Rabbit myocardial membrane Ca2+-adenosine triphosphatase activity: stimulation in vitro by thyroid hormone

The in vitro stimulation of human and rabbit erythrocyte membrane Ca2+-ATPase activity by physiological concentrations of thyroid hormone has recently been described. To extend these observations to a nucleated cell model, Ca2+-ATPase activity in a membrane preparation obtained from rabbit myocardium has been studied. Activity of 5'-nucleotidase in the preparation was increased 26-fold over that of myocardial homogenate, consistent with enrichment by sarcolemma. Mean basal enzyme activity in membranes from nine animals was 20.8 +/- 3.3 mumol Pi mg membrane protein-1 90 min-1, approximately 20-fold the activity described in rabbit red cell membranes. Exposure of heart membranes in vitro to L-thyroxine (T4) (10(-10)M) increased Ca2+-ATPase activity to 29.2 +/- 3.8 mumol Pi (P less than 0.001). Dose-response studies conducted with T4 showed that maximal stimulatory response was obtained at 10(-10) M). Hormonal stimulation was comparable for L-T4 and triiodo-L-thyronine (T3) (10(-10) M). Tetraiodothyroacetic acid was without biological activity, whereas triiodothyroacetic acid and D-T4, each at 10(-10) M, significantly decreased enzyme activity compared to control (basal) levels. The action of L-T4 on myocardial membrane Ca2+-ATPase activity was inhibited by trifluoperazine (100 microM) and the naphthalenesulfonamide W-7 (50-100 microM), compounds that block actions of calmodulin, the protein activator of membrane-associated Ca2+-ATPase. Radioimmunoassay revealed the presence of calmodulin (1.4 micrograms/mg membrane protein-1) in the myocardial membrane fraction and 0.35 micrograms/mg-1 in cytosol. Myocardial Ca2+-ATPase activity, apparently of sarcolemmal origin, is thus thyroid hormone stimulable. The hormonal responsiveness of this calcium pump-associated enzyme requires calmodulin.     

Calmodulin-dependent regulation of Ca,Mg-ATPase activity in plasma membranes of the swine myometrium

Highly purified plasma membrane (PM) preparations of pig myometrium were found to contain 0.91 +/- 0.22 microgram calmodulin per mg of PM protein. Treatment of membranes with 1 mM EGTA in the presence of 0.2 M NaCl causes the diminution of the calmodulin content down to 3% of the original level. The activity of Ca, Mg-ATPase is thereby decreased by 40%. Exogenous calmodulin restores the enzyme activity up to 1.94 +/- +/- 0.30 mumol Pi/mg protein/hour. The maximal activation of Ca, Mg-ATPase is observed with 10(-7) M calmodulin. Calmodulin increases the total ATPase activity of myometrium PM without affecting the Mg-ATPase activity. Trifluoroperazine (20 microM) diminishes the activating effect of exogenous calmodulin on Ca, Mg-ATPase. Calmodulin stimulates Ca, Mg-ATPase at low concentrations of Ca2+(10(-8)-10(-6) M) by decreasing Km for Ca2+ from 0.4.10(-6) M to 2.10(-8) M as well as by increasing Vmax--from 0,8 to 1.42 mumol Pl/mg protein/hour. It is supposed that the activating effect of calmodulin on Ca, Mg-ATPase is based on electrostatic interactions of Ca2+-free calmodulin with the enzyme.     

Effect of pentobarbital dependence on adenylate cyclase activity and calmodulin levels in rat cerebral cortex

The effect of calcium (Ca2+) on the adenylate cyclase activity and calmodulin level of cerebral cortex was determined in pentobarbital dependent rats and age matched controls. Female Sprague-Dawley rats were made dependent and maintained on pentobarbital by eating a mixture of pentobarbital and rat chow (350 mg pentobarbital/30 g chow). Ca2+ activated then inhibited the adenylate cyclase activity associated with a 20,000 X g particulate fraction from pentobarbital dependent and age matched control rats. The values for one-half maximal stimulation and inhibition by Ca2+ did not differ significantly in either cortical preparation. However, the ability of Ca2+ to activate adenylate cyclase from pentobarbital dependent animals was significantly decreased (p less than 0.05) when compared to control animals. Pentobarbital (10(-4) - 10(-3) added to particulate fractions from naive control rats did not alter the ability of Ca2+ to activate adenylate cyclase. The calmodulin levels in the particulate fraction from pentobarbital dependent animals (30.2 +/- 6.7 ng calmodulin/mg protein) did not differ significantly when compared to control (33.0 +/- 4.7 ng/mg). By contrast, the calmodulin levels (37.9 +/- 5.9 ng/mg) in the 20,000 X g supernatant from cortex of pentobarbital dependent animals was significantly greater than the level in the supernatant from control animals (28.6 +/- 2.6 ng/mg). The ability of forskolin, dopamine, GTP or forskolin plus GTP (all at a concentration of 100 microM) to activate adenylate cyclase was significantly decreased in particulate preparations from pentobarbital dependent animals. In summary, our data show that alterations in calmodulin levels and a decreased responsivity of adenylate cyclase occur in animals physically dependent on pentobarbital.     

