Bahjat A. Faraj

Expression of a high-affinity serotonin transporter in human lymphocytes

This study was undertaken to assess the capability of lymphocytes to actively transport serotonin (5-HT). The data we obtained showed that lymphocytes isolated from the blood of normal human subjects contained a high-affinity uptake system for [3H]5-HT. Kinetic analysis of the uptake data as computed by regression analysis from Lineweaver--Burk plots, yielded a Km of 180 +/- 20 nM and Vmax of 94 +/- pmole/10(7) cells. The uptake of [3H]5-HT was temperature, sodium and chloride dependent and was potently inhibited by the antidepressants clomipramine, imipramine, fluoxetine and fluvoxamine, which are specific for the 5-HT transporter. Compounds that are more selective for norepinephrine and dopamine transporters such as mazindol, desipramine, and GBR 19209 had a lower inhibitory effect on the uptake of [3H]5-HT in human lymphocytes. The expression of a 5-HT transporter in human lymphocytes that resembles 5-HT uptake by platelets and brain synaptosomes may provide insights into the potential role of 5-HT in immune function and its relationship to the neurobiology of affective and addictive disorders.

Hypertyraminemia in Cirrhotic Patients

To evaluate the role of tyramine in hepatic disorders, we used a radioimmunoassay to study plasma concentration of tyramine in eight healthy subjects, 20 hospitalized patients without liver disease, and 13 cirrhotic patients of whom seven had hepatic encephalopathy. The effect of increasing dietary protein on tyramine level of cirrhotic patients was also assessed. No significant difference in plasma tyramine concentration was seen between normal subjects, 1.3 +/- 0.1 ng per milliliter (average +/- S.E.), hospitalized patients without hepatic disease (1.4 +/- 0.1 ng per milliliter) and cirrhotic patients without encephalopathy (2.7 +/- 0.5 ng per milliliter). However, the tyramine level in cirrhotic patients with encephalopathy, 6.4 +/- 0.1 ng per milliliter, was significantly (P less than 0.001) higher than in normal subjects or in cirrhotic patients without encephalopathy. Increasing dietary protein from 40 to 80 g per day raised fasting tyramine concentration by 30 to 70 per cent within three days in both encephalopathic and non-encephalopathic cirrhotic patients. Concentration of plasma tyramine in cirrhotic subjects was significantly correlated with that of plasma tyrosine (P less than 0.001).

Active [3H]-Dopamine Uptake by Human Lymphocytes: Correlates with Serotonin Transporter Activity

The main objective of the present investigation was to determine whether the uptake of [3H]-dopamine in human lymphocytes is mediated through a serotonin transporter. This was examined by studying the effects of various monoamine uptake inhibitors on the uptake of [3H]-dopamine in human lymphocytes. Among the compounds tested, indatraline, imipramine and fluoxetine, selective inhibitors of neuronal serotonin transporter, were the most potent inhibitors of [3H]-dopamine uptake in lymphocytes. The 50% inhibiting concentration (IC50) for these inhibitors was in the range of 3.5-17 nmol/l. Bupropion, GBR 12909, nomifensine and xylamine, selective inhibitors of dopamine and norepinephrine transporters, had low affinity for the dopamine uptake system in human lymphocytes with IC50 values ranging between 1,000 and 40,000 nmol/l. These findings provide supportive evidence for the participation of a serotonin transporter in the uptake of [3H]-dopamine in human lymphocytes. The existence of a high affinity transport system for dopamine and serotonin in human lymphocytes may serve as a readily accessible model to detect changes in the neuronal uptake of dopamine and serotonin in addictive and psychiatric disorders.

Binding of [3H]-dopamine to human lymphocytes: possible relationship to neurotransmitter uptake sites.

Freshly isolated human lymphocytes from 11 healthy subjects had specific binding sites for dopamine which were dependent on time, temperature and sodium, and appeared to follow Michaelis-Menten kinetics. The apparent affinity constant (KD) of human lymphocytes for dopamine and the maximal number of binding sites (Bmax) were 109 +/- 21 nM and 2.66 +/- 1.75 pmol/10(7) cells, respectively. Dopamine binding was markedly affected by cocaine (IC50 = 150 nM) and other inhibitors of biogenic amine uptake. The relatively high potency of cocaine in competing for dopamine binding suggested that human lymphocytes may serve as a readily accessible model to detect changes in the neuronal uptake of dopamine and perhaps other monoamine neurotransmitters.

Prevalence of high serotonin uptake in lymphocytes of abstinent alcoholics.

An impairment in serotonergic neurotransmission may be associated with alcoholism. We recently identified a high-affinity serotonin transporter (5-HTT) in human peripheral blood lymphocytes (PBLs). Moreover, molecular analysis of RNA samples of human lymphocytes using reverse transcription, coupled with polymerase chain reaction, enabled us to confirm the expression of a 5-HTT identical to the one reported in neuronal tissues, as evidenced by hybridization and sequence analysis. In this investigation, we measured the serotonin (5-HT) uptake in PBLs of recovering alcoholics (N = 10) with long-term abstinence (2-10 years) and non-alcoholic controls (N = 10). 5-HT uptake was measured by incubating 1 x 10(7) cells of PBLs with [3H]5-HT (3-1000 nM; sp. act. 23 Ci/mmol) for 10 min at 37 degrees. The results of this preliminary study revealed that abstinent alcoholics had significantly (P < 0.01) increased uptake of 5-HT (43.6 +/- 5.70 pmol/10(7) cells) as compared with controls (23.33 +/- 2.50 pmol/10(7) cells). An enhanced uptake of 5-HT in PBLs of abstinent alcoholics agrees with previously reported observations of increased 5-HT uptake in brain and platelets of former alcoholics and their descendants. This suggested that a serotonergic mechanism may be linked to the heredity of alcoholism.

