Propofol acts at the sigma-1 receptor and inhibits pentazocine-induced c-Fos expression in the mouse posterior cingulate and retrosplenial cortices.

BACKGROUND: The sigma-1 receptor is functionally linked with psychotomimetic effects of various drugs. A sigma-1 receptor agonist enhances bradykinin-induced intracellular Ca(2+) concentration ([Ca(2+)]i) increase and induces c-Fos expression in a part of the brain. The aim of this study was to investigate the effects of several intravenous anaesthetics on the sigma-1 receptor. METHODS: First, using Wistar rat brains, (+)[(3)H]SKF-10,047, a selective sigma-1 receptor agonist was displaced by propofol, dexmedetomidine, droperidol, and thiopental. Second, Fura-2 loaded NG-108 cells were incubated with (+)pentazocine, a selective sigma-1 receptor agonist, and propofol and then its fluorescence was observed after stimulation with bradykinin. Third, male ICR mice received Intrafat or propofol intraperitoneally (i.p.), followed by pentazocine i.p. Brain slices were prepared and Fos-like immunoreactivity was detected using an immunohistochemical method. results: Propofol, droperidol, and dexmedetomidine displaced (+)[(3)H]SKF-10,047 binding in a concentration-dependent manner with Ki50s of 10.2 +/- 0.6, 0.17 +/- 0.03, 5.73 +/- 1.2 microM, respectively. Thiopental sodium was practically ineffective. Propofol produced a statistically significant reduction in the maximal binding capacity (Bmax) but did not affect the dissociation constant (K(d)). (+)Pentazocine significantly enhanced bradykinin-induced [Ca(2+)]i increases, but propofol did not affect it. Pentazocine induced marked Fos-LI positive cells in the posterior cingulate and retrosplenial cortices (PC/RS), which was significantly reduced by propofol. CONCLUSIONS: These results suggest that propofol may be a sigma-1 receptor antagonist, and that various effects of propofol on the brain may be mediated, at least partly, by the sigma-1 receptor.

Nonneurogenic hypoxia sensitivity in rat adrenal slices.

A change in the intracellular Ca(2+) ([Ca(2+)](i)) level induced by hypoxia was detected in rat adrenal slices by use of fura-2/AM. After hypoxic stress, an increase in [Ca(2+)](i) was observed only in the adrenal medulla. This increase was inhibited by nifedipine, but not modified by the cholinergic receptor blockers. The hypoxia-induced increase in [Ca(2+)](i) was observed in all postnatal developmental stages to a similar extent, whereas the nicotine and high K(+) sensitivities increased along with postnatal development. A 10 nM ryanodine enhanced the hypoxia-induced [Ca(2+)](i) increase in adult but not in neonatal rat slices. These results suggest the existence of an oxygen-sensing mechanism in adult rat adrenals even after sympathetic innervation. Hypoxic responses seemed to be similar both in neonate and in adult rat adrenals and were triggered by the influx of Ca(2+) via L-type voltage-sensitive Ca(2+) channels. However, the sustained [Ca(2+)](i) increase caused by hypoxia might depend on postnatal development and be triggered by Ca(2+)-induced Ca(2+) release (CICR).

Hypoxia-induced catecholamine release and intracellular Ca2+ increase via suppression of K+ channels in cultured rat adrenal chromaffin cells.