Tissue calmodulin levels in normal and Graves' thyroids

Calmodulin levels in normal human thyroids and Graves' disease thyroids were measured by specific radioimmunoassay in the presence of ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The calmodulin levels in tissues from patients with Graves' disease treated with thionamide drugs were significantly higher than those in normal tissues from euthyroid patients with solitary cold nodules (normal: 484 +/- 50 ng/mg protein, mean +/- SE, n = 15; Graves': 901 +/- 54 ng/mg protein, n = 48, p less than 0.001). Such a rise in calmodulin levels in Graves' disease thyroids was also present even after the administration of 50 micrograms of T3 for 5 days before operation (828 +/- 137 ng/mg protein, n = 6, p less than 0.01). Calmodulin levels in Graves' disease thyroids were closely related to the cell height of follicular epithelium. Calmodulin levels in a columnar cell predominant group were significantly higher than those in a flat cell predominant or a cuboidal cell predominant group (columnar cell predominant: 1150 +/- 118 ng/mg protein, n = 13; flat cell predominant: 561 +/- 125 ng/mg protein, n = 3, p less than 0.05; cuboidal cell predominant: 596 +/- 40 ng/mg protein, n = 25, p less than 0.001). The increase in calmodulin content in Graves' disease thyroid could therefore possibly be attributed to the stimulation of the thyroid gland by the thyroid stimulating antibody. An immunofluorescence study demonstrated the presence of calmodulin immunoreactivity in the thyroid epithelial cells, particularly enriched in the apical border in the form of a granulated structure.     

Calmodulin Affinity for Brain Coated Vesicle Proteins

A systematic characterization of the affinity of calmodulin for brain coated vesicles was undertaken. Binding of 125I-labeled calmodulin to coated vesicles was saturable and competed with unlabeled calmodulin, but not with troponin-C. Scatchard analysis revealed one high-affinity, low-capacity binding site, KD = 3.9 +/- 0.6 nM, Bmax = 16.3 +/- 2.4 pmol/mg, and one low-affinity, high-capacity binding site, KD = 102 +/- 15.0 nM, Bmax = 151 +/- 23.0 pmol/mg. Radioimmunoassay revealed that coated vesicles contain 1.05 microgram calmodulin/mg protein. Because this value remained constant even after removal of clathrin, the major coat protein, from the coated vesicle, it is apparent that calmodulin is associated with the vesicle per se rather than with its clathrin lattice. When a Triton X-100-treated extract of coated vesicles was passed through a Sepharose 4B-calmodulin affinity column, polypeptides with Mrs (molecular weights) of 100,000, 55,000, and 30,000 bound in a Ca2+-dependent manner. A 30,000 Mr protein doublet purified from coated vesicles was completely eluted by EGTA from the calmodulin affinity column, confirming that this protein doublet represents one of the coated vesicle calmodulin binding sites. Because calmodulin stimulated [Ca2+-Mg2+]-ATPase activity as well as Ca2+ uptake in coated vesicles, it is postulated that the 100,000 and 55,000 Mr calmodulin binding proteins represent the [Ca2+-Mg2+]-ATPase complex, the other coated vesicle calmodulin binding site.     

