Drugs altering insulin secretion: effects on plasma and brain concentrations of aromatic amino acids and on brain 5-hydroxytryptamine turnover.

1 An investigation was made into the effects of drugs which alter insulin secretion on the concentrations of tryptophan and other aromatic amino acids in plasma and brain and on 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in brain. Drugs used were streptozotocin, propranolol, tolbutamide and phentolamine. 2 Tolbutamide and phentolamine increased the plasma insulin concentrations by 100% and 300% respectively but with little effect on the brain/plasma ratios for the aromatic amino acids. Previously propranolol was found to decrease plasma insulin by 50% without altering the above ratios. The ratios were decreased by streptozotocin but only when plasma insulin fell by more than 50%. 3 Phentolamine and propranolol did not alter the brain/plasma ratios for the aromatic amino acids in streptozotocin-treated rats. 4 The results suggest that only large changes of insulin secretion e.g. those associated with food intake or aminophylline injection are likely to alter appreciably the brain/plasma ratios for the aromatic amino acids. 5 Tolbutamide displaced tryptophan from its binding to plasma albumin and increased brain 5-HIAA probably by inhibiting 5-HIAA efflux from brain. The other drugs did not alter brain 5-HT or 5-HIAA concentrations.     

Effect of aminophylline on tryptophan and other aromatic amino acids in plasma, brain and other tissues and on brain 5-hydroxytryptamine metabolism.

1 Aminophylline and other methylxanthines increase brain tryptophan and hence 5-hydroxytryptamine turnover. The mechanism of this effect of aminophylline was investigated. 2 At lower doses (greater than 100 mg/kg i.p.) the brain tryptophan increase could be explained by the lipolytic action of the drug, i.e. increased plasma unesterified fatty acid freeing plasma tryptophan from protein binding so that it became available to the brain. 3 Plasma unesterified fatty acid did not increase when aminophylline (109 mg/kg i.p.) was given to nicotinamide-treated rats but as both plasma total and free tryptophan rose, a tryptophan increase in the brain still occurred. 4 The rise in brain tryptophan concentration following the injection of a higher dose of the drug (150 mg/kg i.p.) could no longer be explained by a rise of plasma free tryptophan as the ratio of brain tryptophan to plasma free tryptophan rose considerably. Plasma total tryptophan fell and the plasma insulin concentration rose. 5 The increase of brain tryptophan concentration after injection of 150 mg/kg aminophylline appeared specific for this amino acid as brain tyrosine and phenyllanine did not increase. However as their plasma concentrations fell the brain/plasma ratio for all three amino acids rose. 6 The higher dose of aminophylline increased the muscle concentration of tryptophan but that of tyrosine fell and that of phenylalanine remained unaltered. The liver concentrations were not affected. 7 The aminophylline-induced increase of the ratio of brain tryptophan of plasma free tryptophan no longer occurred when the drug was given to animals injected with the beta-adrenoreceptor blocking agent propranolol or the diabetogenic agent streptozotocin. 8 The changes in brain tryptophan upon aminophylline injection may be explained by (a) increased availability of plasma tryptophan to the brain due to increased lipolysis and (b) increased effectiveness of uptake of tryptophan by the brain due to increased insulin secretion.     

Effect of isoprenaline infusion on the distribution of tryptophan,tyrosine and isoleucine between brain and other tissues

Intravenous infusion of anaesthetized rats with the β-adreno-receptor agonist isoprenaline decreased plasma total tryptophan concentration and increased both plasma free and brain tryptophan concentrations. Muscle tryptophan and also tyrosine concentrations showed moderate significant decreases, but concentrations in liver and kidney did not alter significantly. Plasma tyrosine concentration fell and brain tyrosine concentration rose, but these changes were less marked than those of tryptophan. Isoprenaline infusion considerably increased egress of ¹⁴C-tryptophan from plasma and moderately increased egress of ¹⁴C-isoleucine, but did not alter egress of ¹⁴C-tyrosine. However, 5 min after pulse injection of any of the above ¹⁴C-labelled amino acids, the isoprenaline-infused rats had higher brain counts than control animals. Results are consistent with previous evidence that increased availability of tryptophan to the brain can occur in stressful situations.     

