The interaction of U-73122 with the histamine H1 receptor: implications for the use of U-73122 in defining H1 receptor-coupled signalling pathways.

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.

SK&F 96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenylethyl]-1H- imidazole hydrochloride) stimulates phosphoinositide hydrolysis in human U373 MG astrocytoma cells.

SK&F 96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenylethyl]-1H-imi dazole hydrochloride) stimulated the accumulation of [3H]inositol monophosphates ([3H]IP1) in human U373 MG astrocytoma cells prelabelled with [3H]inositol (EC50 15 +/- 1 microM, Hill coefficient 3.8 +/- 0.4). SK&F 96365-induced accumulation of [3H]IP1 increased linearly with time, but there was no initial rapid formation of [3H]IP3. SK&F 96365 also stimulated [3H]IP1 accumulation in human HeLa cells, but only to a small extent in slices of rat cerebral cortex and guinea-pig cerebellum. SK&F 96365-induced accumulation of [3H]IP1 in U373 MG cells increased as extracellular Ca2+ was increased from nominally zero to 4 mM, but there was no evidence that SK&F 96365 induced any marked entry of Ca2+ into cells; only an inhibition of store-refilling-induced Ca2+ entry was apparent. Further, the response to SK&F 96365 was additive with that to the Ca2+ ionophore ionomycin. Depolarization of the cells with raised K+ produced only a small stimulation of phosphoinositide hydrolysis. SK&F 96365 caused the release of Ca2+ from intracellular stores in U373 MG cells (EC50 26 +/- 14 microM), but thapsigargin induced only a small accumulation of [3H]IP1. Miconazole, another N-substituted imidazole, also stimulated [3H]IP1 accumulation in U373 cells.

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

1. In human U373 MG astrocytoma cells agonist-induced increases in intracellular Ca2+ ([Ca2+]i) are rapidly returned towards prestimulated levels. Examination of the effect of histamine and substance P on [Ca2+]i in thapsigargin-treated cells has allowed a mechanism contributing to this effect to be characterized. 2. Histamine and substance P stimulated [3H]-inositol monophosphate ([3H]-IP1) accumulation in U373 MG cells. Concentration-response curves of [3H]-IP1 accumulation in suspensions of U373 MG cells in HEPES buffer containing 30 mM Li+ yielded best-fit EC50 values of 19.1+/-1.5 microM for histamine and 5.7+/-1.3 nM for substance P. 3. In confluent monolayers of fura-2 loaded U373 MG cells perfusion with 100 microM histamine resulted in a transient 597+/-50 nM increase in [Ca2+]i. The best-fit EC50 for histamine was 4.6+/-2.2 microM. The initial, transient, histamine response was often followed by further small transient increases in [Ca2+]i. 4. Treatment of U373 MG cells with 5 microM thapsigargin, followed by the readdition of 1.8 mM Ca2+ to the perfusion buffer, resulted in a steady-state level of [Ca2+]i 97+/-5 nM above pretreated levels (measured 400 s after readdition of Ca2+). Perfusion of histamine (100 microM, 100 s) caused a rapid decline in the thapsigargin-induced steady state level of [Ca2+]i. This effect of histamine was normally reversible upon washout. The best-fit EC50, for the histamine response was 0.8+/-0.2 microM. Substance P (10 nM, 100s) also caused a reduction in thapsigargin-induced steady-state levels of [Ca2+]i. 5. Neither 100 microM histamine nor 10 nM substance P inhibited the rate of quench of fura-2 fluorescence by Mn2+ in U373 MG cells pretreated with 5 microM thapsigargin, indicating that the depressant effect on steady-state raised [Ca2+]i was probably not due to a block of Ca2+ entry. 6. The depressant effect of histamine on [Ca2+]i was blocked by 1 microM mepyramine, and was partially reduced by pre-incubation with 1 microM staurosporine (61+/-7% reduction) and with Ro 31-8220 (24+/-10% and 50+/-6% reduction by 1 and 10 microM Ro 31-8220, respectively). Pre-incubation with H-89 did not alter the depressant effect of histamine. 7. Neither 1 microM staurosporine nor 10 microM KN-62 inhibited the binding of [3H]-mepyramine to guinea-pig cerebellar membranes, whereas it was reduced by 17+/-1% and 55+/-2% by 1 and 10 microM Ro 31-8220, respectively. However, [3H]-IP1 accumulation stimulated by histamine in U373 MG cells was not inhibited by 1 or 10 microM Ro 31-8220 and in 2 out of 3 experiments there was a significant potentiation of the response to histamine with both concentrations of Ro 31-8220. Staurosporine, 1 microM, similarly potentiated the response to 100 microM histamine in 3 out of 4 experiments. KN-62 (10 microM) did not stimulate histamine-induced [3H]-IP1 accumulation. 8. In HEPES buffer to which no Ca2+ had been added, histamine stimulated a transient 451+/-107 nM increase in [Ca2+]i. Pretreatment with 1 microM and 10 microM Ro 31-8220 did not significantly alter the initial peak response to histamine, but slowed the rate at which histamine-induced increases in [Ca2+]i were returned to prestimulated levels. Pretreatment with KN-62 had no significant effect on the response to histamine, but consistently inhibited the secondary slower phase of the decline in [Ca2+]i. H-89 did not alter the histamine response. 9. The effect of histamine in stimulating Ca2+ extrusion was not confined to U373 MG cells, since 100 microM histamine also caused a rapid decrease in steady-state levels of [Ca2+]i in thapsigargin-treated human HeLa cells. 10. The results indicate that agonists which increase [Ca2+]i via activation of phosphoinositide metabolism can also stimulate a homeostatic mechanism which acts to reduce [Ca2+]i. The balance of the evidence indicates that in U373 MG cells the latter effect most likely involves a PKC-mediated stimulation of a Ca2+-extrusion pump.