Further characterization of the membrane-bound (Ca2+ + Mg2+)-ATPase from porcine erythrocytes

1. The kinetic and physicochemical properties of the calcium-pumping protein, (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) were studied in ghost membranes isolated from porcine erythrocytes. 2. The membrane-bound enzyme in situ has a specific activity of 3.12 +/- 0.08 micron/mg protein/hr and a Vmax of 3.47 +/- 0.21 mumol/mg protein/hr in the absence of calmodulin. 3. Its activity was stimulated by calmodulin about 5-fold. The enzyme is also highly sensitive to inhibition by vanadate (Ki = 1.6 +/- 0.2 microM). 4. Calmodulin also affects the pH- and Ca2+-sensitivity of the enzyme. The optimum pH, in the presence of calmodulin, is 7.5 and the optimum temperature is 38 degrees C with an activation energy of 11.9 kcal/mol.     

Calmodulin regulation of Ca2+ transport in human erythrocytes.

Inside-out vesicles of human erythrocytes took up Ca2+ against an electrochemical gradient. This Ca2+ uptake was dependent on ATP and was stimulated by calmodulin. Treatment of vesicles with 1 mM-EDTA exposed an apparent low-CA2+-affinity Ca2+-transport component with Kd of about 100 microM-Ca2+ or more. This was converted into a single high-Ca2+-affinity transport activity of Kd about 2.5 microM-Ca2+ in the presence of 2 micrograms of calmodulin/ml, showing that the decrease in transport activity after EDTA treatment was reversible. Vesicles not extracted with EDTA showed mainly apparent high-Ca2+-affinity kinetics even in the absence of added calmodulin. Trifluoperazine (30 microM) and calmodulin-binding protein (20 micrograms/ml) inhibited about 50% of the high-affinity Ca2+ uptake and (Ca2+ + Mg2+)-ATPase (Ca2+-activated, Mg2+-dependent ATPase) activity of these vesicles, indicating that the vesicles isolated by the procedure used retained some calmodulin from the erythrocytes. Comparison of Ca2+ transport and (Ca2+ + Mg2+)-ATPase activities in inside-out vesicles yielded a variable Ca2+/P1 stoichiometric ratio. At low free Ca2+ concentrations (below 20 micro-Ca2+), a Ca2+/P1 ration of about 2 was found, whereas at higher Ca2+ concentrations the stoichiometry was approx. 1. The stoichiometry was not significantly altered by calmodulin.     

Developmental Changes in the NGF Content in the Brain of Young,Growing, Low-Birth-Weight Rats

The NGF content in each region of the brain of four-week-old rats was ranked in the decreasing order of cerebral cortex, hippocampus, cerebellum, midbrain/diencephalon, and pons/medulla ob-longata, and the NGF concentration, in the decreasing order of hippocampus, cerebral cortex, cerebellum, midbrain/diencephalon, and pons/medulla oblongata in both AFD and SFD groups. The NGF content and concentration in the cerebral cortex were about the same value at each age between those in the AFD and SFD groups. Those in the hippocampus were a little higher in the SFD group than in the AFD group at the ages of three and four weeks, unlike those in the other regions, where the values for the cerebellum, midbrain/diencephalon and pons/medulla oblongata tended to be somewhat higher in the AFD group than in the SFD group. The NGF concentrations in the hippocampus and cerebral cortex increased with growth: the concentration in the hippocampus at four weeks of age was about 4-fold of that at one week in the AFD group and about 5.7-fold of that at one week in the SFD group; and likewise the concentration in the cerebral cortex at four weeks of age was about 5.3-fold in the AFD group and about 7-fold in the SFD group. The NGF concentrations in the cerebellum decreased, and those in midbrain/diencephalon and pons/medulla oblongata hardly changed with growth in either AFD or SFD group. From these results NGF may have stronger implications for the neuronal growth in the hippocampus compared with those in the lower brain regions of the SFD rats.     

Thyrotropin releasing hormone (TRH) binding sites in the adult human brain: localization and characterization