Hypoxia (5% O2) enhanced catecholamine release in cultured rat adrenal chromaffin cells. Also, the intracellular free Ca2+ concentration ([Ca2+]i) increased within 3 min in approximately 50% of the chromaffin cells under hypoxic stimulation. The increase depended on the presence of extracellular Ca2+. Nifedipine and omega-conotoxin decreased the population of the cells that showed the hypoxia-induced [Ca2+]i increase, showing that the Ca2+ influx was attributable to L- and N-type voltage-dependent Ca2+ channels. The membrane potential was depolarized during the perfusion with the hypoxic solution and returned to the basal level following the change to the normoxic solution (20% O2). Membrane resistance increased twofold under the hypoxic condition. The current-voltage relationship showed a hypoxia-induced decrease in the outward K+ current. Among the K+ channel openers tested, cromakalim and levcromakalim, both of which interact with ATP-sensitive K+ channels, inhibited the hypoxia-induced [Ca2+]i increase and catecholamine release. The inhibitory effects of cromakalim and levcromakalim were reversed by glibenclamide and tolbutamide, potent blockers of ATP-sensitive K+ channels. These results suggest that some fractions of adrenal chromaffin cells are reactive to hypoxia and that K+ channels sensitive to cromakalim and glibenclamide might have a crucial role in hypoxia-induced responses. Adrenal chromaffin cells could thus be a useful model for the study of oxygen-sensing mechanisms.

Prostaglandin F2 alpha receptor is coupled to Gq in cDNA-transfected Chinese hamster ovary cells.

We examined intracellular signal transduction of prostaglandin F2 alpha (PGF2 alpha) receptors by transfection and stable expression of the receptor cDNA in Chinese hamster ovary (CHO-PGF2 alpha) cells. The binding to the membrane was specific for PGF2 alpha, and the Scatchard plot analysis showed a single high-affinity binding site (Kd = 25.2 nM). PGF2 alpha gradually elevated intracellular Ca2+ and stimulated phosphoinositide metabolism in CHO-PGF2 alpha cells. In whole-cell clamp recordings, PGF2 alpha induced an outward current in the presence of external Ca2+, but it induced a long-lasting inward Ca2+ current in a Na(+)-free solution containing K+ channel blockers. Gq alpha antibody applied intracellularly blocked both outward and inward currents induced by 1 microM PGF2 alpha. These results demonstrate that the PGF2 alpha receptor is coupled to phosphoinositide metabolism in CHO-PGF2 alpha cells via Gq.

Characterization of the substance P receptor-mediated calcium influx in cDNA transfected Chinese hamster ovary cells. A possible role of inositol 1,4,5-trisphosphate in calcium influx.

In Chinese hamster ovary cells expressing the substance P (SP) receptor clone (CHO-SPR cells), we examined SP-stimulated [Ca2+]i changes by microscopic fluorescence analysis and electrophysiological recordings. In fura-2-loaded cells, SP (1 microM) induced a prolonged elevation of [Ca2+]i, which comprised a rapid and transient Ca2+ mobilization and a prolonged phase of Ca2+ entry, but thrombin (1 unit/ml) induced only transient elevation of [Ca2+]i. The formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) was stimulated to 230% above the control by SP but 10% by thrombin 10 s after stimulation. In whole cell clamp recordings, SP induced a long lasting inward current, whereas thrombin did not evoke an inward current. Gq alpha antibody applied intracellularly blocked the SP-induced current, but GS alpha antibody did not block it. Furthermore, decavanadate and heparin, inhibitors of Ins(1,4,5)P3 binding to its receptor, suppressed the SP-induced current. In cell-attached patch, bath-applied SP activated channel currents carried by Ba2+, Ca2+, or Na+. In inside-out patches, Ins(1,4,5)P3, but neither inositol 1,3,4-trisphosphate nor inositol 1,3,4,5-tetrakisphosphate, activated channel currents carried by Ba2+, Ca2+, or Na+. The channel activity induced by Ins(1,4,5)P3 was abolished by heparin. These results demonstrate that SP induces Ca2+ entry through activation of cation channels and suggest that Ins(1,4,5)P3 may regulate both SP-induced Ca2+ mobilization and Ca2+ entry in CHO-SPR cells.

Arachidonic acid activates cation channels in bovine adrenal chromaffin cells.

