High affinity dopamine D2 receptor radioligands. 2. [125I]epidepride, a potent and specific radioligand for the characterization of striatal and extrastriatal dopamine D2 receptors.

Epidepride, (S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-iodo-2,3-dimethoxybenzamide+ ++, the iodine analogue of isoremoxipride (FLB 457), was found to be a very potent dopamine D2 receptor antagonist. Optimal in vitro binding required incubation at 25 degrees C for 4 h at pH 7.4 in a buffer containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2 and 1 mM MgCl2. Scatchard analysis of in vitro binding to striatal, medial frontal cortical, hippocampal and cerebellar membranes revealed a KD of 24 pM in all regions, with Bmax's of 36.7, 1.04, 0.85, and 0.37 pmol/g tissue, respectively. The Hill coefficients ranged from 0.91-1.00 in all four regions. The IC50's for inhibition of [125I]epidepride binding to striatal, medial frontal cortical, and hippocampal membranes for SCH 23390, SKF 83566, serotonin, ketanserin, mianserin, naloxone, QNB, prasozin, clonidine, alprenolol, and norepinephrine ranged from 1 microM to greater than 10 microM. Partial displacement of [125I]epidepride by nanomolar concentrations of clonidine was noted in the frontal cortex and hippocampus, but not in the striatum. Scatchard analysis of epidepride binding to alpha 2 noradrenergic receptors in the frontal cortex and hippocampus revealed an apparent KD of 9 nM. At an epidepride concentration equal to the KD for the D2 receptor, i.e. 25 pM, no striatal alpha 2 binding was seen and only 7% of the specific epidepride binding in the cortex or hippocampus was due to binding at the alpha 2 site. Correlation of inhibition of [3H]spiperone and [125I]epidepride binding to striatal membranes by a variety of D2 ligands revealed a correlation coefficient of 0.99, indicating that epidepride labels a D2 site. In vitro autoradiography revealed high densities of receptor binding in layers V and VI of prefrontal and cingulate cortices as well as in striatum. In vivo rat brain uptake revealed a hippocampal:cerebellar and frontal cortical:cerebellar ratio of 2.2:1 which fell to 1.1:1 following haloperidol pretreatment. These properties suggest that [125I]epidepride is a superior radioligand for the in vitro and in vivo study of striatal and extrastriatal dopamine D2 receptors.     

Vascularized bone allografts: in vitro assessment of cell-mediated and humoral responses

The immunologic consequences of transplantation of vascularized bone allografts have not been previously characterized. In this study, knee allografts, both vascularized and nonvascularized, were transplanted from Lewis rats to Brown Norway rats across a strong histocompatibility barrier. A total of 66 transplants and 8 control animals were evaluated. The vascularized knee grafts consisted of 1 cm of proximal tibia and distal femur with a minimal muscular cuff isolated on the femoral vessels, and these were transplanted to a heterotopic, subcutaneous position on the abdominal wall of the recipient rat. Nonvascularized allografts (identical but without anastomoses) were transplanted for comparison. The cell-mediated response was measured by lymphocytotoxicity assay, and the humoral response was measured by cytotoxic antibody assay, both employing 51Cr-labeled target cells. The timing and intensity of the immune response differed according to the type of graft. The vascularized bone allografts generated significant cell-mediated and humoral responses as early as 5 days posttransplant. A significant humoral response in nonvascularized bone allografts was not apparent until day 14, while cell-mediated response in these grafts was variable. These findings were correlated with the histologic appearance of the grafted tissue. Cyclosporine, which was administered to one group of vascularized bone allografts, resulted in the suppression of both types of immune responses. The histologic appearance of this group resembled that of isografts transplanted as controls. The clinical application of vascularized bone allografts may offer significant advantages over nonvascularized allografts in the reconstruction of massive bone defects. Complications such as nonunion, fracture, and collapse of articular segments seen in nonvascularized allograft transplantation may be avoided by preservation of the blood supply to the graft. Characterization of the immune response to vascularized bone allografts may subsequently allow the manipulation of the host and/or graft tissue and promote graft incorporation.     

