Stereospecific (--)-[3H]norepinephrine binding to bovine hypothalamus. Possible identification of the catecholamine uptake site in synaptic vesicles.

A (--)-[3H]norepinephrine binding site was identified in a crude synaptosomal fraction isolated from bovine hypothalamus which bound norepinephrine rapidly, reversibly, and stereospecificially. The results were most consistent with binding of (-)-[H]norepinephrine to the carrier molecule used to translocate biogenic amines into synaptic vesicles. The binding studies indicated that specific binding of (--)-[3H]norepinephrine to the crude synaptosomal fraction was greatly enhanced by 4 mM MgCl2 pand 1 mM ATP. The increased binding of (--)-[3H5norepinephrine also occurred in the presence of MgCl2 and GTP, but AMP, adenosine and adenyl-5'-yl imidodiphosphate would not substitute for ATP. Neither CaCl2 nor ZnSO4 could be substituted for the MgCl2. In the presence of MgCl2 and ATP, the dissociation constant for (--)-[3H]norepinephrine was 280 nM with a specific binding site density of 4.8 pmol/mg protein. Binding was stereospecific with ratios of 15, 4, and 6.5 for the affinities of (--)-isomers to (+)-isomers for norepinephrine, epinephrine and isoproterenol, respectively. Drug competition studies, conducted in the presence of Mg2+ and ATP, indicated that (--)-epinephrine, (--)-norepinephrine, dopamine and serotonin had inhibitory constants ranging from 0.25 to 0.8 micron with (--)-isoproterenol and tyramine having inhibitory constants around 2 micron. Reserpine was the most potent inhibitor having an inhibition constant of 8.6 +/- 0.3 nM. The binding data were not consistent with the specific site being the alpha- or beta-receptors for norepinephrine, the Uptake1 Site for norepinephrine into synaptosomes or the metabolizing enzymes for norepinephrine.

Catecholamine content of chromaffin granule "ghosts' isolated from bovine adrenal glands.

Studies on the mechanism of catecholamine transport into chromaffin granules is complicated by the release of endogenous catecholamines. To overcome this problem chromaffin granule ghosts have been prepared by many investigators by osmotic lysis of the granules which results in a loss of over 90% of the endogenous catecholamine. However, in the studies reported here, the resulting ghosts still contained 36 +/- 3.9 nmol epinephrine/mg of protein if they were lysed by passage through a Sephadex G-50 column preequilibrated with hypoosmotic media. This residual catecholamine was found to slowly diffuse out of the ghosts in a temperature-dependent process at a rate sufficient to interfere with kinetic analysis of catecholamine transport. Attempts to remove the endogenous catecholamine from the ghosts indicated that most of it could not be removed by further osmotic shock of freeze-thaw treatments, but that over 85% of it was released from the granules by incubating them at 30 degree C for 90 min or by dialysis with a 35 and 86% loss of rate of catecholamine transport into the ghosts, respectively. If the endogenous catecholamine was removed from chromaffin granule ghosts by preincubating them for 90 min at 30 degree C, the resulting ghosts transported catecholamine with a linear Lineweaver-Burk plot indicating a Km of 12 +/- 2 microM. In addition, the resulting ghosts did not leak catecholamines over a 10 min period at 30 degree C, and the transport of catecholamines was blocked by reserpine and enhanced with increasing pH from 6.0 to 8.5.

Interaction of neuropeptides and biogenic amines on cyclic adenosine monophosphate accumulation in hypothalamic nuclei.

Neuropeptides and biogenic amines known to be present in neurons or afferent terminals in the paraventricular nucleus (PVH), supraoptic nucleus (SON) and/or lateral hypothalamus (LH) were added to small areas of these structures obtained by micropuncture and cyclic adenosine monophosphate (cAMP) levels were measured. cAMP accumulation occurred in PVH, SON and LH in response to neuropeptides of the secretin family, such as vasoactive intestinal peptide (VIP) and in response to catecholamines. Bradykinin, alpha-melanocyte-stimulating (alpha-MSH), luteinizing hormone-releasing hormone (LH-RH), oxytocin and carbamylcholine stimulated cAMP accumulation selectively in one or two of the above structures. Glucagon, cholecystokinin (CCK), somatostatin (SRIF), corticotropin-releasing factor (CRF), thyrotropin-releasing hormone (TRH), adrenocorticotropin (ACTH), melanocyte-stimulating hormone (MSH), methionine enkephalin (Met-Enk), beta-endorphin, neurotensin, bombesin and angiotensin II did not effect cAMP levels while leucine enkephalin (Leu-Enk), arginine vasopressin and gamma-aminobutyric acid (GABA) elicited regionally selective decreases in basal levels of cAMP. When interactions between some of these compounds were measured, VIP and norepinephrine exerted a more than additive effect on cAMP elevation in the PVH, while the effect on cAMP of the SON and LH was additive.