In the current study, we found evidence for the existence of binding sites for TRH in synaptic membrane preparations of several regions of the postmortem adult human brain. High levels of specific binding (fmol [3H]Me-TRH/mg protein/2 hr) were found in limbic structures: amygdala (7.1 +/- 0.6, Mean +/- SE), hippocampus (2.8 +/- 0.3), and temporal cortex (2.4 +/- 0.8). Intermediate levels of binding were found in the hypothalamus and nucleus accumbens whereas binding was low to undetectable in frontal and occipital cortex, cerebellum, pons, medulla and corpus striatum. Binding of the radioligand was linear over protein concentrations of 0.05-1.5 mg, and greater than 6 hr of incubation was required to achieve maximal binding. In the amygdala, binding was inhibited in the presence of TRH and Me-TRH but not in the presence of up to 1 microM concentrations of cyclo (His-Pro), TRH-OH, pGlu-His or peptides unrelated to TRH. Pretreatment of amygdala synaptic membranes with detergents, proteases or phospholipases disrupted [3H]Me-TRH binding; pretreatment with DNase or collagenase had no effect on binding. Saturation and association/dissociation analyses of the binding of [3H]Me-TRH to purified amygdala synaptic membranes revealed the presence of a high affinity (KD = 2.0 nM), low capacity (Bmax = 180 +/- 16 fmoles/mg protein) binding site. These results demonstrate that a highly specific membrane associated receptor for TRH is present in the adult human brain. The specific role that this receptor plays in brain function remains to be elucidated.     

Beta-endorphin and neurotensin in brainstem and cerebrospinal fluid in the sudden infant death syndrome.

Beta-endorphin (BE) and neurotensin (NT) are two neuropeptides which induce apneas. In infants who died of Sudden Infant Death Syndrome (SIDS) we measured, in brainstem and CSF, BE and NT by IRMA and RIA respectively. BE and NT levels are compared to same aged infant and adult controls. CSF BE level was significantly higher in SIDS than in the two control groups (86 +/- 14 vs 33 +/- 13 and 16 +/- 5 pmol/l). In 6 SIDS victims NT and BE were assayed in 5 brainstem sections, each of them divided in median, intermediate and lateral parts. We found high levels of BE in every fragment (3-11 pmol/mg protein) while NT elevated values were restricted to the mesencephalic regions (1.4-12 pmol/mg), the medial pons (6 pmol/mg) and the intermediate parts of the medulla (including the olive: 1.3-1.6 pmol/mg). These results support the hypothesis that NT and/or BE could induce or participate to the fetal issue of SIDS.     

Calmodulin inhibitors protect against cadmium-induced testicular damage in mice

Recent reports showing that cadmium can interact with calmodulin and activate calmodulin-sensitive enzymes have lead us to examine the effects of calmodulin inhibitors on cadmium-induced testicular toxicity in mice. Male CF-1 mice were pretreated with the various calmodulin inhibitors or inactive analogs and then, one hour later, given CdCl2 X 2 1/2 H2O (32 mumoles/kg). After 24 hours, the mice were killed and the testes were removed and weighed. The extent of hemorrhaging was quantified by determining the absorbance of hemoglobin at 414 nm in the soluble fraction of testicular homogenates. Exposure to Cd2+ increased the mean testicular weight from 118 +/- 5 mg to 146 +/- 4 mg and the hemoglobin absorbance from 0.096 +/- 0.006 to 0.767 +/- 0.138. Pretreatment with the calmodulin inhibitors, trifluoperazine (40 mumoles/kg), chlorpromazine (40 mumoles/kg) or W-7 (140 mumoles/kg) greatly attenuated the CD2+-induced increase in both parameters, whereas pretreatment with chlorpromazine sulfoxide (140 mumoles/kg), pentobarbital (140 mumoles/kg), verapamil (80 mumoles/kg) or ethylenediaminetetraacetate (140 mumoles/kg) did not. These results indicate that calmodulin inhibitors can protect against certain toxic effects of cadmium and are consistent with the hypothesis that some of the effects of cadmium may result from the improper activation of calmodulin-dependent enzymes.     

Studies on rat parotid-cell actomyosin

Actomyosin was partially purified from rat parotid cells dispersed by collagenase digestion and found to possess different solubility characteristics from that from (undispersed) rat parotid tissue. This is attributed to the decrease in vascular contamination effected by the isolation of parotid cells, yielding a non-muscle actomyosin [Adelstein, Conti, Johnson, Pastan & Pollard (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 3693-3697]. Myosin light-chain kinase was partially purified from dispersed rat parotid cells by calmodulin affinity chromatography and shown to be activated by Ca2+-calmodulin. The calmodulin content of dispersed rat parotid cells was shown to be 6.50 +/- 0.59 ng of calmodulin/micrograms of rat parotid-cell protein (mean +/- S.E.M.), as determined by the activation of purified bovine brain phosphodiesterase by heat-treated extracts of dispersed rat parotid cells.     