Increased insulin is not required for beta2-adrenoceptor-induced increases in mouse brain tryptophan

The current study tested the hypothesis that beta(2)-adrenoceptor-mediated increases in brain tryptophan are caused by increased insulin secretion. Male mice were treated with streptozotocin (40 mg/kg) for 5 days to induce experimental diabetes. Control and diabetic mice were treated with the beta(2)-adrenoceptor agonist, clenbuterol (0.1 mg/kg), 1 h before selected brain regions were dissected for analysis by high performance liquid chromatography (HPLC) with electrochemical detection for tryptophan content, and plasma was collected for analysis of total and free tryptophan and glucose concentrations. Clenbuterol increased brain tryptophan and plasma glucose and decreased plasma total tryptophan but did not alter plasma free tryptophan. There were no significant differences in brain or plasma tryptophan between control and streptozotocin-treated mice. In a separate experiment, pretreatment of the mice with an insulin antibody did not prevent the clenbuterol-induced increases in brain tryptophan. These results suggest that beta(2)-adrenoceptor agonists increase brain tryptophan by a mechanism that does not involve changes in insulin.     

Fatty acid and tryptophan changes on disturbing groups of rats and caging them singly

The effects of disturbing groups of 24 hr fasted rats on plasma unesterified fatty acid (UFA) and tryptophan concentrations and brain tryptophan concentrations were investigated. Removing rats from cages rapidly increased plasma UFA and corticosterone and decreased plasma and whole blood tryptophan of cage mates. The disturbance also appeared to influence biochemical values of rats in other cages within the same chamber. Effects specific to individual cages were also suggested. In subsequent experiments 24 fasting rats caged together were rapidly transferred to 24 separate cages and killed at intervals. Plasma UFA rose to a maximum by 12 min and then fell toward initial values. Plasma total tryptophan concurrently fell then rose. Its percentage in the free (ultrafilterable) state, and in some experiments the absolute values of free tryptophan rose then fell. When the latter rise was marked then brain tryptophan and the 5-HT metabolite 5-hydroxyindoleacetic acid rose. Tyrosine changes were negligible. Thus altered brain tryptophan level and 5-HT metabolism may be associated with plasma tryptophan changes caused by brief environmental disturbance.     

The roles of glucagon, insulin and glucocorticoid hormones in the effects of sublethal doses of endotoxin on glucose homeostasis in rats

The effects of sub-lethal doses of endotoxin on plasma glucose, glucagon, insulin, glucocorticoids and non-esterified fatty acids (NEFA) were determined in rats. Endotoxin did not change the plasma concentration of glucocorticoids, but blocked the effects of elevated glucocorticoid hormone concentrations on both plasma glucose and hepatic tryptophan dioxygenase activity. Endotoxin increased the plasma concentrations of glucose, glucagon and insulin in rats with basal glucocorticoid concentrations, and changed the observed relationships between glucose, glucagon and insulin in a manner consistent with an increased sensitivity of glucagon secretion to lowered glucose concentrations. At the highest dose of endotoxin used, 20 mg/kg over 6 hr, a substantial decrease (greater than 7-fold) in the insulin/glucagon ratio provides evidence for changes in basal (as opposed to hormone-stimulated) glucose production and/or utilisation in vivo.     

Tryptophan and tyrosine disposition and brain tryptophan metabolism in acute carbon tetrachloride poisoning.