Binding sites for 125I-neuropeptide Y (NPY) on membranes from bovine adrenal medulla.

Bovine adrenal medulla membranes were examined for the presence of specific 125I-neuropeptide Y (125I-NPY) binding sites using rapid centrifugation to measure the amount of bound ligand. Specific binding was determined from the difference between 125I-NPY bound in the presence and absence of 10(-7) M unlabeled NPY. The binding was saturable and reached equilibrium within 5 min at 0 degrees C. Analysis of specific 125I-NPY binding using the LIGAND computer program indicated a best fit for a two site model with a Kd of 0.26 nM and a Bmax of 12 fmol/mg protein for the high affinity site and a Kd of 170 nM and a Bmax of 6 pmol/mg protein for the low affinity site. The rate of dissociation (k-1) was 0.071/min with a t1/2 of 9 min. Displacement curves for avian or human pancreatic polypeptide revealed that these peptides displaced 125I-NPY from both sites with IC50 values greater than 10 nM.

Dopamine receptors: effects of chronic L-dopa and bromocriptine treatment in an animal model of Parkinson's disease.

Male albino rats received a stereotaxic injection of 6-hydroxydopamine (6-OHDA) into the right substantia nigra. Animals demonstrating contralateral rotations 2 weeks postoperatively with apomorphine (0.5 mg/kg i.p.) were treated with L-Dopa (55 mg/kg i.p.), bromocriptine (2 mg/kg i.p.), or polyethylene glycol (vehicle) every 12 h for 30 days. Striatal dopamine (DA) receptors were analyzed by Scatchard plot using 3H-spiroperidol (3H-SP) as ligand. 3,4-Dihydroxyphenylacetic acid (DOPAC) and DA were measured by use of high pressure liquid chromatography. 6-OHDA lesions produced a supersensitivity in striatal DA receptors. Chronic L-Dopa or bromocriptine treatment reversed this supersensitivity. Neither lesion nor drug treatment alone or together produced a significant change in affinity (KD) for 3H-SP. Drug treatment alone also had no effect on Bmax. DA and DOPAC levels were reduced by greater than 98% in lesioned striata. Neither drug treatment affected DA or DOPAC levels as compared with controls. These results indicate that chronic administration of either bromocriptine or L-Dopa will reverse the DA receptor denervation supersensitivity in striatum seen following 6-OHDA lesion. This reversal may play a role in the clinical changes seen in Parkinson's disease patients following chronic use of these drugs.

Possible involvement of alpha-2A adrenergic receptors in attention deficit hyperactivity disorder: radioligand binding and polymorphism studies.

Neuropharmacological and genetic association studies have implicated norepinephrine and adrenergic receptors in the pathogenesis of ADHD. The purpose of this study was to compare genetic association studies of three polymorphisms of the alpha-2A adrenergic receptor gene (ADRA2A) with radioligand binding studies of the alpha-2A adrenergic receptor protein in platelets from a sample of children without or with ADHD. The pediatric subjects ranged from 6 to 18 years of age. A thorough clinical assessment of each child resulted in one of the following DSM-IV ADHD diagnoses: inattentive, hyperactive/impulsive, combined, or no ADHD. No significant linkage was found between the ADRA2A polymorphisms (MspI, HhaI, and DraI) and any of the phenotypes tested. Association analysis, however, did detect significant linkage disequilibrium for the DraI polymorphism. Association was also evaluated considering the three ADRA2A single nucleotide polymorphisms as haplotypes. The HhaI-DraI and the MspI-HhaI-DraI haplotypes were significantly associated with ADHD. The platelet alpha-2 adrenergic receptor density did not differ between children without or with ADHD. The affinity of the receptor for the radioligand however, differed significantly between those without and with ADHD. In addition, there were some significant correlations between binding parameters and severity of ADHD in this well-characterized clinical population, and significant association was found between these measures of receptor function and MspI and DraI polymorphisms. Thus, both the genetic and the binding studies indicate that the alpha-2 adrenergic receptor may play a role in ADHD.

