John S. Partilla

Evidence for the Involvement of Dopamine Transporters in Behavioral Stimulant Effects of Modafinil

Modafinil is prescribed for numerous medical conditions, but the drugs mechanism of action is unclear. Here, we examined the interaction of modafinil with receptors and transporters in vitro and compared pharmacological effects of the drug with those produced by indirect dopamine (DA) agonists 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (GBR12909) and (+)-methamphetamine (METH). Modafinil was screened at various receptors and transporters using binding assays. Transporter-mediated uptake and release were examined in rat brain synaptosomes. Effects of modafinil on motor activity and neurochemistry were determined in rats undergoing in vivo microdialysis in nucleus accumbens. Of the receptors and transporters assayed, modafinil displayed measurable potency only at DA transporters (DAT), inhibiting [3H]DA uptake, with an IC50 value of 4.0 μM. Accordingly, modafinil pretreatment (10 μM) antagonized METH-induced release of the DAT substrate [3H]1-methyl-4-phenylpyridinium. Intravenous modafinil (20 and 60 mg/kg) produced dose-dependent increases in motor activity and extracellular DA, without affecting serotonin (5-HT). Analogous results were observed for GBR12909 (1 and 3 mg/kg), whereas METH (0.3 and 1 mg/kg) increased DA and 5-HT. Locomotor effects of all drugs were positively correlated with dialysate DA (P < 0.001). Interestingly, modafinil pretreatment reduced METH-induced ambulation and DA release. Our data show that modafinil interacts with DAT sites in rat brain, a property shared with agonist medications under investigation for treating cocaine dependence. Nondopaminergic mechanisms may also contribute to the pharmacology of modafinil. Finally, the results suggest that modafinil should be tested as an adjunct for treating METH addiction.

Loss of rat ventricular α1-adrenergic receptors during aging

Ventricular alpha 1-adrenergic receptor concentration, measured by specific binding of [3H]-prazosin, decreases by 33% as rats age from 3 to 24 months. No age changes occur in binding affinity for [3H]-prazosin or potency of various alpha-adrenergic agonists and antagonists to displace [3H]-prazosin. The ratio of membrane protein to ventricular wet weight also does not change significantly with age. These results suggest a possible mechanism for loss of cardiovascular alpha-adrenergic responsiveness during aging.

Increased sensitivity to epinephrine stimulated lipolysis during starvation: tighter coupling of the adenylate cyclase complex.

Abstract Adipocytes of rats starved for 72 hr showed three-fold increase in sensitivity of the lipolytic response to epinephrine. Adenylate cyclase sensitivity in adipocyte membranes from starved rats was increased almost seven-fold over control rats. Binding studies using [ 3 H]-dihydroalprenolol showed that the number of β-adrenergic receptors per fat cell (1.1 × 10 5 and 1.1 × 10 5 receptors/cell, starved vs control) and the dissociation constant (5 vs 4 nM) of the receptor for the antagonist did not change following starvation. Increased sensitivity of adenylate cyclase activity in response to epinephrine without changes in the β-adrenergic binding parameters suggests that tighter coupling of adenylate cyclase with the receptor may mediate the more sensitive lipolytic response of starved rat adipocytes to epinephrine.

Quantitation of beta adrenergic receptors in rat liver: confounding effect of displaceable but nonstereospecific antagonist binding.

In rat liver membranes three types of ligand binding were seen using [3H]-dihydroalprenolol (DHA) and [125I]-hydroxybenzylpindolol (HYP): binding stereospecifically displaced by beta-adrenergic agonists and antagonists, binding nonstereospecifically displaced by beta-adrenergic antagonists, and binding which was not displaced by beta-adrenergic agonists or antagonists. The magnitude of the nonstereospecific displaceable binding varied with the physiological state of the animal. It was sufficient to prevent the quantitation of the stereospecific displaceable binding in some preparations from young rats but in all preparations of rats greater than 150 g or more than about 6 weeks of age. In adrenalectomized weanling rats 10-30% of the total binding was of nonstereospecific displaceable type while in control rats it comprised up to 60% of the total binding. Addition of 5 X 10(-6) M phentolamine to the assay eliminated a large proportion of the nonstereospecific displaceable binding. When phentolamine was included in the assay, liver membranes from weanling rats stereospecifically bound 30-35% of total binding; membranes from adrenalectomized rats showed stereospecific binding of up to 50 to 80%. Because the amount of displaceable, nonstereospecific binding varied greatly depending on the physiologic state of the animals, stereospecific displacement should be monitored for every type of liver membrane preparation. Furthermore, animal age is an important variable. Using the published antagonist binding methodology (DHA or HYP) in liver membranes, it is not presently possible to quantitate liver beta-adrenergic receptors in normal rats that have reached maturity.