Microscopic fluorescence analysis of fura-2-loaded bovine adrenal chromaffin cells demonstrates that approximately 70% of the cells responded to arachidonic acid in increasing the intracellular Ca2+ concentration. Because this increase was markedly less in the absence of external Ca2+, we examined the effect of arachidonic acid on Ca2+ influx electrophysiologically. Bath application of 10 microM arachidonic acid induced a long-lasting inward current when the cell was clamped at -50 mV. Other fatty acids, such as oleic acid, linoleic acid, eicosatrienoic acid, and eicosapentaenoic acid, were all ineffective. The current-voltage relationships suggest that arachidonic acid may activate voltage-insensitive channels. Arachidonic acid (> or = 2 microM) activated a single-channel current in the inside-out patch, even in the presence of inhibitors of cyclooxygenase and lipoxygenase, possibly suggesting that arachidonic acid could activate channels directly. The onset delay of the inward channel current in the outside-out patch configuration (54.2 +/- 63.5 s; mean +/- SD) was significantly shorter than that in the inside-out patch one (197.3 +/- 177.7 s). Washout of arachidonic acid decreased the probability of channel openings in the outside-out patch but not in the inside-out one. These results suggest that arachidonic acid activates channels reversibly from outside of the plasma membrane. The unitary conductance for Ca2+ of arachidonic acid-activated channel was approximately 17 pS. The arachidonic acid-activated channel was permeable to Ba2+, Ca2+, and Na+ but not to Cl-. The opening probability of the arachidonic acid-activated channel did not depend on membrane potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostaglandin E2 activates Ca2+ channels in bovine adrenal chromaffin cells.

We have demonstrated that prostaglandin E2 (PGE2) treatment of bovine adrenal chromaffin cells results in a sustained elevation of intracellular Ca2+ concentration ([Ca2+]i) in these cells. Because the continued elevation of [Ca2+]i was dependent on extracellular Ca2+ concentration, it can be assumed that the PGE2-induced [Ca2+]i increase is due, at least in part, to an opening of membrane Ca2+ channels. In this study, we used electrophysiological methods to examine the mechanism of the PGE2-induced [Ca2+]i increase directly. Puff application of PGE2 to the external medium resulted in a prolonged depolarization in about half of the chromaffin cells examined. In whole-cell voltage-clamp recordings, an increase in inward current was observed over a 6-7 min period following bath application of PGE2 (greater than or equal to 10 microM), even in the absence of external Na+. This inward current was abolished when the recordings were made with the cells in a Ca2(+)-free medium, but it was not inhibited by Mn2+, a blocker of voltage-dependent Ca2+ channels. In cell-attached patch-clamp configuration, PGE2 produced an increase in the opening frequency of inward currents. The reversal potential of the PGE2-induced currents was about +40 mV, which is close to the reversal potential of the Ca2+ channel. The opening frequency was not affected by membrane potential changes. In inside-out patch-clamp configuration, inositol 1,4,5-trisphosphate (2 microM) added to the cytoplasmic side activated the Ca2(+)-channel currents, but PGE2 was ineffective when applied to the cytoplasmic side. These results suggest that PGE2 activates voltage-independent Ca2+ channels in chromaffin cells through a diffusible second messenger, possibly inositol 1,4,5-trisphosphate.

Characterization of prostaglandin E2-induced Ca2+ mobilization in single bovine adrenal chromaffin cells by digital image microscopy.

