System In Slices Research Articles

Action of various antidepressant treatments reduces reactivity of noradrenergic cyclic AMP-generating system in limbic forebrain

STUDIES on the pharmacology of the noradrenergic cyclic AMP-generating system in slices from the limbic forebrain of the rat1 and on adaptive properties of this system in conditions of persistent changes in the availability of nor-adrenaline (NA) have revealed that the system may serve as a model for the central NA receptor in this area, and that its sensitivity to NA increases or decreases when the availability of NA at the receptor site decreases or increases respectively2,3. Thus, hypersensitivity of the system has been achieved by treatment with reserpine2, a drug known to precipitate occasionally severe depressive reactions in man4, and the syndrome of which, when elicited in animals, is widely used as a model for depression5–7. Conversely, the monoamine oxidase (MAO) inhibitors, pargyline and nialamide, caused a marked reduction in the reactivity of the cyclic AMP-generating system to NA after chronic administration3. To determine whether or not antidepressant drugs which do not elevate the level of monoamines in brain share the effect of MAO inhibitors on the noradrenergic cyclic AMP-generating system, we studied the effect of the tricyclic antidepressants, desipramine and iprindole, on the reactivity of the system to NA. Desipramine blocks the uptake of NA through the neuronal membrane8,9 whereas iprindole neither blocks the neuronal uptake of NA nor alters its metabolism or turnover10,11, but is nevertheless a potent antidepressant12–14. In addition, we have tested the effect of electroconvulsive treatment (ECT), as it is generally accepted to be one of the most effective treatments for severe depression15.

Studies on the phenylalanine hydroxylase system in liver slices.

A method was developed to study the unsupplemented phenylalanine hydroxylase system in rat liver slices. All of the components of the system--tetrahydrobiopterin, dihydropteridine reductase, and the hydroxylase itself--are present under conditions which should be representative of the actual physiological state of the animal. The properties of the system in liver slices have been compared to those of the purified enzyme in vitro. The three pterins, tetrahydrobiopterin, 6,7-dimethyltetrahydropterin, and 6-methyltetrahydropterin, all stimulate the hydroxylation of phenylalanine when added to the liver slice medium in the presence of a chemical reducing agent. The relative velocities found at 1 mM phenylalanine and saturating pterin concentrations are: tetrahydrobiopterin, 1; 6,7-dimethyltetrahydropterin, 2.5; 6-methyltetrahydropterin, 13. This ratio of activities is similar to that found for the purified, native phenylalanine hydroxylase and indicates that the enzyme in vivo is predominantly in the native form. Rats pretreated with 6-methyltetrahydropterin showed enhanced phenylalanine hydroxylase activity in liver slices demonstrating for the first time that an exogenous tetrahydropterin can interact with the phenylalanine hydroxylase system in vivo. This finding opens up the possibility of treating phenylketonurics who still possess some residual phenylalanine hydroxylase activity with a tetrahydropterin like 6-methyltetrahydropterin which can give a large increase in rate over that seen with the natural cofactor, tetrahydrobiopterin.

Kinetic analysis of phenylalanine-induced inhibition in the saturable influx of tyrosine, tryptophan, leucine and histidine into brain cortex slices from adult and 7-day-old rats.

Abstract—An attempt was made to isolate the saturable uptake from the unidirectional influx of amino acids into tissue slices and to estimate the transport constants and maximal velocities of saturable transport. The method was applied to studies on the inhibition of phenylalanine in the saturable influx of tyrosine, tryptophan, histidine and leucine into brain cortex slices from adult and 7‐day‐old rats. In both age groups phenylalanine inhibited the influx of the other amino acids, and vice versa. The apparent transport constants of the other amino acids increased in the presence of phenylalanine more noticeably in the slices from 7‐day‐old rats than in those from adult rats, whereas the concomitant influx of phenylalanine was inhibited less in the slices from 7‐day‐old rats. In immature animals in vivo competition between amino acids may play a more marked role in the supply of amino acids from plasma to brain, as the transport systems in brain slices from 7‐day‐old rats become saturated with extracellular amino acids more readily than do the transport systems in brain slices from adult rats.

