Modulation of antagonist binding to histamine H₁‐receptors by sodium ions and by 2‐amino‐2‐hydroxymethyl‐propan‐1,3‐diol HCl

Abstract

NaCl (100 mm) reduced the potency of (+)‐N‐methyl‐4‐methyldiphenhydramine ((+)‐QMDP) as an inhibitor of the binding of [3H]‐mepyramine to histamine H1‐receptors on guinea‐pig cerebellar membranes to a greater extent than that of mepyramine, consistent with the greater inhibitory effect of Na+on the binding of [3H]‐QMDP than on the binding of [3H]‐mepyramine. The concentration of 2‐amino‐2‐hydroxymethyl‐propan‐1,3‐diol HCl (Tris, HCl) buffer, pH 7.5, present had little effect on the temelastine‐insensitive binding of [3H]‐mepyramine, but caused a concentration‐dependent inhibition of the binding of [3H]‐mepyramine sensitive to 1 μm temelastine (H1‐receptor binding), with an approximate IC50 of 75 mm, assuming that complete inhibition would have been achieved. Inhibition of [3H]‐mepyramine binding by Na+was more marked in 10 mm than in 50 mm Tris HCl and was not evident in 200 mm Tris HCl. The Kd for the temelastine‐sensitive binding of [3H]‐mepyramine measured in 10 mm Tris HCl, 0.24 ± 0.01 nm, was increased by 2.2 ± 0.2 fold by 100 mm NaCl, without any significant change in the maximum binding (Bmax). The Bmax for [3H]‐mepyramine was similarly unchanged in 50 mm Tris HCl, but the Kd was increased 2.5 ± 0.2 fold. The Kd for the temelastine‐sensitive binding of [3H]‐mepyramine was also increased in 50 mm, compared with 10 mm, N‐[2‐hydroxyethyl]piperazine‐N′‐[2‐ethanesulphonic acid] KOH (HEPES.KOH) buffer (Kd 0.25 ± 0.02 nm in 10 mm HEPES), but the evidence for an interaction between HEPES and Na+was less clear. The effect of 100 mm NaCl on the inhibition of [3H]‐mepyramine binding in 10 mm Tris HCl was examined for a range of antagonists. The decrease in potency caused by Na+was greatest for triprolidine, (+)‐chlorpheniramine and benzilylcholine (9.6–10.3 fold increase in Kd values) but the binding of mepyramine and promethazine was much less affected (1.8 and 1.9 fold increase in Kd respectively). The Kd for temelastine was not significantly changed. In contrast to the general decrease in antagonist affinity in the presence of Na+, the Kd for MDL 16,455A (4‐[1‐hydroxy‐4‐[4‐(hydroxydiphenylmethyl)‐1‐piperidinyl]butyl]‐α,α‐dimethylbenzene acetic acid) was increased, but only by 1.5 fold. It is concluded that Na+can act as an allosteric effector of the binding of antagonists at the histamine H1‐receptor. Tris HCl also appears to have an allosteric action at the H1‐receptor.

Dual effects of histamine and substance P on intracellular calcium levels in human U373 MG astrocytoma cells: role of protein kinase C

“…Membrane preparations from cerebella of Dunkin‐Hartley strain guinea‐pigs (males, 250–600 g; Tucks, Battlebridge, Essex) were prepared as described previously (Gibson et al , 1993). Incubations in 10 m m Tris‐HCl buffer, pH 7.5, contained 0.65 n m [ 3 H]‐mepyramine, inhibitor and cerebellar membranes (0.16 mg protein) in a total volume of 1.00 ml (4 replicates at each inhibitor concentration, 8 replicates in the absence of inhibitor).…”

The interaction of U-73122 with the histamine H 1 receptor: implications for the use of U-73122 in defining H 1 receptor-coupled signalling pathways

“…For experiments with U-73343 and experiments with U-73122 in which the maximum level of the binding of [ 3 H]mepyramine sensitive to U-73122 was ill-defined, NS was set to the percentage of non-specific binding defined by 2 µM temelastine, measured in the same experiment. Statistical comparison of IC 50 values from two inhibition curves was made as described previously (Gibson et al 1994) by fitting the curves simultaneously and assessing the significance of the increase in the residual sum of squares when the IC 50 was constrained to be the same for both curves.…”

“…250-400 g; Tuck's, Battlebridge, Essex, UK) were prepared as described previously (Gibson et al 1994). Incubations in 10 mM Tris-HCl buffer, pH 7.5, containing inhibitor, [ 3 H]mepyramine (usually 0.3-0.5 nM) and cerebellar membranes (ca.…”

Analysis of an H1 receptor‐mediated, zinc‐potentiated vasoconstrictor action of the histidyl dipeptide carnosine in rabbit saphenous vein

“…These experiments were carried out essentially as described by Gibson et al (1994). Dunkin-Hartley guinea-pigs (male, 300 ± 400 g) were killed by a blow to the head and the cerebella removed.…”