No abstract

Because dihydroergocryptine (DHE) (13,14) have found that DHE acts as a pure dopamine-like agonist on pituitary cells in culture. It is also known that 2-bromocryptine, which is chemically similar to DHE, has dopamine-like agonist activity on rat behavior (15), mimicks the effect of L-3,4-dihydroxyphenylalanine on patients with Parkinson disease (16), and reduces prolactin secretion in patients in a fashion similar to other dopamine-mimetic drugs (17).These previous studies on the ergot compounds (13)(14)(15)(16)(17) METHODS AND MATERIALS Preparation of Calf Caudate Homogenates. The experiments were done on crude homogenates of calf caudate, prepared as described (6). In order to retain all the dopamine receptors, the homogenates were not purified or subfractionated. Calf brains were obtained fresh from the Canada Packers Hunnisett plant (Toronto, Canada). The caudates were removed within 2 hr after death, pooled, sliced into small cubes, and suspended in buffer at an approximate concentration of 50 mg of wet weight per ml of buffer. The buffer contained 15 mM Tris-HCI (pH 7.4), 5 mM Na2EDTA, 1.1 mM ascorbate, and 12.5 ,uM nialamide. A preliminary crude homogenate of the suspension was made with a glass homogenizer with a Teflon piston (0.13-0.18 mm clearance). This piston, rotating at 500 rpm, was passed up and down 20 times in the homogenizer. The crude homogenate was first incubated at 370 for 60 min and then stored in 3-ml aliquots at -20°for future use. Before using, the samples were thawed, resuspended in the glass-Teflon homogenizer and homogenized by hand (10 upand-down passes), and centrifuged at 39,000 X g for 15 min at 40; the supernatant was discarded and the pellet was resuspended in 10 ml of buffer. The suspension was finally homogenized by a Polytron homogenizer (Brinkman Instrument Co.) at a setting of 7 (full range = 10) for 20 sec, by using a PT-10 homogenizer probe and a 50-ml polycarbonate tube to contain the suspension. Homogenization at settings higher than 7 led to a loss of protein through the glass fiber GF/B filters used in the radioreceptor assays. Except were indicated, the homogenates were always kept chilled on ice.[

This study was done to obtain direct in vitro evidence for the possible existence of more than one type of dopaminergic binding site in homogenates of the caudate nucleus from calf brain. Five MATERIALS AND METHODSPreparation of Calf Caudate Nucleus Homogenates. The experiments were done on crude homogenates of calf caudate nucleus, prepared as previously described (16). In order to retain all the dopamine receptors, the homogenates were not purified or subfractionated. Calf brains were obtained fresh from the Canada Packers Hunnisett plant (Toronto). The caudates were removed within 2 hr after death, pooled, sliced into small cubes, and suspended in buffer at an approximate concentration of 50 mg of wet weight per ml of buffer. The buffer contained 15 mM Tris-HCI (pH 7.4), 5 mM Na2EDTA, 1.1 mM ascorbate, and 12.5 ,uM nialamide. A preliminary crude homogenate of the suspension was made using a glass homogenizer with a Teflon piston (0. 12-0.18 mm clearance). This piston, rotatingThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.at 500 rpm, was passed up and down 20 times. The crude homogenate was incubated at 370 for 60 min and then stored in 3-ml aliquots at -200. Before using, the samples were thawed, resuspended in the glass-Teflon homogenizer (10 up-and-down passes) by hand, and centrifuged at 39,000 X g for 15 min at 40; the supernatant was discarded and the pellet was resuspended in 10 ml of buffer. The suspension was finally homogenized by a Polytron homogenizer (Brinkmann Instruments) at a setting of 7 (full range = 10) for 20 sec, using a PT-10 homogenizer probe and a 50-ml polycarbonate tube to contain the suspension. Homogenization at settings higher than 7 led to a loss of protein through the glass-fiber GF/B filters used in the radioreceptor assays. Except where indicated, the homogenates were always kept chilled on ice.[3H]Apomorphine Binding Assays. The [3H]apomorphine had been custom-prepared for this laboratory by New England Nuclear Corp., Boston, and used without further purification; the specific activity was 14.1 Ci/mmol (in 1975). The material was stored in ethanol at -20°. The [3H]apomorphine binding assays were done using 12-X 75-mm glass test tubes in which the following aliquots were placed (using Eppendorf Brinkmann pipettes with polypropylene tips): 0.2 ml of [3H]apomorphine (final concentration of 0.7 nM); 0.2 ml of brain homogenate (always added last, and containing between 0.2-and 0.3 mg of protein); 0.1 ml of drug solution; and 0.1 ml of a second drug solution or buffer. Each determination was always done in sextuplicate. After the sample was incubated for 30 min (220), an aliquot of 0.5 ml was removed (polypropylene pipette tip) from the mixture and filtered under reduced pressure through a glass-fiber filter (GF/B, Whatman, 24-mm diameter) using a Millipore stainless steel mesh support for the filte...

No abstract

No abstract