamprey Lampetra fluviatilis is studied in dynamics of the pre-spawning period as well as under effects of insulin and IGF-1. It is shown that GS exists in the muscles in two forms, the active (I-form) and inactive (D-form), the I-form prevailing during all studied time periods. With approaching the spawning, the GS activity fell 1.5–2 times due to a decrease of the I-form activity. From October to January, both insulin and IGF-1 stimulated GS at concentrations of 10^–10–10^–8 M and 10^–9–10^–8 M, respectively. The maximally effective concentrations (10^–9 M insulin, 10^–8 M IGF-1) produced a 2.5–3-fold rise of the I-form activity of GS at the period from October to December. In January the stimulating effect of these peptides decreased. In March the GS was insensitive both to insulin and to IGF-1. The obtained data indicate participation of insulin and IGF-1 in regulation of glycogen synthesis in lamprey skeletal muscles, the ability of IGF-1 to stimulate the enzyme activity being shown in the lower vertebrates for the first time. It is concluded that IGF-1 takes part in regulation of the carbohydrate metabolism already at early stages of evolution of vertebrates.     

Michaelis constant (K m ) of acid phospatase as affected by montmorillonite,illite, and kaolinite clay minerals

The influence of Ca homoionic clay minerals (montmorillonite, illite, and kaolinite) on the activity,K _m , andV _m values of acid phosphatase was examined in model experiments. At each substrate (p-nitrophenyl phosphate) level tested, the addition of increasing amounts of clays (50, 100, and 150 mg, respectively) decreased the activity and increased theK _m value from 1.43×10^–3 m PNP (in the soluble state) to 82.3×10^–3M (montmorillonite), 8.02×10^–3 m (kaolinite), and 7.65×10^–3 m (illite) at the 150 mg level. The maximum enzyme reaction velocity (V _m ) remained nearly constant at different amounts of kaolinite and illite, but increased remarkably with rising quantities of montmorillonite. Apparently, the substrate affinity of sorbed acid phosphatase is significantly lower with montmorillonite than with kaolinite or illite. This may be ascribed to an intensive sorption of both substrate and enzyme to the surface as well as to interlattice sites of montmorillonite.     

Subcellular distribution of glycoside hydrolase and polysaccharide depolymerase enzymes in the rumen entodiniomorphid ciliatePolyplastron multivesiculatum

The distribution of 12 acid hydrolase and two polysaccharide depolymerase enzymes in the rumen entodiniomorphid ciliatePolyplastron multivesiculatum, isolated from the ovine rumen 2 h after feeding, was examined by differential and density-gradient centrifugation. Approximately 60%–70% of the recovered activity was sedimentable in fractions prepared by centrifugation at 10³ g for 10 min (F1) and 10⁴ g for 10 min (F2) with 25%–35% of the acid hydrolases and 15%–20% of acid phosphatase and the polysaccharidases remaining nonsedimentable (in fraction F5) after centrifugation at 10⁵ g for 60 min. Approximately 60% of the sedimentable activity was located in fraction F1. Latency of the hydrolase activity was demonstrated. After isopycnic centrifugation in sucrose density gradients, the hydrolytic enzymes cosedimented in acid phosphatase-containing, membrane-bound, pleomorphic lysosomelike vesicles 0.1–1.0 m in size, with a mean equilibrium density of 1.17 (1.15–1.19) g/ml.     

Studies of nitrate reductase in the fresh water dinoflagellatePeridinium cinctum

Nitrate reduction was studied in the dinoflagellatePeridinium cinctum collected from extensive algal blooms in Lake Kinneret (Israel).Among several methods tested for the preparation of cell free extracts, only the use of a ground-glass tissue culture homogenizer was found to be efficient. The assimilatory nitrate reductase ofP. cinctum was located in a particulate fraction. In this respect,P. cinctum did not behave like other eukaryotes, such as green algae, but as a prokaryote. Nitrite reductase activity was found in the soluble fraction.Nitrate reductase used NADH as a preferable electron donor; it reacted also with NADPH but only to give 16.5% of the NADH dependent rate. Methyl viologen and benzyl viologen could also serve as electron donors, with rates higher than the NADH dependent activity (3–6 times and 1.5–3 times, respectively). The Km of nitrate reductase for NADH was 2.8×10^–4 M and for NO₃-1.9×10^–4 M. Flavins did not stimulate the activity, nor was ferricyanide able to activate it. Carboxylic anions stimulated nitrate reductase activity 3–4 fold, an effect which was not mimicked by other anions.Chlorate, azide and cyanide were competitive inhibitors ofP. cinctum, nitrate reductase withK _i values of 1.79×10^–3 M, 2.1×10^–5 M and 8.9×10^–6 M respectively.     