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

1. 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. 2. 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 microM temelastine (H1-receptor binding), with an approximate IC50 of 75 mM, assuming that complete inhibition would have been achieved. 3. 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. 4. 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. 5. 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.6. 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 K1 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 for MDL 16,455A (4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]butyl]-alpha,alpha-dimethylbenzene acetic acid) was increased, but only by 1.5 fold.7. It is concluded that Na+ can act as an allosteric effector of the binding of antagonists at the histamine HI-receptor. Tris HCl also appears to have an allosteric action at the H1-receptor.

Sulfated O-glycoproteins secreted by guinea pig trachea in organ culture.

Organ culture of guinea pig trachea was performed in the presence of [35S]sulfate in order to characterize the sulfated glycoproteins released from the respiratory epithelium and mucosa. The sulfated macromolecules that were synthesized during a 6-h incorporation were separated by CsBr density-gradient centrifugation and gel-filtration chromatography successively. Most of the sulfated secreted macromolecules corresponded to a population of glycoproteins sensitive to reductive beta-elimination but resistant to both chondroitinase ABC and heparinase. These glycoproteins had different buoyant densities (ranging from 1.48 g/ml to 1.16 g/ml) and could be subfractionated according to molecular mass. A major part of the radioactivity was incorporated into high-molecular-mass mucins that were excluded from a Sepharose CL-2B column and did not penetrate into polyacrylamide gel in PAGE. However, a mixture of sulfated O-glycoproteins of much lower molecular mass was also characterized in addition to low amounts of chondroitin sulfate. Epithelial goblet cells are the predominant mucin-containing cells of the respiratory guinea pig trachea. Our results suggest that a wide range of sulfated O-glycoproteins are secreted by the guinea pig tracheal mucosa.

Effect of pyocyanin and 1-hydroxyphenazine on in vivo tracheal mucus velocity.

