Mazindol to Cardiac Norepinephrine Transporters: Kinetic and Equilibrium Studies
Total Page：16
File Type：pdf, Size：1020Kb
Naunyn-Schmiedeberg's Arch Pharmacol (2004) 370: 9–16 DOI 10.1007/s00210-004-0949-y ORIGINAL ARTICLE David M. Raffel . Wei Chen Binding of [3H]mazindol to cardiac norepinephrine transporters: kinetic and equilibrium studies Received: 27 February 2004 / Accepted: 27 May 2004 / Published online: 22 July 2004 # Springer-Verlag 2004 Abstract The norepinephrine transporter (NET) is the values. These studies demonstrate the high selectivity of carrier that drives the neuronal norepinephrine uptake [3H]mazindol binding for the norepinephrine transporter in mechanism (uptake1) in mammalian hearts. The radioli- membrane preparations from mammalian hearts. gand [3H]mazindol binds with high affinity to NET. In this study, the kinetics of [3H]mazindol binding to NET were Keywords Sympathetic nervous system . Norepinephrine measured using a rat heart membrane preparation. Results transporter . Positron emission tomography . meta- from these studies were used to set up saturation binding iodobenzylguanidine . meta-hydroxyephedrine assays designed to measure cardiac NET densities (Bmax) and competitive inhibition assays designed to measure inhibitor binding affinities (KI) for NET. Saturation Introduction binding assays measured NET densities in rat, rabbit, and canine hearts. Assay reproducibility was assessed and The autonomic nervous system plays a central role in the the effect of NaCl concentration on [3H]mazindol binding regulation of cardiac function. Abnormalities of cardiac to NET was studied using membranes from rat and canine autonomic innervation have increasingly been implicated hearts. Specificity of [3H]mazindol binding to NET was as a major underlying factor contributing to the high determined in experiments in which the neurotoxin 6- morbidity and mortality associated with sudden cardiac hydroxydopamine (6-OHDA) was used to selectively death (Schwartz 1998), congestive heart failure (Packer destroy cardiac sympathetic nerve terminals in rats. 1992), diabetic autonomic neuropathy (Ewing 1996), Competitive inhibition studies measured KI values for myocardial ischemia (Armour 1998) and cardiac arrhyth- several NET inhibitors and substrates. In kinetic studies mias (Zipes 1995). With more than 300,000 deaths/year in using rat heart membranes, [3H]mazindol exhibited a the United States attributed to sudden cardiac death alone −1 dissociation rate constant koff=0.0123±0.0007 min and (Virmani et al. 2001), increasing our understanding of the an association rate constant kon=0.0249 role of autonomic dysfunction in these diseases is an − − ±0.0019 nM 1min 1. In saturation binding assays, [3H] important research goal. mazindol binding was monophasic and saturable in all In an effort to provide clinicians with better tools for cases. Increasing the concentration of NaCl in the assay investigating autonomic dysfunction, several radiotracers buffer increased binding affinity significantly, while only have been developed at our institution for non-invasive modestly increasing Bmax. Injections of 6-OHDA in rats imaging studies of the sympathetic branch of cardiac decreased measured cardiac NET Bmax values in a dose- autonomic innervation. Radiotracers that have been dependent manner, verifying that [3H]mazindol binds successfully used as markers of cardiac sympathetic specifically to NET from sympathetic nerve terminals. neurons include the single-photon imaging agents [131I] Competitive inhibition studies provided NET inhibitor and meta-iodobenzylguanidine and [123I]meta-iodobenzylgua- substrate KI values consistent with previously reported nidine (MIBG; Wieland et al. 1981), and the positron emission tomography (PET) radiotracers [11C]meta-hydro- 11 *) . xyephedrine (HED; Rosenspire et al. 1990), [ C]epineph- D. M. Raffel ( W. Chen 11 Division of Nuclear Medicine, Department of Radiology, rine (Chakraborty et al. 1993), and [ C]phenylephrine University of Michigan Medical School, (Del Rosario et al. 1996). All of these imaging agents are 3480 Kresge III Building, transported into sympathetic neurons by the neuronal Ann Arbor, MI, 48109-0552, USA e-mail: [email protected] norepinephrine transporter (NET) and subsequently stored Tel.: +1-734-9360725 in vesicles by the vesicular monoamine transporter Fax: +1-734-7640288 (VMAT; Raffel and Wieland 2001). 