Forty-eight hours after injecting rats with carbon tetrachloride there were large increases of plasma non-esterifed fatty acid and free tryptophan concentrations. Total plasma tryptophan was not significantly altered. Brain, liver and muscle tryptophan concentrations increased significantly with percentage changes in the order: brain > liver > muscle > kidney. The increased brain tryptophan was associated with a small but significant increase of 5-hydroxytrptamine and a larger increase of its metabolite 5-hydroxyindolylacetic acid. Tyrosine concentrations increased significantly in plasma, brain, liver, muscle and kidney with percentage changes in the same order as those of tryptophan. Food intake was decreased and this correlated significantly with brain tryptophan and 5-hydroxyindolylacetic acid concentrations. Control rats kept for 48 hr on restricted intake did not show the above brain changes though plasma non-esterified fatty acid concentration was increased as in the drug treated rats. Results are discussed in relation to previous findings in acute liver failure and to the possible roles of brain tryptophan and 5-hydroxytryptamine in liver failure and appetite control.     

Rapid effects of environmental disturbance on rat plasma unesterified fatty acid and tryptophan concentrations and their prevention by antilopolytic drugs.

1 Changes of plasma unesterified fatty acid (UFA) and tryptophan concentration in group-housed rats following removal of their cage-mates and the effects of antilipolytic drugs on these changes were investigated. 2 Removal of group-housed 24 h fasted rats but not fed rats from cages resulted in increased plasma UFA concentration in the remaining rats which was associated with significant increases of the proportion of free tryptophan but significant falls of total tryptophan concentration. These rapid changes were not associated with brain tryptophan changes. Plasma tyrosine concentration was unaffected. 3 The fall of plasma tryptophan did not appear to be due to passage into red cells as erythrocyte tryptophan concentration remained unchanged. 4 Plasma UFA concentrations correlated positively and significantly with corticosterone concentrations which were also increased following removal of cage-mates. 5 Plasma UFA increases and tryptophan changes in the fasting rats were both prevented by nicotinic acid or propranolol. Corticosterone concentration was increased by nicotinic acid but unaffected by propranolol. 6 The possible importance of these rapid changes of plasma tryptophan and of their prevention by antilipolytic drugs is discussed.     

Effects on plasma and brain tryptophan in the rat of drugs and hormones that influence the concentration of unesterified fatty acid in the plasma

1 The effects on tryptophan distribution and metabolism of drugs altering plasma unesterified fatty acid (UFA) concentration were investigated in the rat.2 UFA and plasma free (i.e. ultrafilterable) tryptophan altered in the same direction.3 Catecholamines and L-DOPA increased both plasma UFA and free tryptophan. L-DOPA also increased brain tryptophan and 5-hydroxyindoleacetic acid (5-HIAA) but decreased brain 5-hydroxytryptamine (5-HT).4 Aminophylline increased plasma UFA and free tryptophan and also brain tryptophan, 5-HT and 5-HIAA. Food deprivation had qualitatively similar effects.5 Insulin decreased plasma UFA and free tryptophan in both fed and food-deprived rats. However, while in fed rats these changes were associated with small decreases of brain indoles, in food-deprived animals small increases occurred.6 Nicotinic acid had only small effects in fed rats but it opposed both the UFA and indole changes in food-deprived animals. Total plasma tryptophan increased in nicotinic acid treated, food-deprived rats.7 There was a tendency towards inverse relations between changes of plasma free and total tryptophan.8 The results suggest that drugs which influence plasma UFA through actions on cyclic AMP thereby alter the binding of tryptophan to plasma protein and that this leads to altered distribution and metabolism of tryptophan.     

Attenuation of intravenous amphetamine reinforcement by central dopamine blockade in rats

Norepinephrine (NE) and dopamine (DA) receptor blockade differentially affected amphetamine self-administration. DA blockade (pimozide, 0.0625 to 0.5 mg/kg, or (+)-butaclamol, 0.0125 to 0.1 mg/kg) caused periods of increased rate of responding for amphetamine which were followed, in the case of higher doses, by response cessation. The response cessation produced by 0.5 mg/kg pimozide was not reversed by non-contingent amphetamine injections until well after the peak effect of the pimozide was over. When access to amphetamine injections was delayed until 4 h after animals received 0.5 mg/kg pimozide, rate of responding was elevated. Thus DA seems to be critically involved in mediation of the reinforcing effects of amphetamine. Alpha-NE blockade with phentolamine (2.5–10 mg/kg) produced dose-related decreases in responding; blockade with phenoxybenzamine (1.25–10 mg/kg) had no effect. Beta-NE blockade with l-propranolol (2.5–10 mg/kg) decreased responding, although probably not through a beta-blocking action. The effects of phentolamine and propranolol do not appear to result from attenuation of the reinforcing effects of amphetamine.     

