Proc. Natl. Acad. Sci. USA Vol.74, No. 12, pp. 5697-5701, December 1977 Medical Sciences Antagonism of histamine-activated adenylate cyclase in brain by D-lysergic acid diethylamide (histaminergic antagonists/adenosine 3':5'-cyclic monophosphate/H2-receptors/ergots/D-2-bromolysergic acid diethylamide) JACK PETER GREEN, CARL LYNN JOHNSON, HAREL WEINSTEIN, AND SAUL MAAYANI Department of Pharmacology, Mount Sinai School of Medicine of the City University of New York, 100th Street and Fifth Avenue, New York, New- York 10029 Communicated by Vincent P. Dole, August 19, 1977 ABSTRACT D-Lysergic acid diethylamide and D-2-bro- (ED50; amount necessary to produce half-maximal response) molysergic acid diethylamide are competitive antagonists of and antagonist affinities (pA2) were not altered. the histamine activation of adenylate cyclase [ATP pyrophos- Adenylate Cyclase Assay. The assay system has been de- phate-lyase (cyclizing); E.C. 4.6.1.11 in broken cell preparations in All additions of the hippocampus and cortex of guinea pig brain. The ade- scribed (8). All assays were performed triplicate. nylate cyclase is linked to the histamine H2-receptor. Both D- were made to the assay tubes on ice. They were then transferred lysergic acid diethylamide and D-2-bromolysergic acid dieth- to a 30° shaking incubator and preincubated for 5 min to allow ylamide show topological congruency with potent H2-antago- the enzymatic activity to reach a steady state and to eliminate nists. D-2-Bromolysergic acid diethylamide is 10 times more the influence of any lag periods in hormone activation. After potent as an H2-antagonist than cimetidine, which has been the the preincubation period, 25 of [a-32PJATP (1-2 gCi) were most potent H2-antagonist reported, and D-lysergic acid di- pl ethylamide is about equipotent to cimetidine. Blockade of added and in most cases the reaction was allowed to proceed H2-receptors could contribute to the behavioral effects of D-2- for 10 min, when it was stopped by adding 100 ,il of 1% sodium bromolysergic acid diethylamide and D-lysergic acid dieth- dodecyl sulfate. After addition of 650 gl of [3H]cyclic AMP ylamide. (13H]cAMP; 5000-10,000 cpm) to monitor recovery, the labeled cAMP was isolated with an alumina and Dowex column (9). Evidence is growing that histamine may function as a neuro- The reaction was linear with protein concentration (10) in the transmitter (see reviews, refs. 1-4). Histamine has a nonuniform range used and for at least 15 min after addition of the [a- regional distribution in brain. Most of the histamine is found 32pJATP. in subcellular fractions containing nerve endings. Brain contains Treatment of the Data. Curve fitting techniques (11) were specific enzymes for histamine formation and metabolism. used to estimate the apparent ED50 values, maximum stimu- Histamine appears to turn over rapidly. Potassium ions release lation by agonists, and parallelism of the dose-response curves. histamine from brain slices by a calcium-dependent process. Antagonism was analyzed by Schild plots (12) in which an- Brain lesions result in a fall in the activity of histidine decar- tagonism is expressed by the dose-ratios .(DR) of agonist needed boxylase in areas distal to the lesion. Neurons respond to his- to produce half-maximal responses in the presence and absence tamine. Histamine stimulates the activity of adenylate cyclase of different concentrations of antagonists (B). Simple compet- JATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1] and this itive antagonism results in a straight line of slope 1 when log effect is blocked by histamine HI-antagonists (5, 6) and hista- (DR - 1) is plotted against log B; and the intercept with the mine H2-antagonists (6, 7). We show here that D-lysergic acid abscissa is -log KB, where KB is the apparent dissociation diethylamide (D-LSD) and D-2-bromo-LSD (D-BrLSD) are constant for the antagonist-receptor interaction; -log KB is competitive antagonists of histamine in the activation of the referred to as pA2. H2-receptor linked to adenylate cyclase in the hippocampus arid cortex of the brain. RESULTS MATERIALS AND METHODS Histamine Receptor Linked to Adenylate Cyclase. Hista- Preparation of Tissue. Membrane-bound adenylate cyclase mine stimulated adenylate cyclase activity in all areas of the was prepared from brain by homogenization in a Potter-El- guinea pig brain that were examined-cortex, hippocampus, vejhem glass-Teflon vessel in 0.32 M sucrose/5 mM Tris-HCl/1 thalamus, striatum, hypothalamus, and central grey; cortex and mM ethylene glycol bis(3-aminoethyl ether)-N,N'-tetraacetate hippocampus were more sensitive than the other regions, as (EGTA)/1 mM dithiothreitol pH 7.4. The homogenate