Amino acid transport and turnover of a transport system in liver slices from rats treated with glucagon and antibiotics

Experiments have been conducted in which glucagon-induced stimulation of α-aminoisobutyric acid (AIB) transport in rat liver, presumably mediated by cyclic AMP, was markedly inhibited by actinomycin D, cycloheximide or puromycin. Inhibition of transport occurred despite the presence of high concentrations of cyclic AMP, suggesting that the nucleotide may act prior to, or at the same point as, the antibiotics. Kinetic studies showed that 1) the half-life of the glucagon-stimulated system(s) was less than 1 hour, and 2) glucagon treatment doubled V max without having any effect on the apparent K m for hepatic AIB transport. The results suggest that glucagon stimulates hepatic AIB transport by increasing the synthesis of some critical protein having a rapid turnover rate; this effect may be due to a prior increase in RNA synthesis.

Inhibitory Action of Thyroid Hormone on the Activation of Adenyl Cyclase-Cyclic AMP System by Thyroid-Stimulating Hormone in Human Thyroid Tissues from Euthyroid Subjects and Thyrotoxic Patients

ABSTRACT The regulatory role of thyroid hormone on the adenyl cyclase-adenosine 3′,5′-monophosphate (cyclic AMP) system in human thyroid slices and plasma membranes from euthyroid subjects and thyrotoxic patients were studied by measuring the formation and accumulation of labeled and cold cyclic AMP. TSH stimulated adenyl cyclase-cyclic AMP system of the thyroid from euthyroid subjects and thyrotoxic patients, but the response was less in the thyroid from thyrotoxic patients. Small (10−9M) or large (10−4M) doses of thyroxin (T4) and triiodothyronine (T3) decreased the TSH-stimulated elevation of labeled and cold cyclic AMP level in the thyroid from euthyroid subjects. In contrast, graded doses of T4 (10−5 to 10−8M) and T3 (10−6 to 10−7M) failed to depress TSH-stimulated elevation of 3H cyclic AMP level in the thyroid from thyrotoxic patients. Large doses of T4 (10−4M) and T3 (10−4 and 10−5M) depressed TSH-stimulated adenyl cyclase activity, however. Since large doses of iodide and d-T4 have no effect on a...

Norepinephrine-sensitive adenylate cyclases in rat brain: relation to behavior and tyrosine hydroxylase.

Responses of norepiniephrine sensitive adeniosinie 3',5'-monophosphate (cyclic AMP)-generating systems in combined midbrain-striatal slices of four rat strains correlate positively with spontaneous behavioral activity and negatively with levels of midbrain and striatal tyrosine hydroxylase. Responses of cerebral cortical norepinephrine-sensitive cyclic AMP systems correlate negatively with spontaneous behavioral activity antd positively with midbrain and striatal tyrosine hydroxylase. Such correlations were not found with responses of the cyclic AMP- generatinlg systems to isoproterenol, adenosine. veratridine or of an adenosne and norepinephrine combination.

Effects of sera and sera fractions from spontaneously hypertensive rats on renal organic anion and cation transport.

SummarySera taken from spontaneously hypertensive Wistar rats compared to sera from normotensive Wistar rats depressed both PAH and TEA systems in kidney slices from normotensive Sprague-Dawley rats. This depression was found to reside in a specific fraction of sera separated on Sephadex columns, the same fraction which in azotemic sera suppresses both PAH transport and sodium transport in vitro. The observation that factors in sera affect organic anion and cation transport early in the development of hypertension may have implications in the pathogenesis of hypertension.

High affinity uptake of transmitters: studies on the uptake of L-aspartate, GABA, L-glutamate and glycine in cat spinal cord.