Resistance of Pediococcus cerevisiae to amethopterin as a consequence of changes in enzymatic activity and cell permeability : II. Permeability changes to amethopterin and other folates in the drug-resistant mutant

Pediococcus cerevisiae/AMr, resistant to amethopterin, possesses a higher dihydrofolate reductase (5, 6, 7, 8-tetrahydrofolate: NADP⁺ oxidoreductase, EC 1.5.1.3) activity than the parent, a folate-permeable and thus amethopterin-susceptible strain and than the wild-type. The properties of dihydrofolate reductase from the three strains have been compared. Temperature, pH optima, heat stability, as well amethopterin binding did not reveal significant differences between the enzymes from the susceptible and resistant strains. The enzyme from the wild-type was 10 times more sensitive to inhibition by amethopterin and more susceptible to heat denaturation. The apparent K_m values for dihydrofolate in enzymes from the three strains were in the range of 4.8–7.2 μM and for NADPH 6.5–8.0 μM. The amethopterin-resistant strain exhibited cross-resistance to trimethoprim and was about 40-fold more resistant to the latter than the sensitive parent and the wild-type. The resistance to trimethoprim appears to be a direct result of the increased dihydrofolate reductase activity. Inhibition of dihydrofolate reductase activity by this drug was similar in the three strains. 10–20 nmol caused 50% inhibition of 0.02 enzyme unit. Trimethoprim was about 10 000 times less effective inhibitor of dihydrofolate reductase than amethopterin. The cell extract of the AMr strain possessed a folate reductase activity three times higher than that of the sensitive strain. The activities of other folate-related enzymes like thymidylate synthethase and 10-formyltetra-hydrofolate synthetase (formate: tetrahydrofolate ligase (ADP)-forming), EC 6.3.4.3) were similar in the three strains studied.     

(Na + K)-ATPase activity of crude homogenates of rat skeletal muscle as estimated from their K-dependent 3-O-methylfluorescein phosphatase activity

A highly sensitive fluorimetric assay using 3-O-methylfluorescein phosphate as substrate was used in the determination of K⁺-dependent phosphatase activity in preparations of rat skeletal muscle. The gastrocnemius muscle was chosen because of mixed fibre composition. Crude, detergent treated homogenate was used so as to avoid loss of activity during purification. K⁺-dependent phosphatase activities in the range 0.19–0.37 μmol · (g wet weight)⁻¹ · min⁻¹ were obtained, the value decreasing with age and K⁺-deficiency. Complete inhibition of the K⁺-dependent phosphatase was obtained with 10⁻³ M ouabain. Using a KSCN-extracted muscle enzyme the intimate relation between K⁺-dependent phosphatase activity and (Na⁺ + K⁺)-activated ATP hydrolysis could be demonstrated. A molecular activity of 620 min⁻¹ was estimated from simultaneous determination of K⁺-dependent phosphatase activity and [³H]ouabain binding capacity using the partially purified enzyme preparation. The corresponding enzyme concentration in the crude homogenates was calculated and corresponded well with the number of [³H]ouabain binding sites measured in intact muscles or biopsies hereof.     

Redox modulation of reductase and phosphatase activities in human erythrocytes

Summary We have previously reported that ferricyanide reductase activity in human erythrocytes depended on glycolysis and could be modulated by several compounds including oxidants and hormones like insulin. Insulin could activate glycolysis, probably as a consequence of tyrosine phosphorylation of protein band 3, implicating phosphorylation reactions as an important signal for activation of the reductase by insulin. Reversible phosphorylation of cellular proteins is also believed to play a key role in the action of insulin. Cytosolic acid phosphatase activity has been found in human erythrocytes. To further extend initial reports, we studied the effect of modulators on the cytosolic erythrocyte acid phosphatase. Mild oxidants like ferricyanide (1 mM), vanadate (1 mM), Mn²⁺ (0.5 and 1 mM), and phenylarsine oxide (10 and 100 M) inhibited the phosphatase activity. Similarly, insulin at concentrations that stimulate ferricyanide reduction (500, 1000 IU/ml) inhibited the activity of the phosphatase enzyme. The overall results indicated that oxidants are able to inhibit the acid phosphatase and stimulate the redox enzyme. In addition, a significant negative correlation (r = –0.400; P = 0.006) was observed between phosphatase and reductase activities. The observations discussed here, together with previous ones, emphasize that a close association between reductase and phosphatase enzymes may exist and also suggest a role for redox reactions in tyrosine phosphorylation/dephosphorylation-mediated signal transduction pathways.     