Glycated phosphatidylethanolamine promotes macrophage uptake of low density lipoprotein and accumulation of cholesteryl esters and triacylglycerols.

Non-enzymatic glycation of low density lipoprotein (LDL) has been suggested to be responsible for the increase in susceptibility to atherogenesis of diabetic individuals. Although the association of lipid glycation with this process has been investigated, the effect of specific lipid glycation products on LDL metabolism has not been addressed. This study reports that glucosylated phosphatidylethanolamine (Glc-PtdEtn), the major LDL lipid glycation product, promotes LDL uptake and cholesteryl ester (CE) and triacylglycerol (TG) accumulation by THP-1 macrophages. Incubation of THP-1 macrophages at a concentration of 100 micrograms/ml protein LDL specifically enriched (10 nmol/mg LDL protein) with synthetically prepared Glc-PtdEtn resulted in a significant increase in CE and TG accumulation when compared with LDL enriched in non-glucosylated PtdEtn. After a 24-h incubation with LDL containing Glc-PtdEtn, the macrophages contained 2-fold higher CE (10.11 +/- 1.54 micrograms/mg cell protein) and TG (285.32 +/- 4.38 micrograms/mg cell protein) compared with LDL specifically enriched in non-glucosylated PtdEtn (CE, 3.97 +/- 0.95, p < 0.01 and TG, 185.57 +/- 3.58 micrograms/mg cell protein, p < 0.01). The corresponding values obtained with LDL containing glycated protein and lipid were similar to those of LDL containing Glc-PtdEtn (CE, 11.9 +/- 1.35 and TG, 280.78 +/- 3.98 micrograms/mg cell protein). The accumulation of both neutral lipids was further significantly increased by incubating the macrophages with Glc-PtdEtn LDL exposed to copper oxidation. By utilizing the fluorescent probe, 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate (DiI), a 1.6-fold increase was seen in Glc-PtdEtn + LDL uptake when compared with control LDL. Competition studies revealed that acetylated LDL is not a good competitor for DiI Glc-PtdEtn LDL (5-6% inhibition), whereas glycated LDL gave an 80% inhibition, and LDL + Glc-PtdEtn gave 93% inhibition of uptake by macrophages. These results indicate that glucosylation of PtdEtn in LDL accounts for the entire effect of LDL glycation on macrophage uptake and CE and TG accumulation and, therefore, the increased atherogenic potential of LDL in hyperglycemia.     

Characterization of high mobility group protein levels during spermatogenesis in the rat

The distribution, quantitation, and synthesis of high mobility group (HMG) proteins during spermatogenesis in the rat have been determined. HMG1, -2, -14, and -17 were isolated from rat testes by Bio-Rex 70 chromatography combined with preparative gel electrophoresis. Amino acid analysis revealed that each rat testis HMG protein was similar to its calf thymus analogue. Tryptic peptide maps of somatic and testis HMG2 showed no differences and, therefore, failed to detect an HMG2 variant. Testis levels of HMG proteins, relative to DNA content, were equivalent to other tissues for HMG1 (13 micrograms/mg of DNA), HMG14 (3 micrograms/mg of DNA), and HMG17 (5 micrograms/mg of DNA). The testis was distinguished in that it contained a substantially higher level of HMG2 than any other rat tissue (32 micrograms/mg of DNA). HMG protein levels were determined from purified or enriched populations of testis cells representing the major stages of spermatogenesis; spermatogonia and early primary spermatocytes, pachytene spermatocytes, early spermatids, and late spermatids; and testicular somatic cells. High levels of HMG2 in the testis were due to pachytene spermatocytes and early spermatids (56 +/- 4 and 47 +/- 6 micrograms/mg of DNA, respectively). Mixtures of spermatogonia and early primary spermatocytes showed lower levels of HMG2 (12 +/- 3 micrograms/mg of DNA) similar to proliferating somatic tissues, whereas late spermatids had no detectable HMG proteins. The somatic cells of the testis, including isolated populations of Sertoli and Leydig cells, showed very low levels of HMG2 (2 micrograms/mg of DNA), similar to those in nonproliferating somatic tissues. HMG proteins were synthesized in spermatogonia and primary spermatocytes, but not in spermatids. Rat testis HMG2 exhibited two bands on acid-urea gels. A "slow" form comigrated with somatic cell HMG2, while the other "fast" band migrated ahead of the somatic form and appeared to be testis-specific. The "fast" form of HMG2 accounted for the large increase of HMG2 levels in rat testes. These results show that the very high level of HMG2 in testis is not associated with proliferative activity as previously hypothesized.     