Abstract— The uptake of l‐aspartate, l‐glutamate and glycine each appeared to be mediated by two kinetically distinct systems with apparent Km's of the order of 10 ('high affinity') and 100 μM ('low affinity') in slices of cat spinal cord, whereas the uptake of GABA appeared to be mediated by a single system of high affinity. The high affinity uptake of these amino acids in slices of spinal grey matter was approximately 5 times faster than that in slices of spinal white matter. The high affinity uptake systems in the cord slices survived homogenisation of the tissue under conditions known to preserve nerve terminals. Subcellular fractionation studies indicated that osmotically‐sensitive particles of equilibrium density equivalent to that of 1.0 m‐sucrose were at least in part responsible for the uptake of these amino acids.Inhibition studies indicated that three structurally specific systems of high affinity transported these amino acids: specific for glycine—not inhibited by GABA or any of the other depressant amino acids found in cat spinal cord; specific for GABA—not inhibited by glycine, taurine, l‐aspartate or l‐glutamate and (3) specific for l‐aspartate and l‐glutamate—not inhibited by glycine or GABA but strongly inhibited by various acidic amino acids such as l‐cysteate and l‐cysteine sulphinate. The high affinity uptake of these amino acids was not inhibited by any of the known antagonists of the postsynaptic actions of these amino acids—strychnine (glycine), bicuculline and benzyl penicillin (GABA), methioninesulphoximine and l‐glutamate diethyl ester (l‐aspartate and l‐glutamate). p‐Chloromercuriphenylsulphonate strongly inhibited the high affinity uptake of glycine and GABA but was much less effective as an inhibitor of l‐aspartate/l‐glutamate high affinity uptake. This is in good agreement with microelectrophoretic studies in which this mercurial was found to potentiate depression of neuronal firing induced by glycine and GABA much more readily than excitation induced by l‐aspartate or l‐glutamate. These findings suggest the importance of high affinity transport processes in the removal of amino acids from the synaptic environment.

GLYCINE UPTAKE IN RAT CENTRAL NERVOUS SYSTEM SLICES AND HOMOGENATES: EVIDENCE FOR DIFFERENT UPTAKE SYSTEMS IN SPINAL CORD AND CEREBRAL CORTEX

Abstract— Evidence is presented that glycine is taken up by two different transport systems in rat CNS tissue slices; one system has relatively low affinity for glycine (Km= 300 μm) and predominates in cerebral cortex, cerebellum and mid‐brain, the other has a higher affinity for glycine (Km= 40 μm) and is detectable only in spinal cord, medulla and pons. The low affinity transport system appears to be shared by other small neutral amino acids, whereas the high affinity system is very specific for glycine. Both transport systems were shown to be present in particles in homogenates of CNS tissue by incubation with glycine in vitro, and subcellular fractionation studies suggested that synaptosomes were partly responsible for such uptake. Various substances were tested as inhibitors of the high affinity uptake system for glycine in spinal cord slices; the most potent inhibitors were p‐chloro‐mercuriphenylsulphonate, N‐ethylmaleimide, chlorpromazine, imipramine, desipramine, hydrazinoacetic acid and haloperidol. No competitive inhibitors of the high affinity glycine uptake were found. It is suggested that the high affinity transport system is associated with inhibitory synapses where glycine is a transmitter.

Effects of ions on thyrotrophin and prostaglandin E 1 stimulation of glucose oxidation and adenyl cyclase-cyclic AMP system in dog thyroid slices