Products of the bacterium Pseudomonas aeruginosa have been shown to slow the beating of human respiratory tract cilia in vitro. We have tested the effects of two of these compounds, pyocyanin and 1-hydroxyphenazine (given as a bolus dose dissolved in 2 microliters Ringer solution), on tracheal mucus velocity of radiolabeled erythrocytes in anesthetized guinea pigs. 1-Hydroxyphenazine (200 ng) caused a rapid slowing of tracheal mucus velocity (maximum fall 47% at 20 min) with recovery by 1 h. The effect of pyocyanin was slower in onset, 600 ng causing 60% reduction in tracheal mucus velocity at 3 h, and no recovery occurred. A combination of pyocyanin and 1-hydroxyphenazine produced an initial rapid slowing equivalent to the same dose of 1-hydroxyphenazine given alone, but the later slowing attributed to pyocyanin was greater than the same dose administered alone. This study demonstrates one mechanism by which products of P. aeruginosa may facilitate its colonization of the respiratory tract.

The effect of acetylsalicylate on aggregation and arachidonate metabolism by human platelets suspended in plasma or buffer.

Acetylsalicylate inhibits prostaglandin and thromboxane production by human platelets suspended in plasma or buffer. Acetylsalicylate inhibits arachidonate-induced aggregation of human platelets suspended in plasma, but the effect of acetylsalicylate on arachidonate-induced aggregation of human washed platelets in buffer has not been reported. We have therefore studied this in relation to arachidonate metabolism in human platelets suspended in plasma or buffer. Platelets suspended in plasma and in buffer were both prepared from each donor, who had not taken acetylsalicylate or like-acting drugs for at least 7 days. Acetylsalicylate was 1500 times less potent in inhibiting arachidonate-induced aggregation in buffer (IC50 = 27.3 +/- 7.5 (s.e.m.)mM) than it was in plasma (IC50 = 18.3 +/- 6.0 microM); whereas it was only 4 times less potent in inhibiting thromboxane production in buffer (IC50 = 110 +/- 51.0 microM) than in plasma (IC50 = 25.3 +/- 8.9 microM). The acetylsalicylate concentration required to inhibit aggregation in buffer was sufficient to inhibit 12-hydroxyeicosatetraenoic acid production whereas the concentration that inhibited thromboxane production in buffer was not. These results indicate that arachidonate-induced aggregation of platelets in buffer may depend on product(s) of lipoxygenase rather than of cyclooxygenase, and is hence insensitive to inhibition by acetylsalicylate compared with arachidonate-induced aggregation of platelets in plasma.

Comparisons between the abilities of various human and ovine plasmas to inhibit prostaglandin synthesis.

The ability of various human and ovine blood plasmas to inhibit prostaglandin synthesis in vitro has been tested. Human plasmas were significantly more potent in their ability to inhibit prostaglandin synthesis than their counterpart ovine plasmas. In general, female plasma had greater inhibitory activities than male plasmas and adult plasmas were more active than fetal plasmas. There was no simple correlation between the activity of plasmas as inhibitors of prostaglandin synthesis and their respective albumin or haptoglobin contents.

Prostaglandin synthesis inhibition by maternal and fetal sheep plasma and its relation to haptoglobin and albumin levels.

Mammalian plasmas and sera have been reported to contain endogenous inhibitors of prostaglandin synthesis (EIPS), but the identity and role of these suggested inhibitors is as yet undetermined. Albumin and haptoglobin have been proposed as possible inhibitors, and it has been suggested that EPIS may have a part to play in the control of PG production during pregnancy and in the neonatal period. As part of a series of studies aimed at elucidating the identity and role of EIPS, maternal and fetal blood samples were collected from chronically catheterized pregnant ewes, and plasma levels of albumin, haptoglobin and EIPS activity determined. Pregnant ewe plasma possessed high EIPS activity and fetal lamb plasma little or no EIPS activity. Levels of albumin and/or haptoglobin did not consistently parallel that of EIPS activity. A post-operative rise (4 sheep studied) and a pre-parturition nadir (1 sheep studied) in maternal plasma EIPS activity were also noted. The possible physiological significance of these results is discussed.