Localization of ( 3 H)pimozide in the rat brain in relation to its anti-amphetamine potency

The pharmacological activity of the neuroleptic agent pimozide is well correlated with the concentration of [³H]pimozide in the brain of the Wistar rat. There is an enhanced uptake and retention of [³H]pimozide in the caudate nucleus, an area in which amphetamine and apomorphine act. Its concentration in the caudate nucleus is not correlated with its anti-apomorphine and anti-amphetamine potency. There are, however, significant shifts in the subcellular distribution of [³H]pimozide in the caudate nucleus, from the mitochondrial fraction to the fraction consisting mainly of submicroscopic nerve endings. These shifts correlate well with its pharmacological activity. This points to the small nerve-endings as being the site of action of pimozide. The drug does not influence either the penetration of (+)-[¹⁴C]amphetamine into the brain, or its metabolism, indicating that the anti-amphetamine action cannot be due to a decreased penetration of amphetamine into the brain, although a subcellular redistribution of amphetamine induced by pimozide cannot be excluded.     

Involvement of central dopamine in the hyperthermia in rats produced by d-amphetamine.

The effect of amphetamine on the body temperature was studied in 6-hydroxydopamine and pimozide pretreated rats. Amphetamine alone (1, 2.5 and 5 mg/kg) produced a fairly dose-dependent increase in body temperature. The effect was almost totally antagonized by pimozide, and also reduced after pretreatment with 6-hydroxydopamine. These results are discussed with regard to the possible role of central dopamine in mediating amphetamine-induced hyperthermia in rats.     

Neuropharmacology: Amitriptyline and Clomipramine Increase the Concentration of Administered L-Tryptophan in the Rat Brain

The tricyclic antidepressant amitriptyline has been shown to reduce concentrations of large neutral amino acids (LNAA) in rat plasma. Compounds with that property might interact with such amino acids used as therapeutic agents with a central site of action by causing a change in the relationship between the administered LNAA and its endogenous LNAA competitors for carrier-mediated transport through the blood–brain barrier into the brain. This study was performed to investigate if the antidepressant agents amitriptyline and clomipramine could, by such a mechanism, increase brain concentrations of administered tryptophan. Intraperitoneal administration of L-tryptophan alone (100 mg kg^?1) resulted in an increase in the concentration of tryptophan in the rat brain from 14 ±0.7 to 100 ± 4.3 nmol g^?1 compared with rats given saline only. When rats were given tryptophan with amitriptyline (25 mg kg^?1, i.p.) or clomipramine (25 mg kg^?1, i.p.) brain concentrations of tryptophan were increased even further, to 150 ±4.5 and 157 ± 10.2 nmol g^?1, respectively. Administration of L-tryptophan alone resulted in an increase in the rat plasma tryptophan ratio [(concentration of tryptophan)/(total concentration of LNAAs)] from 0.14±0.003 to 0.42±0.011 compared with rats given saline only. When rats were given tryptophan with amitriptyline or clomipramine the plasma tryptophan ratios were increased even further to 0.52 ±0.017 and 0.54 ±0.025, respectively. All these effects were statistically significant (P < 0.001). These findings support the hypothesis that tricyclic antidepressants could interact with administered tryptophan by changing the relationship in plasma between tryptophan and its endogenous LNAA competitors for transport into the brain, resulting in higher concentrations of tryptophan in the brain. It is possible that this could be the mechanism of the previously reported finding that clomipramine and tryptophan potentiate each other in the treatment of depression.     

Narcotic withdrawal like mouse jumping produced by amphetamine and L-DOPA.