Substitution of K + or choline for Na + in Krebs-Ringer bicarbonate buffer increased basal glucose oxidation and 3H-adenine incorporation into 3H-CAMP but had no effect on CAMP concentrations. In high K + buffer, TSH (3 mU/ml and 50 mU/ml) and prostaglandin E 1 (PGE 1) (0.1 μg/ml and 10 μg/ml) had little or no effect on glucose oxidation while their stimulation of 3H-CAMP formation and CAMP levels was equivalent to that obtained using regular Krebs-Ringer bicarbonate buffer. These results indicated that high K + inhibited the effects of CAMP and not its generation. The increased basal glucose oxidation in high K + buffer probably reflected increased membrane permeability since slices incubated with 14C-1-glucose and regular Krebs-Ringer bicarbonate buffer did not have augmented 14CO 2 production when they were later transferred to high K + buffer. The increased 3H-adenine incorporation into 3H-CAMP cannot be explained by increased membrane permeability, but appeared to reflect an effect on the intracellular conversion of 3H-adenine to 3H-ATP or 3H-ATP to 3H-CAMP. The effect of high K + probably reflected low Na + in the buffer since similar results were obtained when choline chloride was substituted for NaCl in Krebs-Ringer bicarbonate buffer. Exclusion of Ca ++ from Krebs-Ringer bicarbonate buffer decreased basal glucose oxidation, but had no effect on 3H-adenine conversion to 3H-CAMP or CAMP concentrations. Although effects of TSH and PGE 1 on glucose oxidation were diminished in buffer devoid of Ca ++, their stimulation of 3H-CAMP formation and CAMP levels was similar in the presence and absence of Ca ++. These results demonstrate that changes in the ionic composition of the buffer do not interfere with activation of the adenyl cyclase-CAMP system but modify some of the subsequent actions of the CAMP formed.

Effects of chlorpromazine, propranolol and phospholipase C on thyrotropin and prostaglandin stimulation of adenyl cyclase-cyclic AMP system in dog thyroid slices

The effect of chlorpromazine, propranolol and phospholipase C on the TSH and PGE 1, stimulation of the adenyl cyclase-cyclic AMP system and 14C-1-glucose oxidation was studied in dog thyroid slices. Chlorpromazine (2 × 10 −4 M) completely inhibited TSH (3 mU./ml.) stimulation of glucose oxidation, 3H-adenine incorporation into 3H-cyclic AMP and cyclic AMP concentrations. The inhibition could be partially overcome by increasing the amount of TSH to 50 mU./ml. Chlorpromazine also reduced the stimulation of 3H-cyclic AMP formation and cyclic AMP concentrations induced by prostaglandin E 1 (1 μg./ml.). Propranolol (10 −3 M) markedly diminished the TSH (5 mU./ml.) augmentation of adenyl cyclase activity, 3H-cyclic AMP formation and cyclic AMP levels while 10 −4 M propranolol had no effect. Neither concentration of propranolol nor chlorpromazine influenced basal levels of these parameters. Incubation of thyroid slices with phospholipase C was associated with some increase in basal glucose oxidation but marked reduction of the subsequent TSH (10 mU./ml.) stimulation of 14CO 2 production, cyclic AMP levels and 3H-cyclic AMP formation. These results are all compatible with the concept that the chlorpromazine, propranolol and phospholipase C inhibition of TSH stimulation of glucose oxidation reflect and effect on adenyl cyclase and cyclic AMP generation. Prostaglandin E 1 and F 1α (100 mg./ml.) increased 32P incorporation into phospholipids while an equivalent amount of prostaglandin B 1 had no effect and A 1 inhibited 32P incorporation. These results provide further evidence that stimulation of 32P incorporation into phospholipids may be independent of changes in cyclic AMP concentrations.

A comparison of inhibition of steady state, new transport, and exchange fluxes of amino acids in brain slices.