Regional Distribution of Neuropeptide Y and Its Receptor in the Porcine Central Nervous System

The regional distribution of neuropeptide Y (NPY) immunoreactivity and receptor binding was studied in the porcine CNS. The highest amounts of immunoreactive NPY were found in the hypothalamus, septum pellucidum, gyrus cinguli, cortex frontalis, parietalis, and piriformis, corpus amygdaloideum, and bulbus olfactorius (200-1,000 pmol/g wet weight). In the cortex temporalis and occipitalis, striatum, hippocampus, tractus olfactorius, corpus mamillare, thalamus, and globus pallidus, the NPY content was 50-200 pmol/g wet weight, whereas the striatum, colliculi, substantia nigra, cerebellum, pons, medulla oblongata, and medulla spinalis contained less than 50 pmol/g wet weight. The receptor binding of NPY was highest in the hippocampus, corpus fornicis, corpus amygdaloideum, nucleus accumbens, and neurohypophysis, with a range of 1.0-5.87 pmol/mg of protein. Intermediate binding (0.5-1.0 pmol/mg of protein) was found in the septum pellucidum, columna fornicis, corpus mamillare, cortex piriformis, gyrus cinguli, striatum, substantia grisea centralis, substantia nigra, and cerebellum. In the corpus callosum, basal ganglia, corpus pineale, colliculi, corpus geniculatum mediale, nucleus ruber, pons, medulla oblongata, and medulla spinalis, receptor binding of NPY was detectable but less than 0.5 pmol/mg of protein. No binding was observed in the bulbus and tractus olfactorius and adenohypophysis. In conclusion, immunoreactive NPY and its receptors are widespread in the porcine CNS, with predominant location in the limbic system, olfactory system, hypothalamoneurohypophysial tract, corpus striatum, and cerebral cortex.     

Activation by calmodulin of inositol-1,4,5-trisphosphate 3-kinase in guinea pig peritoneal macrophages

The activity of inositol-1,4,5-trisphosphate 3-kinase in the cytosol fraction of guinea pig macrophages was assayed with special reference to the dependence on the free Ca2+ concentration. The enzyme activity, as assessed by the production of inositol 1,3,4,5-tetrakisphosphate was reversibly activated by free Ca2+ concentrations ranging from 10(-7) to 10(-6)M. The calmodulin antagonists, W-7 and chlorpromazine, inhibited the Ca2+-activated enzyme activity in a dose-dependent fashion, thereby indicating that calmodulin may be involved in the activation by Ca2+. The content of calmodulin in the cytosol fraction (about 2.8 micrograms/mg of cytosol protein) was markedly reduced to less than 0.03 microgram/mg of proteins by subfractionation by ammonium sulfate, followed by an anion-exchange chromatography. The subfraction obtained by the chromatography showed no Ca2+ dependence in the enzyme activity, while an exogenous addition of calmodulin with 10(-6)M Ca2+ increased the enzyme activity. The enzyme activity was retained on a calmodulin-affinity column in the presence of Ca2+, and was eluted from the column by lowering the free Ca2+ concentration by adding ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. These results clearly indicate that calmodulin activates the inositol-1,4,5-trisphosphate 3-kinase activity.     

Levetiracetam protects against kainic acid-induced toxicity

We investigated the Levetiracetam (LVT) ability to protect the brain against kainic acid (KA) induced neurotoxicity. Brain injury was induced by intraperitoneal administration of KA (10 mg/kg). Sham brain injury rats were used as controls. Animals were randomized to receive either LVT (50 mg/kg) or its vehicle (1 ml/kg) 30 min. before KA administration. Animals were sacrificed 6 hours after KA injection to measure brain malonildialdehyde (MDA), glutathione levels (GSH) and the mRNA for interleukin-1beta (IL-1beta) in the cortex and in the diencephalon. Behavioral changes were also monitored. Intraperitoneal administration of LVT decreased significantly MDA in the cortex (KA + vehicle = 0.25 +/- 0.03 nmol/mg protein; KA + LVT = 0.13 +/- 0.01 nmol/mg protein; P < 0.005), and in the diencephalons (KA + vehicle = 1,01 +/- 0.2 nmol/mg protein; KA + LVT = 0,33 +/- 0,08 nmol/mg protein; P < 0.005), prevented the brain loss of GSH in both cortex (KA + vehicle = 5 +/- 1 micromol/g protein; KA + LVT = 15 +/- 2 micromol/g protein; P < 0.005) and diencephalons (KA + vehicle = 9 +/- 0.8 micromol/g protein; KA + LVT = 13 +/- 0.3 micromol/g protein; P < 0.05), reduced brain IL-1beta mRNA and markedly controlled seizures. Histological analysis showed a reduction of cell damage in LVT treated samples. The present data indicate that LVT displays neuro-protective effects against KA induced brain toxicity and suggest that these effects are mediated, at least in part, by inhibition of lipid peroxidation.     