The effect of total parenteral nutrition (TPN) on the enteroinsular axis in the rat

The effect of 6 days of total parenteral nutrition (TPN) on the enteroinsular axis was studied in vivo and in vitro in the rat. During the TPN period, blood samples were taken from control and TPN animals to determine the comparative pattern of GIP release. Glucose, insulin and GIP responses to oral glucose (OGTT) were compared in TPN and control rats. The effect of glucose and GIP on insulin release from the isolated perfused pancreas of the same animals was investigated to determine if TPN altered the sensitivity of the beta cell. In conjunction with these studies the number and distribution of GIP-containing cells were compared in control and TPN animals. TPN resulted in no change in basal levels of glucose, insulin and IR-GIP. An exaggerated insulin response to OGTT occurred after TPN whereas the glucose response was reduced. The IR-GIP response to glucose was normal following TPN. The isolated perfused pancreas showed a 30% increase in insulin release in response to GIP after TPN. The insulin response to glucose appeared normal as did the number and distribution of GIP cells. Fluctuations in GIP and insulin levels in control animals were diurnal in nature, whereas IR-GIP levels in TPN animals remained near fasting levels. It was hypothesized that the increase in beta cell sensitivity to GIP may be causally connected to the exposure of the pancreas to chronically low levels of GIP during TPN.     

The PEP4 gene encodes an aspartyl protease implicated in the posttranslational regulation of Saccharomyces cerevisiae vacuolar hydrolases.

pep4 mutants of Saccharomyces cerevisiae accumulate inactive precursors of vacuolar hydrolases. The PEP4 gene was isolated from a genomic DNA library by complementation of the pep4-3 mutation. Deletion analysis localized the complementing activity to a 1.5-kilobase pair EcoRI-XhoI restriction enzyme fragment. This fragment was used to identify an 1,800-nucleotide mRNA capable of directing the synthesis of a 44,000-dalton polypeptide. Southern blot analysis of yeast genomic DNA showed that the PEP4 gene is unique; however, several related sequences exist in yeasts. Tetrad analysis and mitotic recombination experiments localized the PEP4 gene proximal to GAL4 on chromosome XVI. Analysis of the DNA sequence indicated that PEP4 encodes a polypeptide with extensive homology to the aspartyl protease family. A comparison of the PEP4 predicted amino acid sequence with the yeast protease A protein sequence revealed that the two genes are, in fact, identical (see also Ammerer et al., Mol. Cell. Biol. 6:2490-2499, 1986). Based on our observations, we propose a model whereby inactive precursor molecules produced from the PEP4 gene self-activate within the yeast vacuole and subsequently activate other vacuolar hydrolases.     

Receptor binding characterization of the benzodiazepine radioligand 125I-Ro16-0154: potential probe for SPECT brain imaging

The binding of an iodinated benzodiazepine (BZ) radioligand has been characterized, particularly in regard to its potential use as a neuroreceptor brain imaging agent with SPECT (Single Photon Emission Computed Tomography). Ro16-0154 is an iodine-containing BZ antagonist and a close analog of Ro15-1788. In tissue homogenates prepared from human and monkey brain, the binding of 125I-labeled Ro16-0154 was saturable, of high affinity (Kd = 0.5 nM at 37 degrees C), and had high ratios of specific to non-specific binding (approximately 40:1). Physiological concentrations of NaCl (150 mM) enhanced specific binding approximately 15% compared to buffer without this salt. Kinetic studies of association and dissociation demonstrated a temperature dependent decrease in affinity with increasing temperature. Drug displacement studies confirmed that 125I-Ro16-0154 binds to the "central" type BZ receptor: binding is virtually identical to that of 3H-Ro15-1788 except that 125I-Ro16-0154 shows an almost 10 fold higher affinity at 37 degrees C. These in vitro results suggest that 123I-labeled Ro16-0154 shows promise as a selective, high affinity SPECT probe of the brain's BZ receptor.     

Immunoaffinity Purification of Epitope-Tagged Human β2-Adrenergic Receptor to Homogeneity

To obtain large quantities of pure human β₂-adrenergic receptor (β₂-AR) needed for structural studies, an efficient method for β₂-AR purification was developed using a recombinant receptor with an eight amino acid epitope at its C-terminus. This epitope is recognized by KT3-monoclonal antibody. The epitope tagged β₂-AR was expressed in Sf9 cells with a specific activity of 5–20 pmol/mg of membrane protein. The epitope-tagged and wild-type receptors had identical ligand binding properties. The tagged receptor was solubilized using dodecyl-β-maltoside with a quantitative yield. Solubilized epitope-tagged receptors were partially purified by KT3-mAb immunoaffinity in 60–70% yield. Further purification of the receptors on an alprenolol-affinity column resulted in a homogenous preparation with an overall yield of >30%. The purified receptor was concentrated to >1 mg/ml without loss of ligand binding activity.