1. The steady state, net transport, and exchange flux at steady state of four amino acids (aminoisobutyric acid, leucine, lysine and d-glutamic acid) whose transport is primarily mediated by different transport systems in brain slices were compared. The effects of various metabolic inhibitors (1 mM cyanide, 10 μM ouabain) and of sub-optimal incubation conditions (sodium- and glucose-free mediums and incubations of 0°) were studied. 2. Steady-state amino acid levels were reduced to a “new” lower level by the above experimental conditions. The changes in steady state appear to be due primarily to changes in net transport. The maintenance of steady state required the expenditure of metabolic energy. 3. The inhibition of influx varied with the particular amino acid studied. This is consistent with differences in sensitivity to the experimental conditions of the transport systems involved. 4. The exit of amino acids was increased by the experimental conditions. The increased exit is not likely to be explained by an inhibition of re-uptake. 5. Exchange influx at steady state compared to influx from identical medium concentrations was significantly greater for lysine. The inhibition of exchange influx by the various experimental conditions was not always identical to that of influx. Exchange influx but not net transport of aminoisobutyric acid and lysine was sodium independent. 6. Exchange efflux was greater than exit for all four amino acids. The ratio of exchange efflux and exchange influx approximated a 1:1 exchange ratio. The inhibition of exchange efflux was similar to the inhibition of exchange influx; inhibition of re-uptake could not explain all the observed data. 7. Whether the differences between exchange flux and net transport were due to exchange diffusion representing different transport systems or to other factors such as variation of properties of the carriers or membrane changes induced by the experimental conditions can not be answered at this time.

Cell-free Hydrogenase from Chlamydomonas

10. LATIES, G. G. 1959. The development and control of coexisting respiratory systems in slices of chicory root. Archiv. Biochem. Biophys. 79: 378-91. 11. LATIES, G. G. 1962. Controlling influence of thickness on development and type of respiratory activity in potato slices. Plant Physiol. 37: 679-90. 12. MAcDONALD, I. R. AND P. C. DEKOCK. 1958. Temperature control and metabolic drifts in aging disks of storage tissue. Ann. Botany NS 22: 429-48. 13. MAcDONALD, I. R., P. C. DEKOCK, AND A. H. KNIGHT. 1960. Variations in the mineral content of storage tissue disks maintained in tap water. Physiol. Plantarum 13: 77-89. 14. SPLITTSTOESSER, W. E. 1963. Some aspects of dark CO2 fixation in storage tissues. Ph.D. thesis. Purdue University, Lafayette, Indiana. 15. ULRICH, A. 1941. Metabolism of non-volatile organic acids in excised barley roots as related to cation-anion balance during salt accumulation. Am. J. Botany. 28: 526-37. 16. WOLF, B. AND V. ICHESAKA. 1947. Rapid chemical soil and plant tests. Soil Sci. 64: 227-44.

Aphid transmission of potato virus Y diminished by electrostatic charge

Ipomoea tyrianthina Lindley (syn. I. orizabensis Pelletan, Lebed. ex Steud., Convolvulaceae) is known as a purgative, but it has been also used in Mexican traditional medicine in the treatment of seizures and pain for their anticonvulsive, hypnotic and sedative properties. Some glycolipids isolated from this plant have shown significant effects on Central Nervous System, modifying inhibitory or excitatory processes. The mechanism for such activity it is not clear; studies with these metabolites have suggested that a pore-forming mechanism is involved in their activity. Therefore, the present work explores a possible not pore-forming mechanism related to the effect of four resin glycosides, Scammonin 1 (S-1), tyrianthin C (T-C), tyrianthin A (T-A) and tyrianthinic acid VI (TA-VI), isolated from Ipomoea tyrianthina root on GABAergic transmission system in cerebral cortex slices of mouse brain in an in vitro model. The results obtained show that all glycolipids tested evoked endogenous GABA release and increased its concentration within the incubation medium compared with controls; T-C demonstrated a dose-dependent effect. Sodium absence and guvacine presence did not affect significantly the activity of S-1 and T-C in contrast to T-A and TA-VI. S-1 and T-C effects were calcium-dependent, where GABA concentrations were considerably reduced. These results suggest that the increase of endogenous γ-aminobutyric acid (GABA) released evoked by these glycolipids is possibly done through a Na+- and/or Ca2+-dependent mechanisms, discarding a pore-forming mechanism.

