TY - JOUR
T1 - A radiolabeled-ligand-binding technique for the characterization of opioid receptors in the intact mouse vas deferens
AU - Cicero, Theodore J.
AU - Bell, Roy D.
N1 - Funding Information:
’ This research was supported in part by USPHS Grants DA-00259, AA-03539, and DA-03833. ’ Recipient of Research Scientist Award, DA-00095. 3 Abbreviations used: MVD, mouse vas deferens; DADL, 2+alanine, 5-D-leucine enkephalin; EKC, ethylketocyclazocine; DHM, dihydromorphine; KREG, Krebs Ringer bicarbonate glucose buffer.
PY - 1985/10
Y1 - 1985/10
N2 - The mouse vas deferens has served as a useful bioassay for examining the properties of opiate receptor subtypes. However, recent data indicate that the response of the vas deferens to opiates may be mediated by one or more of the several opiate receptors found in this preparation. Although a number of techniques can be utilized to assess the relative contribution of these receptors to the response of the mouse vas deferens to opiates (e.g., selective tolerance and naloxone antagonism studies), a radiolabeled-binding technique would provide an independent means of more completely characterizing the opiate receptor profiles in this preparation. Up to the present, however, there has been only limited success in developing a binding assay utilizing crude membrane fractions of the mouse vas deferens. To circumvent these problems, we have developed a binding technique utilizing the intact vas deferens. In contrast to results obtained with membrane fractions, we found highly specific (90-95%) and saturable binding of d-[2-3H]alanine, 5-d-leucine enkephalin, a ligand selective for delta opiate receptors, to the intact vas. Scatchard analyses indicated a single class of binding sites with an apparent Kd of 1.5 nm and a Bmax of approximately 12 pmol/2 vas. The selectivity of binding was also examined. Naltrexone was 40 times less potent than unlabeled 2-d-alanine, 5-d-leucine enkephalin in displacing binding, whereas morphine and ethylketocyclazocine were 300 and 500 times less effective, respectively. This technique, coupled with the mouse vas deferens bioassay, should provide a more complete characterization of opioid receptor populations than has heretofore been possible.
AB - The mouse vas deferens has served as a useful bioassay for examining the properties of opiate receptor subtypes. However, recent data indicate that the response of the vas deferens to opiates may be mediated by one or more of the several opiate receptors found in this preparation. Although a number of techniques can be utilized to assess the relative contribution of these receptors to the response of the mouse vas deferens to opiates (e.g., selective tolerance and naloxone antagonism studies), a radiolabeled-binding technique would provide an independent means of more completely characterizing the opiate receptor profiles in this preparation. Up to the present, however, there has been only limited success in developing a binding assay utilizing crude membrane fractions of the mouse vas deferens. To circumvent these problems, we have developed a binding technique utilizing the intact vas deferens. In contrast to results obtained with membrane fractions, we found highly specific (90-95%) and saturable binding of d-[2-3H]alanine, 5-d-leucine enkephalin, a ligand selective for delta opiate receptors, to the intact vas. Scatchard analyses indicated a single class of binding sites with an apparent Kd of 1.5 nm and a Bmax of approximately 12 pmol/2 vas. The selectivity of binding was also examined. Naltrexone was 40 times less potent than unlabeled 2-d-alanine, 5-d-leucine enkephalin in displacing binding, whereas morphine and ethylketocyclazocine were 300 and 500 times less effective, respectively. This technique, coupled with the mouse vas deferens bioassay, should provide a more complete characterization of opioid receptor populations than has heretofore been possible.
KW - binding assays
KW - bioassays
KW - general
KW - liquid scintillation counting
KW - opiate
KW - opioid
KW - receptors
UR - http://www.scopus.com/inward/record.url?scp=0022227983&partnerID=8YFLogxK
U2 - 10.1016/0003-2697(85)90441-5
DO - 10.1016/0003-2697(85)90441-5
M3 - Article
C2 - 3002205
AN - SCOPUS:0022227983
VL - 150
SP - 67
EP - 75
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 1
ER -