Paracetamol potentiates acetylsalicylate in inhibiting prostaglandin synthesis.

Lower concentrations of paracetamol stimulated, but a higher concentration inhibited prostaglandin synthesis by bull seminal vesicle homogenate in the absence of added co-factors. Admixed with acetylsalicylate or indomethacin, paracetamol strongly potentiated the inhibition of prostaglandin synthesis in vesicle homogenate, and weakly potentiated inhibition in rat gastric fundus strip. We propose that, by acting as a phenolic co-factor, paracetamol stimulates prostaglandin synthesis and thus renders the cyclo-oxygenase more vulnerable to acetylsalicylate or indomethacin.

Endogenous inhibitor of prostaglandin synthetase.

Mammalian serum and plasma contain an endogenous inhibitor of prostaglandin synthetase (EIPS). Human plasma fractions rich in EIPS show anti-inflammatory activity in vivo. In rats, glucocorticoids raise EIPS activity of plasma and serum. These findings suggest the existence of a natural mechanism of controlling prostaglandin synthesis, possibly related to corticosteroid action.

The local antinociceptive and topical anti-inflammatory effects of propyl gallate in rodents.

1 In common with several anti-inflammatory, analgesic, local anaesthetic and antioxidant drugs, propyl gallate in vitro inhibited the biosynthesis of prostaglandin E2 and F2alpha from arachidonic acid by a prostaglandin synthetase from bull seminal vesicles. 2 In common with analgesic drugs, propyl gallate reduced the ability of arachidonic acid, acetylcholine or acetic acid to cause abdominal constriction in mice. 3 Using a new method for evaluating anti-inflammatory activity, we demonstrated the effectiveness of aspirin or indomethacin given subcuteneously before u.v. irradiation of guinea-pig ears, the prophylactic action of topically applied sunscreen agents and the therapeutic value of bufexamac and propyl gallate applied after irradiation.

Stimulation of prostaglandin biosynthesis by drugs: effects in vitro of some drugs affecting gut function.

Low concentrations of several emetic, purgative or irritant drugs in the absence of added co-factors stimulated conversion of arachidonic acid to prostaglandin E2 and F2alpha by prostaglandin synthetase extracted from bull seminal vesicles (BSV prostaglandin synthetase). Their effect was dependent on concentration and time. Stimulation of BSV prostaglandin synthetase by apomorphine, aloes, tyramine or zingerone was increased several-fold by addition of reduced glutathione to the incubation medium, whereas hydroquinone, a phenolic co-factor of prostaglandin synthetase caused slight depression. From this finding and from the observation that many of the stimulant drugs possess a phenolic group, whereas their inactive relatives lack such a group, it is suggested that these stimulant drugs act as co-factors for prostaglandin synthetase in place of hydroquinone. Aloes, tyramine, ethanol and quipazine also produced a dose-related increase in resting tone of the isolated fundus of the rat stomach. This increase occurred at concentrations comparable to those effective in stimulating BSV prostaglandin synthetase, and was abolished by acetylsalicylate. These findings support the view that certain drugs exert some of their pharmacological effects by stimulating prostaglandin synthetase.

Inhibition of prostaglandin biosynthesis by sulphasalazine and its metabolites.

Sulphasalazine (SZ) inhibits prostaglandin (PG) biosynthesis in vitro with a potency comparable to that of aceylsalicylate. The metabolites of SZ, sulphapyridine and 5-aminosalicylic acid, were of considerably lower potency as inhibitors of PG biosynthesis in the synthetase preparations used. Th inhibition of prostaglandin production by SZ could at least partly account for the clinical utility of sulphasalazine in ulcerative colitis. Sulphapyridine may help to maintain inhibitory concentrations of SZ by restraining bacterial breakdown of the active drug.