Invertase and phosphatase of yeast in a phosphate-limited continuous culture

Summary Deficiency of inorganic phosphate caused the hyper production of invertase and the derepression of acid phosphatase in a continuous culture ofSaccharomyces carlsbergensis. The specific invertase activity was 40,000 enzyme units per g dry cell weight at a dilution rate lower than 0.05 h^–1 with a synthetic glucose medium of which the molecular ratio of KH₂PO₄ to glucose was less than 0.006. This activity is eight fold higher than in a batch growth and 1.5 fold as much as the highest enzyme activity observed so far in a glucose-limited continuous culture.For the hyper production of invertase, it is necessary to culture the yeast continuously by keeping the Nyholm's conservative inorganic phosphate concentration at less than 0.2 m mole per g dry weight cell. The derepression of acid phosphatase brought about by phosphate deficiency, was similar in both batch and continuous cultures.Nomenclature D dilution rate of continuous culture (h^–1) - E_i invertase concentration in culture (enzyme unit l^–1) - E_p acid phosphatase concentration in culture (enzyme unit l^–1) - P inorganic phosphate concentration in culture (mM) - S glucose concentration in culture (mM) - X cell concentration in culture (g dry weight cell l^–1) Greek Letter specific rate of growth (h^–1) Suffix f feed - 0 initial value     

The role of cellulase in hormonal regulation of shoot morphogenesis in tobacco callus

A polyclonal antibody raised against cellulase (EC 3.2.1.4.) from callus ofNicotiana tabacum L. cv. Petit Havana SR1 reduced cellulase activity and induced shoot formation in tobacco callus in the presence of callus maintaining concentrations of auxin and cytokinin. Shoot induction as well as reduction of the cellulase activity was also obtained by withdrawing auxin from the callus medium. The effect of the two hormones on cellulase activity in the tobacco tissue was examined by varying the concentration of one of the hormones -naphthylacetic acid (NAA) or benzylaminopurine (BAP) at a time while the other was kept at a level sufficient for either callus growth or shoot induction. While NAA stimulated the enzyme activity increasingly with concentration in the range 5 × 10^–7 M to 5 × 10^–5 M at both levels of BAP, BAP only stimulated the cellulase activity at an optimum concentration of 5 × 10^–6 M when NAA was present at a level sufficient to induce callus growth. The results point to a pivotal role of the downward regulation of cellulase in the initiation of shoot induction. A series of events leading to oriented cell divisions as a result of the lowered cellulase level during the initial phase of the morphogenetic process is discussed.Abbreviations Ab Purified cellulase antibody - BAP benzylaminopurine - MS Murashige and Skoog medium - NAA -naphthylacetic acid - PS Purified preimmune serum We thank Mr. Poul Fabech for constructing the automatic viscosimetric equipment and Mr. Hans Hjorth for making the computer programme. This work was supported by The Danish Veterinary and Agricultural Research Council.     

Some enzymatic properties of vacuolar alkaline phosphatase from yeast

Candida utilis alkaline phosphatase has been detected in vacuoles. Liberation of the vacuoles was carried out by protoplast disruption under isotonic conditions. The polybase DEAE-dextran was used to induce damage to the yeast plasmalemma. The vacuoles were purified by centrifugation on sorbitol-Ficoll gradients. Alkaline phosphatase from a purified fraction of vacuoles was characterized after gel filtration on Sephadex G-200. We have found 15 mU of enzyme activity per 10⁸ vacuoles. This enzyme activity elutes on Sephadex G-200 at a volume-to-void-volume ratio of 1.65. The approximate molecular weight is 1.35×10⁵. TheK _m value forp-nitrophenyl-phosphate is 2.5×10^–3 M. The pH for maximum activity is 8.9, and the enzyme is stable at pH values between 7.0 and 9.0. Rapid inactivation occurs at temperatures above 45°C. The enzyme catalyzes the hydrolysis of phosphomonoester bonds of a wide variety of molecules, especially polyphosphates. Thus, vacuolar polyphosphates are probably the natural substrate of this enzyme. Orthophosphate, arsenate, ethylenediaminetetraacetate, molybdate, and borate act as inhibitors. Fluoride is not an inhibitor, and the activity is not affected byp-hydroxymercuribenzoate. Some metal ions also affect the activity of vacuolar alkaline phosphatase. This may indicate that this enzyme is a metalloprotein.     

