Neuroseience Letters, 13 (1979) 2 4 3 - - 2 4 7
~) Elsevier/North-HollandScientific Publishers Ltd.
BENZODIAZEPINE R E C U R S : AT 37 °C
243
EFFECT OF TISSUE PREINCUB.ATION
R.C. ~PETH, G.J. WASTEK,T.D. REISINE and H.I. YAMAMURA* De~nt of Pharmacology, University of Arizona School of Medicine, Tucson, AZ 86724 (U.S.A.)
(Receivecl~February 19th, 1979) (Revised version received April 17th, 1979) (Accepted May 4th, 1979)
8UMMARY [3H]Flunitrazepam binding to rat brain homogenates was assayed in two ways: by 37--0°C incubations or O°C only incubations. The affinity of brain benzodiazepine receptors for [ 3H ]flunitrazepam was significantly greater in 37--0°C incubated samples than in 0°C incubated samples, while there was no change in the number of benzodiazepine receptors. The possible mechanisms for this enhanced binding affinity as well as the methodological implications for this observation are discussed.
The identification and characterization of specific, pharmacologically relevant, high affinity benzodiazepine receptors suggests that these receptors mediate the psychotropic effects of benzodiazepines [9]. Early in vitro studies of the binding of [ 3H]diazepam to rat brain homogenates revealed that [JHldiazepam binding at 37°C was drastically reduced in comparison to binding at 0*C ['/,9]. However, brain homogenates incubated at 37°C and s u ~ u e n f l y cooled to 0°C, showed increased [aH]diazepam binding compared to binding observed with only 0°C incubation [2]. The lower levels of. [3Hldiazepam [3] and [3H]flunitrazepam [10] b:nding detected at 370C results from a lower affinity of brain tissue homogcnates for benzo-
diazt
es at 370C than at 0°C.
study, h ~ investigated the effects of 37 ° C incubation prior to 0 *~C incUbation on the affinity of [~H]flunitrazepam binding to rat brain homogenate and has demonstrated significant effects which have functional as well as m e t h o d o l o g k ~ relevance.**
* Author to whom reprint ~equests ahtmld ~ sent. i:, A pmliminm'yreport of thue Irmdings was prew.n~d at the Society for Neur~cienc¢: tn~er;mg, St. Louis, MO, Nov., 1978.
244 AMays of [JH]flunitn~q=m binding to rat brain homoSenat~s were performed e~ntial]y m dear'bed p~evicmly [10]. ~ , crude, unwzau~ h o m ~
of w h o l ~ m t bndns equivalent t o 1 . 6 5 - - ~ 5 nag w e t weisht were
incubated with [~Hlflunitmzepmn (85 Ci/mmok 0.1--5.0 nM final eonce~ tratJoll) in sod/um-potass/um phosphate ~ (40.5 HIM N a = ~ 4 - ' - 9 . 5 mM KH2PO4) (pI~ '7.4) in a total volume of 2 m!: Free and bound [3H]fl~pam were sepiuaZed on 10assfiber (GF/B) filtens and filter bound mdiosctiv~ was memured by fiquid . c i n ~ o n ~ e t ~ . V~
presented are for specific (1 .M clonazepsm displaceable) 1~4] f
l
~
binding, When brain homogenates were inculmted for 60 rain at 37"C prior t o incubation for varying time at 0 ~C, there was an mcreme in [3H]flunitrazepam binding with time (Fig. 1). Temperature measurements of the m a y media after placement on ice indicated t h a t tempeml~u~ e q u ~ b m f i o n occurred rapidly: the temperature of the media was IOc after 6 rain on ice. The equilibration of [3H]flunitrazepam binding was increased 33% in 37 ° C preincubated ~ n p l e s in contrast to samptes incubated at 0 °C only (P
~) Elsevier/North-HollandScientific Publishers Ltd.
BENZODIAZEPINE R E C U R S : AT 37 °C
243
EFFECT OF TISSUE PREINCUB.ATION
R.C. ~PETH, G.J. WASTEK,T.D. REISINE and H.I. YAMAMURA* De~nt of Pharmacology, University of Arizona School of Medicine, Tucson, AZ 86724 (U.S.A.)
(Receivecl~February 19th, 1979) (Revised version received April 17th, 1979) (Accepted May 4th, 1979)
8UMMARY [3H]Flunitrazepam binding to rat brain homogenates was assayed in two ways: by 37--0°C incubations or O°C only incubations. The affinity of brain benzodiazepine receptors for [ 3H ]flunitrazepam was significantly greater in 37--0°C incubated samples than in 0°C incubated samples, while there was no change in the number of benzodiazepine receptors. The possible mechanisms for this enhanced binding affinity as well as the methodological implications for this observation are discussed.
The identification and characterization of specific, pharmacologically relevant, high affinity benzodiazepine receptors suggests that these receptors mediate the psychotropic effects of benzodiazepines [9]. Early in vitro studies of the binding of [ 3H]diazepam to rat brain homogenates revealed that [JHldiazepam binding at 37°C was drastically reduced in comparison to binding at 0*C ['/,9]. However, brain homogenates incubated at 37°C and s u ~ u e n f l y cooled to 0°C, showed increased [aH]diazepam binding compared to binding observed with only 0°C incubation [2]. The lower levels of. [3Hldiazepam [3] and [3H]flunitrazepam [10] b:nding detected at 370C results from a lower affinity of brain tissue homogcnates for benzo-
diazt
es at 370C than at 0°C.
study, h ~ investigated the effects of 37 ° C incubation prior to 0 *~C incUbation on the affinity of [~H]flunitrazepam binding to rat brain homogenate and has demonstrated significant effects which have functional as well as m e t h o d o l o g k ~ relevance.**
* Author to whom reprint ~equests ahtmld ~ sent. i:, A pmliminm'yreport of thue Irmdings was prew.n~d at the Society for Neur~cienc¢: tn~er;mg, St. Louis, MO, Nov., 1978.
244 AMays of [JH]flunitn~q=m binding to rat brain homoSenat~s were performed e~ntial]y m dear'bed p~evicmly [10]. ~ , crude, unwzau~ h o m ~
of w h o l ~ m t bndns equivalent t o 1 . 6 5 - - ~ 5 nag w e t weisht were
incubated with [~Hlflunitmzepmn (85 Ci/mmok 0.1--5.0 nM final eonce~ tratJoll) in sod/um-potass/um phosphate ~ (40.5 HIM N a = ~ 4 - ' - 9 . 5 mM KH2PO4) (pI~ '7.4) in a total volume of 2 m!: Free and bound [3H]fl~pam were sepiuaZed on 10assfiber (GF/B) filtens and filter bound mdiosctiv~ was memured by fiquid . c i n ~ o n ~ e t ~ . V~
presented are for specific (1 .M clonazepsm displaceable) 1~4] f
l
~
binding, When brain homogenates were inculmted for 60 rain at 37"C prior t o incubation for varying time at 0 ~C, there was an mcreme in [3H]flunitrazepam binding with time (Fig. 1). Temperature measurements of the m a y media after placement on ice indicated t h a t tempeml~u~ e q u ~ b m f i o n occurred rapidly: the temperature of the media was IOc after 6 rain on ice. The equilibration of [3H]flunitrazepam binding was increased 33% in 37 ° C preincubated ~ n p l e s in contrast to samptes incubated at 0 °C only (P