was shown by others (7). Rat hippocampal adenylate cyclase was centrifuged at 1000 X g and the supernatant fraction was also stimulated by histamine (Table 1), but it was less sensitive centrifuged again at 27,000 X g. The pellet from the second to histamine than guinea pig hippocampal adenylate cy- centrifugation was washed twice by resuspension in the same clase. medium and collected by centrifugation. The final pellet was The ED50 values for histamine, dimaprit, 4-methylhistamine, suspended in the same medium at a protein concentration of 2-methylhistamine, and N't,N't-dimethylhistamine were 14 0.5-0.7 mg/mi. The membranes could be quickly frozen with + 1, 6.4 + 0.45, 24, 120, and 21 + 2.3 MM, respectively. Previous dry ice/acetone and stored at -70°. Freezing and storage in- studies of histamine, 4-methylhistamine, and 2-methylhista- variably resulted in an increase in basal activity and a decrease mine on adenylate cyclase activity in broken cell preparations in maximal histamine activation, but the agonist potencies of the guinea pig hippocampus yielded similar ED50 values (7). It is especially noteworthy that dimaprit, a compound with The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Abbreviations: D-LSD, D-lysergic acid diethylamide; BrLSD, D-2- "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate bromo-LSD; cAMP, cyclic AMP; ED50, amount of agonist necessary this fact. to produce half-maximal response. 5697 Downloaded by guest on September 28, 2021 5698 Medical Sciences: Green et al. Proc. Natl. Acad. Sci. USA -74 (1977' Table 1. Effect of histamine on rat hippocampal adenylate 90 cyclase activity % increase in activity (±SEM) at Animal histamine concentration no. Basal* 10-5 M l0-4 M 10-3 M 1 21.5 + 0.2 13.8 + 2.4t 29.4 ± 1.4t 39.7 ± 2.2t 2 19.6 ± 0.4 9.5 + 0.31 27.6 ± 0.3t 37.8 ± 1.It 3 19.4 + 0.2 14.6 ± 1.41 33.7 ± 2.2t 42.4 i 0.5t * The amount of homogenate protein was 193, 187, and 191 ug, re- spectively, for the three animals. Activity is expressed as pmol of cAMP formed/min per mg of protein (±SEM). t Significantly different (P < 0.001) from basal activity. I lo-3 Significantly different (P < 0.05) from basal activity. Histamine, M considerable H2-agonist activity but with less than 0.0001% of the activity of histamine on HI-receptors (13), was active in the same concentration range as was histamine. Although measuring the relative potencies of agonists has V- been useful in classifying histamine receptors (14), these mea- 0*I1.0 surements are not dependable tools: the potencies of dimaprit and other agonists (13, 15) can vary 50-fold relative to histamine on different H2-receptors. More persuasive evidence for de- ~0 fining receptors comes from studies of antagonists (14). Fig. 1 shows typical dose-response curves for the effect of histamine on guinea pig hippocampal adenylate cyclase activity in the presence and absence of different concentrations of the H2- antagonist, cimetidine. Cimetidine caused a parallel shift in the -6.5 -6.0 -5.5 -5.0 -4.5 dose-response curve. Also shown in Fig. 1 is the Schild plot for log[cimetidine], M cimetidine with histamine or dimaprit as agonists. The fact that of the the dose-response curves of both histamine and the H2-selective FIG. 1. (Upper) Increase in adenylate cyclase activity guinea pig hippocampus with varying concentrations of histamine dimaprit are shifted to the same degree by cimetidine clearly in the absence and presence of cimetidine. Each point is the mean of were H2- establishes that both agonists reacting solely with triplicate determinations. Cimetidine alone did not affect basal ac- receptors linked to adenylate cyclase. The slope of the Schild tivity, which was 79.6 + 1.1 pmol/min per mg of protein. (Louwer) plot (0.90 + 0.07) did not differ significantly from the value of Schild plot for inhibition by cimetidine of histamine-stimulated 1.0 which is predicted by assuming simple competitive kinetics. adenylate cyclase activity in the guinea pig hippocampus. Ten de- The pA2 value was 6.22 + 0.03. The pA2 value for cimetidine terminations were made on four preparations with histamine as the agonist, and two determinations on one preparation with dimaprit on dimaprit-stimulated adenylate cyclase activity in two as agonist. The dose ratios were calculated as the ratio of the ED5o of was preparations of membranes of the guinea pig neocortex the agonist in the presence of cimetidine to the ED50 in the absence similar, 6.45 + 0.19. These are very close (Table 2) to the pA2 of cimetidine. The line has unit slope and an x-intercept (pA2 value) values obtained (16-18) on other H2-receptors. Other H2-an- of 6.22. tagonists caused a parallel shift in the dose-response curves.