The development and control of coexisting respiratory systems in slices of chicory root

1. 1. The respiratory rise which occurs in incubated thin slices of chicory root may be controlled by a variety of environmental factors. By regulating either the lithium- or bicarbonate-ion concentration of the incubation medium, or by controlling the CO 2 or O 2 tension in the gas phase, the respiratory rise may either be prevented, or its nature altered. 2. 2. Whereas the respiratory increment which normally arises on incubation is completely malonate sensitive, it may be predominantly malonate resistant when incubation is carried out in solutions of selected concentrations of lithium or bicarbonate, or at high partial pressures of CO 2, or at low oxygen tensions. 3. 3. Under conditions where an increase in the respiration can occur, the malonate-resistant respiration rises in proportion to the extent to which the development (as distinct from the activity) of the malonate-sensitive respiration is repressed. 4. 4. The malonate-resistant respiration arising upon incubation under selected conditions is apparently different from the malonate-resistant basal respiration. It is suggested that the malonate-sensitive respiration preempts the latter but not the former. 5. 5. Whenever some fraction of the total respiration is malonate sensitive, malate restores the respiration rate of malonate-treated tissue. 6. 6. A suggestion is offered for the means whereby apparently disparate environmental factors may exert a common controlling influence.

Steps in p-aminohippurate transport by kidney slices.

The existence of two intracellular fractions of PAH was demonstrated in renal cortical slices of the rabbit on incubation with C14-labeled PAH. One of these fractions is rapidly diffusible and rapidly equilibrates with extracellular PAH. The other fraction, in contrast, diffuses and equilibrates slowly; it is responsible for the high slice to medium concentration ratio of PAH. On the basis of these results a model of the PAH transport system in slices is proposed. This consists of step I, the diffusion of PAH from the medium into the extracellular space in the tissue; there follows step II, a facilitated diffusion step at the peritubular cell membrane; within the cell step III builds up a high tissue concentration of PAH; finally step IV transfers PAH across the luminal border of the cell into the tubular lumen from which it may diffuse back into the medium. Experiments were designed in which each of these steps could be measured individually and their rate constants determined. Alteration of the value of these rate constants by specific drugs localizes the action of such compounds at the peritubular cell membrane (Benemid, 9-alphafluorohydrocortisone) or at the level of both steps II and III in the case of DNP, octanoate and Diodrast. An explanation is also offered for the effect of cold on PAH influx and efflux. It can be calculated that the contribution of step IV to the turnover of PAH in slices is not quantitatively significant.

The effect of methylthiouracil treatment on the histochemically demonstrable succinic dehydrogenase activity in the organs of the guinea-pig.

It has been shown that the thyroid hormone accelerates the metabolism of all cells in the body. Under the influence of thyroxine, the metabolism of carbohydrates, proteins, fats, minerals and water are all modified (Kendall, 1939). In conjunction with investigations on the influence of the thyroid hormone on the oxidative enzymes, the succinoxidase system – which is an essential link in the citric acid cycle of Krebs, the common pathway for the terminal metabolism of carbohydrates, proteins and fats – has also been investigated. It has been shown by biochemical methods that the administration of desiccated thyroid, thyroxine or thyrotrophic hormone results in a marked rise in the activity of the succinoxidase system in rat liver slices (Tipton & Nixon, 1946, Barker et al., 1949, Tipton, 1950). The increase in enzymatic activity after the administration of thyroid hormone is also noticeable in thyroidectomized animals (Lipner & Barker, 1953); it is closely correlated with the time of administration (Barker & Klitgaard, 1952), but does not occur in vitro (Smith & Williams-Ashman, 1949).

Ageing and enzyme activity

The influence of ageing of normal rats on the activity of some particular enzymes in liver homogenates and of some enzyme systems in liver slices was investigated. In the presence of adequate substrate, the activity of nearly all of the enzymes was found to increase with increasing age.