Jonathan L. Costa

Alpha-adrenergic receptors on human platelets.

Abstract [3H] dihydroergocyrptine, an α-adrenergic antagonist, binds specifically to sites on human platelet membranes. Prostaglandin E1 (PGE1) stimulates the production of cyclic AMP (cAMP) in human platelets. Alpha-adrenergic agonists, 1-epinephrine and 1-norepinephrine, and antagonists, phentolamine, phenoxybenzamine, and dihydroergocyrptine inhibit the binding of [3H] dihydroergocryptine. The α-adrenergic agonists inhibit PGE1-stimulated cAMP production and the α-adrenergic antagonists phentolamine and dihydroergocryptine reverse this inhibition. The β-adrenergic agonist 1-isoproterenol and the β-adrenergic antagonists d1-propranolol and 1-alprenolol do not significantly alter binding or PGE1-stimulated cAMP production. Clonidine, dopamine, and serotonin inhibit binding, but clonidine and dopamine are weak inhibitors of PGE1-stimulated cAMP production, and serotonin is without effect. Tyramine, an amine without direct adrenergic activity fails to inhibit binding. Alpha-adrenergic agonists decrease the apparent affinity of a PGE1-receptor activating cAMP production. The inhibition of PGE1-stimulated cAMP production is a physiological measure of α-adrenergic agonist binding to the α-receptor.

The brain serotonergic system in the affective disorders

Abstract 1. 1. An alteration in the brain serotonergic system in individuals with depression, and possibly mania, has been proposed on the basis that these patients have reduced 5-hydroxyindoleacetic acid levels in cerebrospinal fluid. 2. 2. Reduced brain serotonin concentrations have also been observed in some studies of suicides. 3. 3. Other investigations of serotonin-related functions in depressed patients have yielded equivocal results, although potentiation of the therapeutic effects of antidepressant drugs by L-tryptophan, and their reversal by p-chlorophenylalanine, are in agreement with animal pharmacological studies indicating that antidepressant drugs act in part via serotonergic mechanisms. 4. 4. The biochemical basis for the suggested serotonergic system alterations in the affective disorders remains unknown. 5. 5. Additional basic information is needed on (a) the storage, transport and synthesis of indoleamines, (b) adaptational mechanisms within the system and their relationships to other neurotransmitter systems, and (c) correlations with behavior in animals and man. 6. 6. Such studies may further our understanding of the role which the serotonergic system alterations may play in the affective disorders.

Monoamine oxidase activity in different density gradient fractions of human platelets

Monoamine oxidase (MAO) activity measured in human platelets is reportedly altered by such drugs as epinephrine, lithium carbonate, and imipramine, and also reduced in a number of clinical disorders. To evaluate whether MAO activity might differ in platelet supopulations, density gradient centrifugation with arabino-galactan was used to prepare four platelet fractions that differed in weight and volume. MAO activity in the lightest and smallest platelet subpopulation was approximately one-half that in the heaviest and largest subpopulation. Because platelet weight and size are thought to be related to platelet age, it is possible that some drug effects on platelet MAO activity might represent changes in platelet turnover. Factors other than platelet turnover rates may contribute to individual differences in platelet MAO activity, however, since one group of individuals with markedly reduced platelet MAO activity exhibited no shift in the proportion of lighter versus heavier platelets nor in the relative amount of MAO activity in each density gradient subfraction.

Demonstration and evaluation of apparent cytoplasmic and vesicular serotonin compartments in human platelets

Abstract Thrombin has been used in conjunction with formaldehyde treatment to investigate the distribution of labeled serotonin between releasable and non-releasable compartments during the serotonin uptake process. The data indicate that serotonin accumulates in a predictable fashion in both compartments, which may represent, respectively, vesicular and cytoplasmic amine pools. The results also suggest that the cytoplasmic pool may reach sizable proportions under certain incubation conditions.

Serotonin Storage in Platelets: Estimation of Storage-Packet Size

Storage-body diameter and volume, and the number of molecules of serotonin contained in a storage body, were estimated for blood platelets. In the human, 5.23 x 105 molecules of serotonin are contained in a storage body 198 nanometers in diameter, while in the cat, 31.2 x 105 molecules of this amine are contained in a storage body 298 nanometers in diameter.