Upward jumping in mice that resembled the narcotic-withdrawal syndrome was produced by a combination of amphetamine (4 mg/kg) and L-DOPA (400 mg/kg) administration. Neither drug alone caused that jumping. Pretreatment with haloperidol (0.16 mg/kg) or pimozide (0.08 mg/kg) but not with phentolamine (10mg/kg) effectively blocked amphetamine-dopa jumping. After DOPA there was an increase in the brain levels of DOPA, dopamine and norepinephrine. Amphetamine, ineffective in itself, enhanced DOPA-induced elevation of brain DOPA and dopamine without affecting brain norepinephrine levels.     

Effects of immobilization on rat liver tryptophan pyrrolase and brain 5-hydroxytryptamine metabolism

1. Rat liver tryptophan pyrrolase increased on immobilization. The concentration of 5-hydroxyindoleacetic acid in the brain also rose and that of 5-hydroxytryptamine fell.2. When adrenalectomized rats were immobilized pyrrolase activity did not rise and brain 5-hydroxytryptamine concentration fell to a lesser extent but the 5-hydroxyindoleacetic acid concentration rose as in intact animals.3. When intact rats were injected with the pyrrolase inhibitor Allopurinol both the increase of pyrrolase and the fall of 5-hydroxytryptamine on immobilization were less prominent but the concentration of 5-hydroxyindoleacetic acid rose as before. Allopurinol did not affect the changes in immobilized adrenalectomized rats.4. Immobilization thus appears to cause (a) decreased brain 5-hydroxytryptamine synthesis resulting from pyrrolase induction and (b) increased 5-hydroxytryptamine breakdown by a more direct effect on the brain. Results of experiments on rats injected with lysergic acid diethylamide, and with alpha-methyltryptophan or probenecid are consistent with the above interpretation.5. The 5-hydroxytryptamine and 5-hydroxyindoleacetic acid changes were maximal after 5-6 hours' immobilization and became less on more prolonged immobilization, which suggests regulatory changes.     

Effects of hyper-and hypothyroidism on central nervous system with special reference to its effects on rectal temperature and brain norepinephrine levels in rats]

Infleunce of hyper-and hypothyroidism on amphetamine activity was observed by measuring the effects on hyperthermia and brain amphetamine and norepinephrine levels. Hyperthyroidism was obtained in rats injected with tri-iodothyronine 0.2 mg/kg i.p. every day for 5 days. Controls were treated with the vehicle 1.0 ml/kg i.p. for the same period. On the 6th day, d-amphetamine (10 mg/kg i.p.) was administered to the two groups of animals and the body temperature and brain amphetamine and norepinephrine contents were measured at 0 min, 15 min, 30 min and 60 min. Hyperthyroid rats showed a more marked hyperthermia than did the control animals. On the other hand "amphetamine-induced release of norepinephrine" of hyperthyroid rats was not so marked as in the control rats, however amphetamine levels did not differ in the two groups. Hypothyroidism was evident in the thyroidectomized rats. Controls for this group underwent a sham-operation. All animals were injected with amphetamine 21 days later. Amphetamine did not cause a hyperthermia in thyroidectomized rats. On the other hand, amphetamine levels were considerably higher than in the control rats, but the degree of norepinephrine release was comparable in the two groups.     

Hyperthermia and brain neurotransmitter amino acid levels in infant rats

1. The acute hyperthermia induced by exposure to elevated ambient temperatures (40 degrees C) during 90 min produced dramatic changes in certain brain transmitter amino acid levels in infant rats. 2. All inhibitor transmitter amino acids except taurine, rose significantly in 7 and 14 day-old rats. The effect of acute hyperthermia in excitatory transmitter amino acids was opposite, glutamic acid increased and aspartic acid decreased. 3. Taurine, that does not change during acute hyperthermia is the amino acid whose concentration suffers the greatest change with age. 4. The greater rise of body temperature in 21 day-old rats, was associated to slight changes in brain transmitter amino acid levels. These findings suggested that lower rise on body temperature found in 7 and 14 day-old rats may be related to the higher increase of inhibitor transmitter amino acids.     

