1 In human U373 MG astrocytoma cells agonist-induced increases in intracellular Ca 2+ ([Ca 2+ ] i ) are rapidly returned towards prestimulated levels. Examination of the e ect of histamine and substance P on [Ca 2+ ] i in thapsigargin-treated cells has allowed a mechanism contributing to this e ect to be characterized. 4 Treatment of U373 MG cells with 5 mM thapsigargin, followed by the readdition of 1.8 mM Ca 2+ to the perfusion bu er, resulted in a steady-state level of [Ca 2+ ] i 97+5 nM above pretreated levels (measured 400 s after readdition of Ca 2+ ). Perfusion of histamine (100 mM, 100 s) caused a rapid decline in the thapsigargin-induced steady state level of [Ca 2+ ] i . This e ect of histamine was normally reversible upon washout. The best-®t EC 50 for the histamine response was 0.8+0.2 mM. Substance P (10 nM, 100 s) also caused a reduction in thapsigargin-induced steady-state levels of [Ca 2+ ] i . 5 Neither 100 mM histamine nor 10 nM substance P inhibited the rate of quench of fura-2¯uorescence by Mn 2+ in U373 MG cells pretreated with 5 mM thapsigargin, indicating that the depressant e ect on steady-state raised [Ca 2+ ] i was probably not due to a block of Ca 2+ entry. 6 The depressant e ect of histamine on [Ca 2+ ] i was blocked by 1 mM mepyramine, and was partially reduced by pre-incubation with 1 mM staurosporine (61+7% reduction) and with Ro 31-8220 (24+10% and 50+6% reduction by 1 and 10 mM Ro 31-8220, respectively). Pre-incubation with H-89 did not alter the depressant e ect of histamine. 7 Neither 1 mM staurosporine nor 10 mM KN-62 inhibited the binding of [ 3 H]-mepyramine to guineapig cerebellar membranes, whereas it was reduced by 17+1% and 55+2% by 1 and 10 mM Ro 31-8220, respectively. However, [ 3 H]-IP 1 accumulation stimulated by histamine in U373 MG cells was not inhibited by 1 or 10 mM Ro 31-8220 and in 2 out of 3 experiments there was a signi®cant potentiation of the response to histamine with both concentrations of Ro 31-8220. Staurosporine, 1 mM, similarly potentiated the response to 100 mM histamine in 3 out of 4 experiments. KN-62 (10 mM) did not stimulate histamine-induced [ 3 H]-IP 1 accumulation. 8 In HEPES bu er to which no Ca 2+ had been added, histamine stimulated a transient 451+107 nM increase in [Ca 2+ ] i . Pretreatment with 1 mM and 10 mM Ro 31-8220 did not signi®cantly alter the initial peak response to histamine, but slowed the rate at which histamine-induced increases in [Ca 2+ ] i were returned to prestimulated levels. Pretreatment with KN-62 had no signi®cant e ect on the response to histamine, but consistently inhibited the secondary slower phase of the decline in [Ca 2+ ] i . H-89 did not alter the histamine response. 9 The e ect of histamine in stimulating Ca 2+ extrusion was not con®ned to U373 MG cells, since 100 mM histamine also caused a rapid decrease in steady-state levels of [Ca 2+ ] i in thapsigargin-treated human HeLa cells. 10 The results indicate that agonists which increase [Ca 2+ ] i via activation of phosphoinositide metabolism can also...

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.

U-73122, an N-aminosteroid homologue of N-ethylmaleimide (NEM), widely used as an inhibitor of phospholipase C, was found to be a potent inhibitor (IC50 5.5+/-0.5 microM) of the binding of [3H]mepyramine to guinea-pig cerebellar membranes. The succinimido analogue, U-73343, also inhibited the binding of [3H]mepyramine (estimated IC50 24+/-1 microM), but NEM was only a weak inhibitor, even at 10 mM. The interaction of U-73122 and U-73343 with the H1 receptor was effectively irreversible, on the time-scale of the experiment. There is no indication that reaction with a receptor thiol residue is involved in the binding of U-73122, since preincubation of membranes with 2 mM NEM did not significantly increase the IC50 for the inhibition of [3H]mepyramine binding by U-73122. We conclude that U-73122 binds to the histamine H1 receptor in the concentration range in which it acts as an inhibitor or PLC. This compromises the use of U-73122 to provide evidence that an H1 agonist action is mediated via PLC. The tight binding of U-73343 to the receptor appears to be primarily a function of the hydrophobic nature of the compound.

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