Redox modulation of a phosphatase from Anacystis nidulans

Ascorbic acid (AA) increased the phosphatase activity (pH 6.8) in 10,000 g supernatants from Anacystis nidulans. The enzyme activated by AA was deactivated by dehydroascorbic acid (DHAA). The modulation by AA/DHAA of phosphatase activity in Anacystis appears to be specific; a number of other redox compounds, known to modulate other enzymes, had no effect on the Anacystis phosphatase. A purified phosphatase preparation from Anacystis was also deactivated by DHAA. In contrast, the purified enzyme was not activated by AA, suggesting that a factor mediating the effect of AA was lost during purification. Another factor was found to protect the purified phosphatase against deactivation by DHAA. The enzyme was characterized as a phosphatase with a broad substrate specificity, an apparent molecular weight of 19,000, and a pH optimum of 6.0–7.0. Dialysis of the enzyme preparation against EDTA abolished the phosphatase activity which could be restored by Zn²⁺ ions and partially restored by Co²⁺ ions. Crude extracts also contained a latent enzyme, the phosphatase activity of which could be detected in the presence of Co²⁺ ions only. Zn²⁺ ions did not activate this enzymatically inactive protein. The Co²⁺-dependent phosphatase had an apparent mol. wt. of 40,000, a broad substrate specificity, and an alkaline pH-optimum. Infection of Anacystis cultures by cyanophage AS-1 resulted in a decrease in phosphatase activity. The enzyme present in 10,000 g supernatants from infected cells could not be modulated by the AA/DHAA system.Abbreviations AA ascorbic acid - DEAE diethylamino ethyl - DHAA dehydroascorbic acid - EDTA ethylene-diaminetetra-acetate - G6PDH glucose-6-phosphate dehydrogenase - GSH reduced glutathione - GSSG oxidized glutathione - HMP hexose monophosphate - P _i inorganic phosphorus - pNPP p-nitrophenylphosphate - pNP p-nitrophenol - Tris Tris(hydroxymethyl)-aminomethane     

Time- and dose-dependent effects of protein kinase C on proximal bicarbonate transport

Summary Activation of protein kinase C has been shown to cause both stimulation and inhibition of transport processes in the brush-border membrane and renal tubule. This study was designed to examine the dose-response nature and time-dependent effect of 4 -phorbol-12-myristate-13-acetate (PMA) on the rates of bicarbonate absorption (J _HCO3) and fluid absorption (J _v) in the proximal convoluted tubule (PCT) of rat kidney. Bicarbonate flux was determined by total CO₂ changes between the collected fluid and the original perfusate as analyzed by microcalorimetry. Luminal perfusion of PMA (10^–10 10^–5 M) within 10 min caused a significant increase ofJ _HCO3 andJ _v. A peaked curve of the dose response was observed with maximal effect at 10^–8 M PMA on both bicarbonate and fluid reabsorption, which could be blocked completely by amiloride (10^–3 m) and EIPA (10^–5 M). On the other hand, with an increase of perfusion time beyond 15 min, PMA (10^–8 and 10^–6 M) could inhibitJ _HCO3 andJ _v. Amiloride (10^–3 M) or EIPA (10^–5 M) significantly inhibitsJ _HCO3 andJ _v, while there is no additive effect of PMA and amiloride or EIPA on PCT transport. An inactive phorbol-ester, 4-phorbol, that does not activate protein kinase C, had no effects onJ _HCO3 andJ _v. Capillary perfusion of PMA (10^–8 M) significantly stimulate bothJ _HCO3 andJ _v; however, PMA did not affect glucose transport from either the luminal side or basolateral side of the PCT. These results indicate that activation of endogenous protein kinase C by PMA could either stimulate or inhibit both bicarbonate and fluid reabsorption in the PCT dependent on time and dose, and these effects are through the modulation of Na⁺/H^– exchange mechanism.     