The Ca2+-pumping ATPase of heart sarcolemma. Characterization, calmodulin dependence, and partial purification

The (Ca2+ + Mg2+) ATPase of dog heart sarcolemma (Caroni, P., and Carafoli, E. (1980) Nature 283, 765-767) has been characterized. The enzyme possesses an apparent Km (Ca2+) of 0.3 +/- 02 microM, a Vmax of Ca2+ transport of 31 nmol of Ca2+/mg of protein/min, and an apparent Km (ATP) of 30 microM. It is only slightly influenced by monovalent cations and is highly sensitive to orthovanadate (Ki = 0.5 +/- 0.1 microM). The high vanadate sensitivity has been used to distinguish the sarcolemmal and the contaminating sarcoplasmic reticulum Ca2+-dependent ATPase in heart microsomal fractions. Calmodulin has been shown to be present in heart sarcolemma. Its depletion results in the transition of the Ca2+-pumping ATPase to a low Ca2+ affinity; readdition of calmodulin reverses this effect. The Na+/Ca2+ exchange system was not affected by calmodulin. The results of calmodulin extraction can be duplicated by using the calmodulin antagonist trifluoperazine. The calmodulin-depleted Ca2+-ATPase has been solubilized from the sarcolemmal membrane and "purified" on a calmodulin affinity chromatography column. One major (Mr = 150,000) and 3 minor protein bands could be eluted from the column with ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA). The major protein band (72%) has Ca2+-dependent ATPase activity and can be phosphorylated by [gamma]32P]ATP in a Ca2+-dependent reaction.     

Presence of the Novel Pituitary Protein „7B2” in Bovine Chromaffin Granules: Possible Co-Release of 7B2 and Catecholamine as Induced by Nicotine

We observed the presence of the novel pituitary protein "7B2" and its release in the bovine adrenal medulla. The 7B2 concentration (mean +/- SEM) in extracts of the bovine adrenal medulla was 952 +/- 155 pg/mg tissue (n = 6). 7B2 was distributed in the chromaffin granule fraction prepared from the bovine adrenal medulla and was released by high K+ and/or nicotine from cultured cells of the bovine adrenal medulla. Co-release of 7B2 with catecholamine induced by nicotine from the cultured bovine chromaffin cells was also observed. In an analysis of the bovine adrenal medulla chromaffin granule fraction on gel permeation chromatography, there was a major peak with an apparent molecular weight of 45,000, whereas a major peak with an apparent molecular weight of 20,000 was found in that on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On reverse-phase HPLC, a major peak with a retention time of 35 min was observed in the bovine chromaffin granule fraction and in the bovine anterior pituitary extract. These findings indicate that 7B2 is a secretory protein in the bovine adrenal medulla. The possibility that 7B2 might be released with catecholamine, possibly in response to stress, warrants investigation.     

Amino acid content in several brain regions of the active and hibernating frog, Rana esculenta

1. Total free amino acid contents in the optic lobe and diencephalon increased significantly during hibernation. 2. Free glutamate + glutamine showed significant increases in the cerebral hemisphere, optic lobe, medulla oblongata and diencephalon. 3. Free aspartate + asparagine showed significant increases in the cerebral hemisphere, optic lobe, diencephalon and olfactory lobe. 4. GABA showed a significant change only in the medulla oblongata. 5. Total protein amino acid level in the cerebellum and olfactory lobe decreased significantly during hibernation and most of the amino acids decreased significantly in these regions. 6. The amino acid metabolism during amphibian hibernation differs from that of the mammal.