ABSTRACT
A simple and economical procedure, capable of routine application, is described for the labelling of cerebral phosphoproteins in vivo. [32P]Orthophosphate, in high concentration, was infused into selected brain areas of anaesthetised rats under stereotaxic control. The animals were frozen with liquid N2 and the labelled tissue punched out of frozen thick sections. [32P]Polypeptides were analysed by high-resolution two-dimensional gel electrophoresis. Several phosphoproteins on the gels were provisionally identified, including synapsin I, MAP-2 and an 82-87 kdalton substrate of protein kinase 'C'.
Subject(s)
Brain/metabolism , Nerve Tissue Proteins/metabolism , Animals , Autoradiography , Brain Chemistry , Electrophoresis , Female , Isoelectric Focusing , Male , Phosphoproteins/analysis , Phosphorylation , Rats , Rats, Inbred StrainsABSTRACT
Behavioral changes induced by apomorphine in normal, pseudodeprived (control) and REM sleep-deprived AM-2/TOR inbred rats were investigated. Deprived rats exhibited aggressive behavior for nearly 30 min in the absence of administration of drug, this effect not being observed in normal or control rats. The administration of apomorphine (1, 2 and 5 mg/kg) 5 min before the test elicited short periods of aggressive behavior in normal and control rats, but decreased the total duration of aggressive behavior in deprived rats. However, the deprived rats exhibited a more intense aggressive behavior, since the frequency of real fighting events was enhanced. The administration of apomorphine to deprived rats elicited stereotyped behavior. Enhancement of stereotyped behavior by increasing the dose was correlated with a reduction in the duration of aggressive behavior. Apomorphine also induced short episodes of intense excitability, manifested by increased locomotor activity, jumping and vocalization. This behavioral response was termed "episodic excitation". Deprived rats were significantly more sensitive to apomorphine-induced episodic excitation than normal and control rats. The episodic excitation, stereotyped and aggressive behavior displayed by deprived rats, injected with apomorphine, alternated with time. The results demonstrate increased responsiveness to apomorphine after deprivation of REM sleep. The possible mechanism for such interaction is discussed.
Subject(s)
Aggression/drug effects , Apomorphine/pharmacology , Sleep Deprivation/physiology , Sleep, REM/drug effects , Animals , Humans , Male , Rats , Rats, Inbred Strains , Stereotyped Behavior/drug effectsABSTRACT
Subcellular vesicles present in brain microsomal fraction take up calcium by an ATP-dependent process which is probably one of the mechanisms involved in the regulation of free calcium ions concentratioon in the cytosol of nerve cells. The experiments described in this paper were designed to test the effect of local anesthetics on this transport system since it is known that cytoplasmic calcium concentration interferes with nerve excitability and conduction and transmitter release. It was found that tetracaine increases the rate of calcium uptake in the range of 0.5 to 3 mM and inhibits calcium uptake in the range of 4 to 7 mM. Lidocaine and procaine increase calcium uptake in the range of 5 to 30 mM and inhibit calcium uptake in the range of 40 to 70 mM. The effects of local anesthetics were also tested on th ATP hydrolysis coupled with calcium uptake and on the ATP in equilibrium Pi exchange which represents the reverse reaction of this transport system. It was found that three local anesthetics inhibit ATP in equilibrium Pi exchange in concentrations which increase calcium uptake and inhibit ATP hydrolysis in concentrations which inhibit calcium uptake. These findings indicate that the enhancement of calcium uptake by the lower concentrations of local anesthetics is due to a decrease of the reverse reaction, whereas inhibition of calcium uptake by the higher concentrations of local anesthetics is due to the blockage of the transport adenosine triphosphatase.
Subject(s)
Anesthetics, Local/pharmacology , Brain/metabolism , Calcium/metabolism , Adenosine Triphosphate/metabolism , Animals , Biological Transport, Active/drug effects , Brain/drug effects , Hydrolysis , In Vitro Techniques , Microsomes/metabolism , Phosphates/metabolism , RabbitsABSTRACT
1. ATP-dependent calcium uptake by a rabbit brain vesicular fraction (microsomes) was studied in the presence of phosphate or oxalate. These anions, which are known to form insoluble calcium salts, increased the rate of calcium uptake and the capacity of the vesicles for calcium accumulation. 2. The degree of activation depended on the concentration of phosphate or oxalate. Under optimal conditions, phosphate promoted a 5-fold increase in the amount of calcium stored at steady state. This level was 200-250 nmol Ca-2+/mg protein. 3. Initial rate of calcium uptake followed Michaelis-Menten kinetics with an apparent Km for calcium of 6.7-10-minus 5 M and a V of 44 nmol/min per mg protein. Optimal pH was 7.0. With 2 mM ATP, optimal Mg-2+ concentration was 2 mM. 4. Dintrophenol and NaN3 inhibited calcium uptake in a mitochondria-enriched fraction but not in the microsomal fraction. 5. Calcium uptake activity was compared in the six subfractions prepared from the whole microsomal fraction by means of a sucrose density gradient fractionation. 6. The Mg-2+-dependent ATPase activity of brain microsomes was activated by calcium. Maximal activation was attained with 100 muM CaCl2. Greater calcium concentrations caused a progressive inhibition. 7. The data suggest that the ATP-dependent calcium uptake in brain microsomes, as in muscle microsomes, is brought about by an active transport process, calcium being accumulated as a free ion inside the vesicles.