Biochemical characterisation of extracellular phytase (myo-inositol hexakisphosphate phosphohydrolase) from a hyper-producing strain of Aspergillus niger van Teighem

Aspergillus niger van Teighem, isolated in our laboratory from samples of rotten wood logs, produced extracellular phytase having a high specific activity of 22,592 units (mg protein)^–1 . The enzyme was purified to near homogeneity using ion-exchange and gel-filtration chromatography. The molecular properties of the purified enzyme suggested the native phytase to be oligomeric, with a molecular weight of 353 kDa, the monomer being 66 kDa. The purified enzyme exhibited maximum activity at pH 2.5 and 52–55°C. The enzyme retained 97% activity after a 24-h incubation at 55°C in the presence of 10 mM glycine, while 87% activity was retained when no thermoprotectant was added. Phytase activity was not affected by most metal ions, inhibitors and organic solvents. Non-ionic and cationic detergents (0.1–5%) stabilise the enzyme, while the anionic detergent (SDS), even at a 0.1% level, severely inhibited enzyme activity. The chaotropic agents guanidinium hydrochloride, urea, and potassium iodide (0.5–8 M), significantly affected phytase activity. The maximum hydrolysis rate (V_max) and apparent Michaelis-Menten constant (K_m) were 1,074 IU/mL and 606 M, respectively, with a catalytic turnover number of 3×10⁵ s^–1 and catalytic efficiency of 3.69×10⁸ M^–1 s^–1.     

Stimulatory effect of hormonal signaling factors on (Ca2+−Mg2+)-ATPase activity in rat liver plasma membranes: Cross talk with regucalcin

The effect of hormonal signaling factors on (Ca²⁺–Mg²⁺)-ATPase activity in rat liver plasma membranes was investigated. The presence of inositol-glycan (10^–7–10^–5M), dibutyryl cAMP (10^–4 and 10^–3M) or inositol 1,4,5-trisphosphate (IP₃; 10^–6 and 10^–5 M) in the enzyme reaction mixture produced a significant increase in (Ca²⁺–Mg²⁺)-ATPase activity. These effects were completely inhibited by the presence of vanadate (10^–4 M), an inhibitor of the enzyme phosphorylation, and N-ethylmaleimide (5×10^–3 M), a SH group modifying reagent. Meanwhile, regucalcin, a Ca²⁺-binding protein isolated from rat liver cytosol, increased the enzyme activity by binding to the SH groups of (Ca²⁺–Mg²⁺)-ATPase in liver plasma membranes. The presence of regucalcin (0.25 M) with an effective concentration completely inhibited the effect of inositol-glycan (10^–5 M) to increase (Ca²⁺–Mg²⁺)-ATPase activity, while the effect of dibutyryl cAMP (10^–3M) or IP₃ (10^–5M) was not altered. The inositol-glycan effect was not modulated by the presence of dibutyryl cAMP or IP₃. Now, the preincubation of the plasma membranes with regucalcin did not modify the effect of inositol-glycan on the enzyme activity, suggesting that regucalcin competes with inositol-glycan for the binding to the plasma membranes. The present results suggest that there may be a cross talk with regucalcin and hormonal signaling factors in the regulation of (Ca²⁺–Mg²⁺)-ATPase activity in liver plasma membranes.     

Changes in mediobasal hypothalamic dopamine and GABA release: A possible mechanism underlying taurine-induced prolactin secretion

Summary Taurine (Tau), a putative inhibitory amino acid neurotransmitter, has been shown to stimulate prolactin (PRL) release. Using ovariectomized, estrogen-replaced adult rats we investigated initially the effect of this amino acid, injected by different routes, on PRL secretion in vivo. Tau (100–500 mg/kg) had no effect on PRL release when given i.p.; 15 min after i.c.v. injection of Tau (3moles), a significant increase in serum PRL levels was observed (78 ± 9 ng/ml over basal levels, p < 0.01 vs. controls). In vitro (cultured anterior pituitary cells) PRL release was not affected by a 5 h incubation with Tau (10^–3–10^–8 M). Basal dopamine (DA) or gamma-aminobutyric acid (GABA) output from superfused mediobasal hypothalamic fragments (MBH) was not affected by Tau (10^–3 M or 10^–5 M). However, during stimulation with KCl (50mM), Tau (10^–3 M) significantly lowered DA release, and increased GABA output. It is concluded that Tau acts at a central level to increase PRL secretion, most probably by modulating the hypothalamic release of neurotransmitters controlling lactotroph function.