We recently reported that prostaglandin E2 (PGE2) stimulates phosphoinositide metabolism accompanied by an increase in intracellular free Ca2+ concentration ([Ca2+]i) in cultured bovine adrenal chromaffin cells. In the present study, temporal and spatial changes in [Ca2+]i induced by PGE2 in fura-2-loaded individual cells were investigated by digital image microscopy and were compared with those induced by nicotine and histamine. Image analysis of single cells revealed that responses to PGE2 showed asynchrony with the onset of [Ca2+]i changes. After a lag time of 10-30 s, PGE2-induced [Ca2+]i changes took a similar prolonged time course in almost all cells: a rapid rise followed by a slower decline to the basal level over 5 min. Few cells exhibited oscillations in [Ca2+]i. In contrast, nicotine and histamine induced rapid and transient [Ca2+]i changes, and these [Ca2+]i changes were characteristic of each stimulant. Whereas pretreatment of the cells with pertussis toxin (100 ng/ml, 6 h) did not block the response to any of these stimulants, treatment with 12-O-tetradecanoylphorbol 13-acetate (100 nM, 10 min) completely abolished [Ca2+]i changes elicited by PGE2 and histamine. In a Ca2(+)-free medium containing 3 mM EGTA, or in medium to which La3+ was added, the [Ca2+]i response to nicotine disappeared, but that to histamine was not affected significantly. Under the same conditions, the percentage of the cells that responded to PGE2 was reduced to 37% and the prolonged [Ca2+]i changes induced by PGE2 became transient in responding cells, suggesting that the maintained [Ca2+]i increase seen in normal medium is the result of a PGE2-stimulated entry of extracellular Ca2+. Whereas the organic Ca2(+)-channel blocker nicardipine inhibited [Ca2+]i changes by all stimulants at 10 microM, these [Ca2+]i changes were not affected by any of the organic Ca2(+)-channel blockers, i.e., verapamil, diltiazem, nifedipine, and nicardipine, at 1 microM, a concentration high enough to inhibit voltage-sensitive Ca2+ channels. These results demonstrate that PGE2 may promote Ca2+ entry with concomitant release of Ca2+ from intracellular stores and that the mechanism(s) triggered by PGE2 is apparently different from that by histamine or nicotine.

Sodium fluoride mimics the effect of prostaglandin E2 on catecholamine release from bovine adrenal chromaffin cells.

We have reported recently that prostaglandin E2 (PGE2) stimulated phosphoinositide metabolism in bovine adrenal chromaffin cells and that PGE2 and ouabain, an inhibitor of Na+, K(+)-ATPase, synergistically induced a gradual secretion of catecholamines from the cells. Here we examined the involvement of a GTP-binding protein(s) in PGE receptor-induced responses by using NaF. In the presence of Ca2+ in the medium, NaF stimulated the formation of all three inositol phosphates, i.e., inositol monophosphate, bisphosphate, and trisphosphate, linearly over 30 min in a dose-dependent manner (15-30 mM). This effect on phosphoinositide metabolism was accompanied by an increase in cytosolic free Ca2+. NaF also induced catecholamine release from chromaffin cells, and the dependency of stimulation of the release on NaF concentration was well correlated with those of NaF-enhanced inositol phosphate formation and increase in cytosolic free Ca2+. Although the effect of NaF on PGE2-induced catecholamine release in the presence of ouabain was additive at concentrations below 20 mM, there was no additive effect at 25 mM NaF. Furthermore, the time course of catecholamine release stimulated by 20 mM NaF in the presence of ouabain was quite similar to that by 1 microM PGE2, and both stimulations were markedly inhibited by amiloride, with half-maximal inhibition at 10 microM. Pretreatment of the cells with pertussis toxin did not prevent, but rather enhanced, PGE2-induced catecholamine release over the range of concentrations examined. These results demonstrate that NaF mimics the effect of PGE2 on catecholamine release from chromaffin cells and suggest that PGE2-evoked catecholamine release may be mediated by the stimulation of phosphoinositide metabolism through a putative GTP-binding protein insensitive to pertussis toxin.

Amiloride-sensitive Na+-H+ antiporter in Escherichia coli.

In everted vesicles of Escherichia coli, delta pH caused by H+ efflux through the Na+/H+ antiporter was measured by using a fluorescent dye. Amiloride inhibited the activity of the Na+/H+ antiporter. Kinetic studies showed that amiloride competed with Na+. The inhibition constant of 40 microM was obtained.