A cocaine-sensitive active dopamine transport in human lymphocytes☆

Human lymphocytes possess a cocaine-sensitive high-affinity transport system for [3H]dopamine. [3H]Dopamine uptake was saturated with increasing dopamine concentrations and followed Michaelis-Menten kinetics. The uptake was temperature, sodium, and chloride dependent and was affected by the co-addition of ouabain, phloridzin, potassium cyanide, gramicidin, and other metabolic inhibitors. The uptake of dopamine was blocked significantly in a concentration-dependent manner by cocaine and its congeners. Furthermore, preliminary evidence is presented linking the possible relationship between decreased lymphocyte [3H]dopamine uptake and chronic cocaine abuse in humans.

Effect of astemizole on antigen‐mediated histamine release from the blood of patients with allergic rhinitis

The main objective of this study was to test the effectiveness of astemizole in vitro in blocking the release of histamine from blood of patients with allergic rhinitis. The results of this investigation indicated that astemizole inhibited allergen‐mediated histamine release from blood basophils of patients with this allergic disorder. The inhibition by astemizole (33–156 μmol) was immediate, requiring no pre‐incubation of the cells, and was dose‐dependent, with maximal inhibition of about 91%. The relatively high potency of astemizole in inhibiting the immunologic release of histamine may provide an additional measure in the treatment of allergic rhinitis with this H i‐receptor antagonist.

Tyrosine transaminase activity in normal and cirrhotic liver

Most cirrhotics have tyrosinemia and subnormal tyrosine tolerance; in some the ability to metabolizep-hydroxyphenylpyruvic and homogentisic acids is impaired. In previous studies, the initial transamination appeared to be the rate-limiting step. In this study, hepatic tyrosine transaminase activity was compared in liver biopsies from eight noncirrhotic and ten cirrhotic subjects to determine whether the subnormal tyrosine tolerance was related to decreased maximal activity of this enzyme. Fasting plasma tyrosine in the cirrhotics (133±43 μmol/liter) was significantly higher (P<0.005) than in the noncirrhotic subjects (64±25 μmol/liter). Tyrosine transaminase activity in the cirrhotic livers (42±11 μmol PHPA/g liver/hr, or 0.47±0.1 μmol PHPA/mg protein/hr) was not significantly different from the enzyme activity in the noncirrhotic liver (43±7 μmol PHPA/g liver/hr, or 0.39±.12 μmol PHPA/mg protein/hr.) Thus elevated tyrosine levels in cirrhotics cannot be explained by decreased tyrosine transaminase activity in the liver, and other explanations must be sought.

Determination of Plasma and Tissue Levels of Tyramine by Radioimmunoassay

Summary Antibodies were prepared against tyramine. The antigen was prepared as follows: p-Aminohippuric acid was coupled to mBSA using a carbodiimide reagent. The amino group was diazotized and attached to the aromatic ring of TYR. The immunogen in Freunds complete adjuvant was injected into rabbits. The specificity of the resulting antibody was determined by radioimmunoassay. Using random-labeled TYR-3H, TYR, its metabolites, phenethyla-mine analogs, catecholamines, and certain amino acids were evaluated by a competitive binding assay method. With this technique 4 ng of TYR inhibited the binding of TYR-3H by 50%. The radioimmunoassay of TYR was used to measure the plasma, urine, and tissue levels of TYR in rabbits. The plasma disappearance curve of TYR revealed a biphasic pattern with t 1/2 of 2 min and 54 min. The highest concentration of TYR was found in adrenals and spleen. The factthat the major metabolites of TYR and a series of pharmacologically important sym-pathomimetics and catecholamines did not interfere, makes the radioimmunoassay of TYR a useful, simple, sensitive, and specific method for the direct analysis of TYR in biological materials.

Elevated concentrations of dopamine sulfate in plasma of cocaine abusers

This study investigated the effect of cocaine abuse on peripheral catecholamines. Specifically, we measured the concentration of free dopamine, dopamine sulfate, free norepinephrine, norepinephrine sulfate, free epinephrine and epinephrine sulfate in plasma samples obtained from the blood of a group of patients with cocaine addiction (N = 15). The concentrations of free and sulfoconjugated catecholamines in plasma were measured by a radioenzymatic technique. The results of this study revealed significant (P < 0.0001) elevation in plasma dopamine sulfate (8926 +/- 1204 pg/mL) of cocaine addicts upon admission to an in-patient treatment facility when compared with the level of this dopamine metabolite in plasma of control subjects (2356 +/- 121 pg/mL). Furthermore, there was a significant (P < 0.0001) relationship between elevation in plasma dopamine sulfate levels and severity of cocaine use among these patients, and in the majority of cases the plasma levels of dopamine sulfate declined appreciably in time with abstinence from cocaine. In contrast, no appreciable difference was observed in the concentrations of either free or sulfate-conjugated norepinephrine and epinephrine in plasma of cocaine addicts as compared with controls. Differences in plasma dopamine sulfate among these patients versus controls may be interpreted as a reflection of activation of extracellular dopamine metabolism associated with chronic cocaine exposure in humans.