10 As part of our ongoing efforts to characterize and cold buffer A. The heart chambers were cut open and rinsed of validate these radiotracers in animal models, we were blood. After the aorta and excess vascular tissue were removed the heart was blotted dry and weighed, then placed in a 100-ml beaker interested in implementing an in vitro assay to measure containing 10 ml of ice-cold buffer A. The heart was minced with NET density in heart tissue samples. Measurements of scissors and transferred with the buffer to an ice-chilled 30-ml cardiac NET density in mammalian heart tissue samples borosilicate glass test tube. The heart pieces were homogenized have previously been reported by several laboratories, using a Brinkmann Polytron with a 12-mm sawtooth generator (PT10/35, with Kinematica PCU-2-110 controller, Brinkmann, using [3H]mazindol (Liang et al. 1989; Böhm et al. 1995; 3 Westbury, NY, USA) kept at 4°C in a cold room. Homogenization Ungerer et al. 1996; Mardon et al. 2003), [ H]desipramine was performed on setting 7 for 30 s (3×10-s bursts). The (Raisman et al. 1982; Kiyono et al. 2002a; Kiyono et al. homogenate was centrifuged at 1,000 g (max) for 15 min at 4°C. 3 2002b), or [ H]nisoxetine (Böhm et al. 1998; Leineweber The supernatant fraction (S1) was removed with a polypropylene et al. 2000) as the radioligand in saturation binding assays. transfer pipette and placed in a 10 ml polycarbonate ultracentrifu- gation tube. A fluffy layer of material at the interface between S1 and Cardiac NET density (Bmax) values measured for a given the pellet (P1) was discarded with the P1 fraction. The S1 fraction mammalian species tend to vary from laboratory to was centrifuged at 100,000 g (max) for 30 min at 4°C. The laboratory due to differences in the methods used for supernatant fraction (S2) was discarded and 1 ml of ice-cold buffer B tissue homogenization, subsequent tissue processing, and was added to the tube. The pellet (P2) was removed intact from the tube wall with the tip of a metal spatula and resuspended by saturation binding assay conditions. Furthermore, there repeatedly pipetting the pellet and buffer mixture in and out of a appears to be scant information in the literature regarding 1.25-ml disposable pipettor (Eppendorf Combitip, Brinkmann the kinetics of radioligand binding to cardiac NET. Instruments, Westbury, NY, USA) ∼25 times. Another 5 ml of ice- The present study was undertaken to characterize the cold buffer B was added and the tube vortexed until a uniform 3 mixture was achieved. The resuspended P2 fraction was again binding of [ H]mazindol to cardiac NET and to use this centrifuged at 100,000 g (max) for 30 min at 4°C. The supernatant information in the design of equilibrium binding assays. was discarded and the pellet (P3) resuspended in 1 ml buffer B. An Kinetic studies were performed to measure the dissocia- additional 5 ml of buffer B was added and the solution vortexed −1 until well mixed. Membrane preparations were frozen at −80°C until tion constant koff (min ) and the association constant kon −1 −1 3 used for assays. Protein concentrations in the membrane prepara- (nM min ) for [ H]mazindol binding to NET purified tions were measured with the Lowry spectrophotometric method from rat hearts. These parameters were used to establish (Lowry et al. 1951), using bovine serum albumin for standards. incubation times for saturation binding assays designed to measure cardiac NET density and for competitive inhibi- Binding assays-common methods All assays were performed at tion studies designed to measure equilibrium dissociation room temperature (22°C) in 96-well polystyrene microtiter assay constants (K ) for NET substrates and inhibitors. In plates fabricated with a special non-binding surface designed to I minimize binding of radioligand to the plate wells (Corning addition to control studies, the influence of some COSTAR #3641, Corning Life Sciences, Acton, MA, USA). experimental conditions on saturation binding assay Typically, total binding of [3H]mazindol to NET in cardiac results, including protein concentration and NaCl concen- membranes was determined by incubating 100 μl of membrane tration, were assessed. The reproducibility of the satura- preparation (∼100 μg protein), 100 μl of buffer B and 50 μlof buffer B containing [3H]mazindol at the concentration required for a tion binding assay was also investigated. The specificity of μ 3 given experiment, for a total reaction volume of 250 l. To measure [ H]mazindol binding to NET was determined in studies in non-specific [3H]mazindol binding, identical well contents were which the neurotoxin 6-hydroxydopamine was used to prepared except that the 100 μl aliquot of buffer B contained the chemically denervate the heart. Finally, saturation binding high affinity NET inhibitor desipramine (DMI) at 25 μM (final DMI concentration 10 μM). During incubation, assay plates were assays were performed with canine and rabbit heart tissue continuously shaken on the tray of a shaking water bath. Assays to test the ability of the assay to measure NET density and were terminated by rapid filtration onto Whatman GF/C glass fiber binding affinity in heart samples from other mammalian filters using a Brandel cell harvester equipped