Extended X-ray absorption fine structure (EXAFS) studies on calcium in crystalline and amorphous solids of biological interest

Abstract Using synchroton radiation, the calcium K-edge extended x-ray absorption fine structure (EXAFS) for a number of calcium-containing solids of biological interest was measured. These included synthetic analogues of platelet dense bodies, rat tibia mineral, and synthetic bone-like calcium phosphates. The mean CaO distances in all of the solids examined were essentially the same. However, in contrast to the crystalline calcium solids studied, the dense body analogues showed sufficient structural disorder that CaP and CaCa atom pair contributions to the EXAFS spectra could not be observed. The absence of these second-shell contributions suggest that these analogues are truly amorphous rather than microcrystalline in structure. The EXAFS spectrum of the bone mineral was more similar to carbonate-apatite than to any of the other synthetic apatites studied.

Direct observation of 6-fluorodopamine in guinea pig nerve microsacs by 19F NMR.

19F nuclear magnetic resonance (NMR) has been used to examine the disposition of ring-fluorinated dopamine and norepinephrine in microsacs prepared from striata of guinea pig brains. Following incubation with a 10(-4)M initial concentration of 6-fluorodopamine (6F-DA), intact micmicrosacs at 4 degrees C gave a 19F NMR spectrum in which the 6F-DA present was sufficiently mobile to be visible. Intra-vesicular 6F-DA in striatal nerve terminals thus appears to exist in an environment resembling that in chromaffin vesicles but different from that prevailing inside the amine storage vesicles of platelets. Our data also suggest that the study of fluorinated compounds by 19F NMR can be used to expand our understanding of processes related to amine uptake, metabolism, and storage in nerves.

Purified -SH-activated toxins (streptolysin O, alveolysin): New tools for determination of platelet enzyme activities

The purpose of this study was to examine the lytic effect on human platelet preparations (washed, gel-filtered and dextran-isolated) of two sulfhydryl-activated bacterial protein toxins, streptolysin 0 and and alveolysin, and to compare their efficacy with that of other disruptive procedures (freezing and thawing, ultrasonic, mechanical, or nystatin-toluene treatment) as a method for the determination of various platelet enzyme activities. The enzymes assayed were alkaline and acid phosphatases, monoamine oxidase, phenolsulfotransferase, N-acetyltransferase, hydroxyindole-O-methyltransferase, glutathione peroxidase and lactate dehydrogenase. In all cases, the lowest activities were found after freezing and thawing and/or ultrasonic disruption. The highest activities were always observed in the platelet lysates obtained after toxin, and in some instances after nystatin-toluene treatment. Intermediate values were obtained for mechanical disruption. The -SH-activated cytolysins thus appear to be appropriate and gentle tools for the assay of platelet enzymes when compared to the physical or chemical procedures generally employed.

Limited dermal ossification: Clinical features and natural history

For 9 years we have observed a girl who has ossification in the dermis with a strikingly limited distribution. Recently a second girl with similar dermal ossification restricted to a single extremity was identified. The ectopic bone is histologically identical to normal membranous bone. These two patients have no obvious underlying cause for soft tissue bone formation, and no disorder of calcium or phosphate metabolism. Ossification first involved the dermal and subcutaneous connective tissue, and with time advanced locally in the affected areas to bridge joints and limit mobility. The ossification has now extended to involve muscle fascia but has not involved the muscle itself. This disease appears to represent a heretofore unrecognized disorder of mesenchymal differentiation.

Effects of reserpine and emipramine on vesicular serotonin uptake and storage in intact human platelets

Abstract The effects of reserpine and imipramine on intact human platelets have been investigated, utilizing brief thrombin treatment to evaluate serotonin (5HT) uptake into and loss from the vesicular (thrombin-releasable) compartment. Less than five seconds after its addition, reserpine (10 −6 M) almost completely inhibited the uptake of 5HT into storage vesicles; but induced an outward flux of stored 5HT from vesicles only after more than two minutes following its addition. Imipramine (10 −6 M), acting over a 30-minute period, caused no loss of vesicular 5HT, but acted within five minutes to inhibit markedly the movement of cytoplasmic 5HT into storage vesicles. It thus seems likely that in human platelets, inhibition of vesicular 5HT uptake does not necessarily lead to the loss of vesicular 5HT.