(−) [125I] Iodopindolol Binding to β-Adrenergic Receptors in Tissues of the Rat with Particular Reference to the Characterization in Liver

The high affinity beta-adrenergic antagonist (-)[125I]iodopindolol was used to characterize and quantitate beta-adrenergic binding in liver and other tissues of the rat. Saturable, stereospecific binding with typical characteristics of beta-adrenergic receptors was demonstrated in all tissues examined (liver, lung, heart, kidney, fat and brain). Unlike the case with other radioactive ligands that have been used to measure beta-adrenergic receptors, assays were possible in crude homogenates as well as in purified membranes. Specific binding was defined as [125I]iodopindolol displaced by 0.1 mM isoproterenol. The percentage of [125I]iodopindolol specifically bound was greater than with other labelled antagonist ligands and it ranged from 95-60% of total binding over [125I]iodopindolol concentrations of 15-1000 pM. beta-Adrenergic binding could be quantitated in liver of animals of all ages whereas previously quantitation in liver was possible only using rats less than 2 months of age. Binding capacities ranged from a low of approximately 6 fmol/mg in liver particles precipitated at 4500 g to approximately 550 fmol/mg in 4500 g lung particles. The Kd of binding obtained by kinetic analysis was similar in all tissues. [125I]Iodopindolol is a nearly ideal ligand for the quantitation of beta-adrenergic receptors in various tissues of the rat. Because of its high affinity, stability and availability at high specific activity, this ligand should be especially useful in physiological studies requiring the accurate quantitation of receptor capacities.

Iodate promotes guanosine triphosphate stimulation of human fat-cell adenylate cyclase.

Abstract Iodate promoted GTP activation of human fat-cell adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] and reversed the usual inhibitory effect which GTP presumably exerts through interaction with a distinct regulatory subunit of the enzyme. GTP (0.1 m m ) inhibited adenylate cyclase by 52%, while NaIO3 (0.1 to 5 m m ) was minimally stimulatory. GTP stimulation in the presence of IO3− was dependent on the concentrations of both salt and nucleotide with maximal stimulation (up to 2.5-fold times basal) occurring at 0.5–1.0 m m IO3− and 0.1 m m GTP. Iodate salts of Li+, Na+, K+, and Rb+ all enhanced GTP action with similar salt concentration dependence, although the cation species did affect the magnitude of GTP activation. The action of IO3− in promoting GTP stimulation differed in several respects from that of Na+ cation which allows activation by the nucleotide relatively independent of the accompanying anion ( M. S. Katz, J. S. Partilla, C. R. Schneyer, M. A. Pineyro, R. I. Gregerman, 1981, Proc. Nat. Acad. Sci. USA78, 7417–7421 ). Effective IO3− concentrations were 100-fold lower than those for Na+.IO3−-induced GTP stimulation was not increased by raising the temperature from 30 to 37 °C, in contrast to the temperature dependence of the Na+ effect. Furthermore, unlike Na+, IO3− did not allow stimulation of adenylate cyclase by the nonhydrolyzable GTP analog, 5′-guanylyl-β-γ-imidodiphosphate. Iodate did, however, eliminate inhibition of enzyme by the analog, thereby suggesting that IO3− did not act by inhibiting GTPase activity. Time courses showed that basal adenylate cyclase activity decreased with time but that in the presence of IO3− the initial basal rate of cyclic AMP formation was maintained for at least 10 min. Iodate, when added with GTP, reduced the lag time caused by the nucleotide and produced an increase in enzyme acivity over basal. Vanadate, like IO3−, stimulated adenylate cyclase at millimolar concentrations but had only a minimal effect on the interaction of GTP with the fat-cell enzyme, while molybdates modulation of the nucleotide effect appeared to be entirely attributable to the accompanying Na+ cation. Our results show for the first time modulation of GTP effect by an anion and suggest that IO3− may be a useful probe of the GTP-binding regulatory protein and its interaction with the adenylate cyclase complex.