Effects of nimodipine on the amphetamine- and methamphetamine-induced decrease in tryptophan hydroxylase activity

The effects of nimodipine on the amphetamine- and methamphetamine-induced decrease in central tryptophan hydroxylase activity was examined. Rats were administered 4 or 5 injections of amphetamine or methamphetamine at 6-h intervals with or without nimodipine (1 mg/kg), and killed 1 or 18 h after the last drug administration. The decrease in hippocampal tryptophan hydroxylase activity induced by amphetamine and methamphetamine was potentiated by the administration of nimodipine. Moreover, while hippocampal tryptophan hydroxylase activity was not altered by 2.5 mg/kg methamphetamine alone, the coadministration of 1 mg/kg nimodipine decreased the enzymatic activity to 68% of control. The decrease in striatal tryptophan hydroxylase activity caused by these amphetamine analogues was not significantly altered by the coadministration of nimodipine. Interestingly, nimodipine increased hippocampal and striatal amphetamine concentrations to 187 and 162%, respectively, of the concentrations measured in animals treated with amphetamine alone. Nimodipine also increased by 2-fold the plasma concentration of methamphetamine and amphetamine measured 3 h after a single administration of methamphetamine, whereas the hippocampal concentrations of these compounds were raised to 354 and 516%, respectively, of that in animals treated with methamphetamine alone. These results suggest that nimodipine altered drug distribution and potentiated the methamphetamine-induced decrease in hippocampal tryptophan hydroxylase activity by increasing the cerebral methamphetamine concentration.     

Adrenergic receptors and the onset of streptozotocin-induced diabetes in mice

Treatment with yohimbine, an alpha 2-adrenergic blocker, prior to the injection of a subdiabetogenic dose of streptozotocin (STZ) produced hyperglycemia and hypoinsulinemia in mice 7 days later. Prazosin, an alpha 1-adrenergic blocker, was ineffective. The capacity of phentolamine and phenoxybenzamine to potentiate the diabetogenic effect of STZ was intermediate between that of yohimbine and prazosin. Propranolol and hexamethonium inhibited the potentiating action of yohimbine. Yohimbine enhanced the potentiating effect of isoproterenol on the STZ-induced diabetes. Acute changes in phase glucose and insulin levels induced by STZ were potentiated by yohimbine but not by prazosin. The insulin releasing ability of the pancreatic islets 7 days after STZ was all but lost in mice pretreated with yohimbine but not with prazosin. These results suggest that the beta- and alpha 2-, not alpha 1-adrenergic system which modulates insulin release from pancreatic islets influences the response to the diabetogenic action of STZ in mice.     

PERMEABILITY OF THE DEVELOPING AND MATURE BLOOD-BRAIN BARRIERS TO THEOPHYLLINE IN RATS

1. In the present study, the uptake of theophylline and L-glucose into the adult and neonatal rat brain has been investigated. Steady state cerebrospinal fluid (CSF) and brain concentrations of theophylline were reached within 1 h following a single intraperitoneal (i.p.) injection, whereas steady state CSF and brain concentrations of L-glucose were not approached until after 5 h. 2. Steady state brain:plasma and CSF:plasma concentration ratios for theophylline and L-glucose in neonatal rats were significantly higher than ratios in adult rats. Erythrocyte:plasma ratios for theophylline in neonatal rats were also significantly higher than ratios in adult rats. Steady state ratios for theophylline were significantly higher than those for L-glucose in both neonatal and adult rats. 3. Respiratory acidosis (pH 6.9–7.0) did not affect steady state CSF:plasma or brain.-plasma ratios for theophylline in neonatal or adult rats. In contrast, steady state CSF:plasma and brain:plasma ratios for L-glucose were increased by respiratory acidosis. 4. The lower steady state CSF:plasma, brain:plasma and erythrocyte:plasma ratios for theophylline in adult rats are likely to be due to a higher concentration of plasma proteins in adult blood compared with neonates, with a greater retention of protein-bound (non-exchangeable) theophylline in adult blood, and are unlikely to be due to p-glycoprotein-mediated efflux of theophylline at the adult blood-brain barrier.