The role or non-role of ATPase activation by phenytoin in the stabilization of excitable membranes.

The role or non-role of NaK ATPase, Mg ATPase, and CaMg ATPase involvement in stabilization of excitable membranes by phenytoin is critically evaluated. There is no substantial evidence to indicate that the membrane-stabilizing effect of phenytoin is due to activation of the NaK ATPase. Previous reports of activation of the NaK ATPase at low potassium and high sodium are probably not due to phenytoin but to a potassium contamination in the phenytoin solution. In vitro experiments do not provide any clear evidence of any alterations of NaK ATPase properties by phenytoin. However, one cannot rule out the possibility that phenytoin alters the efficiency of the sodium-potassium pump. Likewise, the Ca ATPase is not inhibited by phenytoin. However, there is some evidence that the Mg ATPase in synaptic vesicles is substantially inhibited by phenytoin. There is substantial evidence indicating that phenytoin partially blocks passive diffusion of sodium into stimulated nerves. The mechanism by which phenytoin blocks sodium influx and the relationship of this effect to the drug's anticonvulsant action remain to be determined.

Interference of biogenic amines with the measurement of proteins using bicinchoninic acid.

The use of bicinchoninic acid (BCA) to measure protein concentrations has received wide acceptance because the reagent is insensitive to many of the buffers, sucrose solutions and detergents used with various tissue and enzyme preparations. However, any compound capable of reducing Cu2+ in an alkaline medium such as biogenic amines will produce a color reaction. The primary objective of this study was to determine whether biogenic amines present in neuronal tissue would interfere with the measurement of protein using the BCA method. Catecholamines were found to produce a linear increase in color of the BCA reagent at concentrations between 1 and 100 nmol/2.1 ml assay volume. Catecholamines appeared to be more sensitive to the BCA reagent than either serotonin or ascorbic acid. Catecholamines at concentrations of 50 nmol/mg of protein or 1 nmol/2.1 ml assay volume or higher will produce significantly (P less than 0.0001) higher color reactions than protein alone. The BCA reagent is not ideal for measuring protein concentrations of intact synaptic vesicles and chromaffin granules since the catecholamine concentrations in these organelles are high enough to increase the color developed by 1.1 to 2.5 times that observed with protein alone. The linearity of the color development produced by catecholamines suggest that BCA could be used to quantitate catecholamine concentrations between 1 and 100 nmol. The BCA reagent will not distinguish between the different catecholamines.

Effects of the sulfhydryl reagent N-ethylmaleimide on reserpine binding to the catecholamine transporter in chromaffin granule membranes.

1. Catecholamines are transported into chromaffin granules via a carrier-mediated, active-transport process which is inhibited by micromolar concentrations of the sulfhydryl reagent, N-ethylmaleimide (NEM). Reserpine is a very potent, competitive inhibitor of the catecholamine transporter and can be used to investigate the characteristics of the catecholamine transporter. 2. The purpose of this study was to determine whether [3H]reserpine binding to the catecholamine transporter present in chromaffin granule membranes isolated from bovine adrenal glands was also inhibited by NEM and, if so, whether this was a direct or an indirect effect of NEM on the catecholamine transporter. 3. Both [3H]norepinephrine transport into and [3H]reserpine binding to the chromaffin granule ghosts isolated from bovine adrenal glands are inhibited by NEM, with IC50 values of 0.63 +/- 0.02 and 2.8 +/- 0.66 microM, respectively. 4. Mg and ATP protected both the [3H]norepinephrine transport into the ghosts and the [3H]reserpine binding to the transporter from inhibition by NEM, shifting the IC50 values to 260 +/- 43 and 120 +/- 29 microM, respectively. 5. NEM inhibition of the catecholamine transport and reserpine binding appears to be due to an action on the proton translocator associated with the Mg ATPase enzyme rather than a direct action on the catecholamine transporter since (a) the concentration of NEM required to inhibit formation of a membrane potential is similar to that required to inhibit [3H]norepinephrine transport into and [3H]reserpine binding to the ghosts and (b) Mg and ATP protected the proton translocation and [3H]norepinephrine transport into the ghosts, and [3H]reserpine binding to the ghosts, from inhibition by NEM.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and characterization of the catecholamine transporter in bovine chromaffin granules using [3H]reserpine.

Characterization of the catecholamine transporter in chromaffin granule membranes has been hampered by the lack of a radioligand with high specific activity which binds selectively to the carrier with high affinity. We report here the identification of a high affinity binding site for [3H]reserpine on chromaffin granule membranes isolated from bovine adrenal gland which has the characteristics expected of the catecholamine transporter. [3H]Reserpine bound predominately to a high affinity site with a Kd for [3H]reserpine of 9 nM and a binding site density of 7.8 pmol/mg of protein. Comparison of the characteristics of the high affinity reserpine binding site to the characteristics of catecholamine transport indicated that (a) the Ki and rank order of potency for inhibition of [3H]reserpine binding by various biogenic amines was similar to their Ki for inhibition of catecholamine transport (b) both the inhibition of (-)-[3H]norepinephrine transport and inhibition of [3H]reserpine binding showed similar stereo-specificity, and (c) Kd for binding of reserpine to chromaffin granule membranes was similar to the Ki for reserpine inhibition of catecholamine transport. These results demonstrate that the high affinity binding site for [3H]reserpine on chromaffin granule membranes is associated with the catecholamine transporter.

Buffers differentially alter the binding of [3H]rauwolscine and [3H]RX821002 to the alpha-2 adrenergic receptor subtypes.

3H-antagonists are known to bind to the alpha-2A adrenergic receptor with higher affinity in glycylglycine buffer than in Tris buffer. The purpose of this study was to examine the effect of buffers on the binding of antagonists to all four subtypes of the alpha-2 adrenergic receptor. Our approach was to examine the effects of glycylglycine, Tris, sodium phosphate (NaPO4) and potassium phosphate buffers on the binding of [3H]rauwolscine, [3H]RX821002, prazosin and oxymetazoline. We found that the affinities for the different subtypes varied with the buffer and the ligands used. Although the Bmax values varied somewhat with the buffers, they were similar for both radioligands for a specific subtype. The highest affinities and Bmax values for both radioligands were generally obtained with NaPO4 buffer. The affinities of antagonists in Tris buffer were always significantly lower than in either NaPO4 or glycylglycine buffer, and the affinities decreased as the concentration of Tris increased. In contrast, the affinity of norepinephrine for the alpha-2B subtype was higher in Tris than in NaPO4 buffer. The buffer effects did not appear to be dependent on the cell membrane composition. There appeared to be some species differences in the effects of buffers on the alpha-2C subtype. These results indicate that buffers affect the binding of antagonists to alpha-2 adrenergic receptors, that not all subtypes are altered in the same manner and that buffers alter the binding of different antagonists differently. It is generally recommended that NaPO4 buffer be used, and that Tris be avoided, when measuring the binding of antagonists to the alpha-2 adrenergic receptor.

Inhibition of catecholamine transport into chromaffin granule ghosts isolated from bovine adrenal glands by phenytoin.

The anticonvulsant drug, phenytoin, has been reported to inhibit both the transport of catecholamines into synaptosomes and monoamine oxidase. The objective of this research was to determine whether phenytoin inhibited the transport of catecholamines into storage granules. This was tested by examining the effects of phenytoin (0.05 to 0.4 mM) on the ability of (-)-[3H] norepinephrine to be transported into chromaffin granule "ghosts" isolated from bovine adrenal glands. Our results indicated that phenytoin, but not phenobarbital, inhibited catecholamine transport in a dose-dependent manner with 50% inhibition occurring at a phenytoin concentration of 0.2 mM. Kinetic analysis of the effects of phenytoin on this transport process indicated that phenytoin was a competitive inhibitor of catecholamine transport with an approximate Ki of 0.3 mM. Furthermore, phenytoin did not inhibit the Mg adenosine triphosphatase required for providing the energy source for the catecholamine transport process, nor did it dissipate the membrane potential generated by this enzyme. The competitive inhibition of catecholamine transport produced by phenytoin is probably not related to the anticonvulsant effects of the drug as it occurred at greater than therapeutic concentrations. However, this effect may be related to the toxic effect of the drug.