Library

Notes: Abstract— Microscopic fluorescence analysis of fura-2-loaded bovine adrenal chromaffin cells demonstrates that ∼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 μM 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 eicosa-pentaenoic acid, were all ineffective. The current-voltage relationships suggest that arachidonic acid may activate voltage-insensitive channels. Arachidonic acid (2μM) 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 (54.02 ± 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 conductarce for Ca2+ of arachidonic acid-activated channel was ∼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. These results demonstrate that arachidonic acid activates cation-selective, Ca2+-permeable channels in bovine adrenal chromaffin cells.

Notes: Abstract: 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-tetra-decanoylphorbol 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 μM, these [Ca2+]i changes were not affected by any of the organic Ca2+-channel blockers, i.e., verapamil, diltiazem, nifedipine, and nicardipine, at 1 μM, 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.

Notes: Abstract: 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 (± 10 μM), 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 μM) 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.

Notes: Abstract: 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 μM PGE2, and both stimulations were markedly inhibited by amiloride, with half-maximal inhibition at 10 μM. 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.

Notes: Abstract: We recently reported that prostaglandin E2 (PGE2) stimulated phosphoinositide metabolism in cultured 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. The effect on catecholamine release was specific for prostaglandin E1 (PGE1) and PGE2 among prostaglandins tested (E1= E2 〉 F2α 〉 D2). The release evoked by PGE2 plus ouabain was greatly reduced in Na+-depleted medium and not observed in Ca2+-free medium. Here we examined the synergistic effect of PGE2 and ouabain on the release with specific reference to ion fluxes. Regardless of the presence of PGE2, ouabain stimulated the release in a dose-dependent manner with half-maximal stimulation at 1 μM, and omission of K+ from the medium, a condition which suppresses the Na+, K+-ATPase activity, also enhanced the release from chromaffin cells exposed to PGE2. Ouabain induced a continuous accumulation of 22Na+ and 45Ca2+, as well as secretion of catecholamines. Although PGE2 itself showed hardly any effects on these cellular responses, PGE2 potentiated all of them induced by ouabain. The time course of catecholamine release was correlated with that of accumulation of 45Ca2+ rather than with that of 22Na+. The release evoked by PGE2 and ouabain was inhibited in a dose-dependent manner by amiloride and the analogue ethylisopropylamiloride, inhibitors of the Na+,H+-antiport, but not by the Na+-channel inhibitor tetrodotoxin nor by the nicotinic receptor antagonist hexamethonium. Ethylisopropylamiloride at 1 μM inhibited PGE2-enhanced accumulation of 22Na+ and 45Ca2+ and release of catecholamine by 40, 83, and 71%, respectively. Activation of the Na+,H+-antiport by elevation of the extracellular pH from 6.6 to 8.0 increased the release of catecholamines linearly. Furthermore, PGE2 induced a sustained increase in intracellular pH by about 0.1 pH unit above the resting value, which was abolished by amiloride or in Na+-free medium. These results taken together indicate that PGE2 activates the Na+,H+-antiport by stimulating phosphoinositide metabolism and that the increase in intracellular Na+ by both inhibition of Na+, K+-ATPase and activation of Na+,H+-antiport may lead to the redistribution of Ca2+, which is the initial trigger of catecholamine release.

Notes: Prostaglandin F2α (PGF2α) binds to its receptor (FP) to increase the intracellular-free calcium concentration ([Ca2+]i) by coupling of FP with Gq protein. Spinal intrathecal administration of PGF2α to mouse induces touch-evoked pain (mechanical allodynia), in which capsaicin-insensitive primary afferent Aβ-fibres and N-methyl-d-aspartate receptor ɛ4 subunit are involved. FP in the spinal cord, however, was not well characterized. Here, we showed constitutive expression of FP mRNA in mouse spinal cord, and functionally characterized spinal FP-expressing cells which were involved in PGF2α-induced mechanical allodynia. The method for repetitive administration of oligodeoxyribonucleotides through tubing to conscious mice was established for mechanical allodynia evaluation. We identified an antisense oligodeoxyribonucleotide targeting FP mRNA, causing both disappearance of PGF2α-induced mechanical allodynia and decrease of FP mRNA. With saline-administered mice, PGF2α rapidly increased [Ca2+]i of the cells in the deeper layer of the dorsal horn. In contrast, when the FP antisense oligodeoxyribonucleotide was repeatedly administered, the population of PGF2α-responsive cells in the slices reduced, and PGF2α-induced [Ca2+]i increase of these cells diminished. These data strongly suggested that, in the dorsal horn of the spinal cord, there are the FP-expressing cells which are involved in PGF2α-induced mechanical allodynia.

Notes: At the spinal level, the involvement of nociceptin/orphanin FQ (N/OFQ) in pain transmission is controversial. JTC-801, a selective nonpeptidergic N/OFQ antagonist, is a good tool to examine the involvement of endogenous N/OFQ in pathophysiological conditions. In the present study, we studied the effect of JTC-801 on neuropathic pain induced by L5 spinal nerve transection in mice. Thermal hyperalgesia was evident on day 3 postsurgery and maintained during the 10-day experimental period. Oral administration of JTC-801 relieved the thermal hyperalgesia in neuropathic mice in a dose-dependent manner. Following L5 nerve transection, the increase in nitric oxide synthase (NOS) activity was observed in the superficial layer of dorsal horn and around the central canal in the spinal cord by NADPH diaphorase histochemistry. Using the novel fluorescent nitric oxide (NO) detection dye diaminofluorescein-FM, we confirmed that NO production increased in the spinal slice prepared from neuropathic mice and that the increase was more prominent in the ipsilateral side to the nerve transection than in the contralateral side. These increases in NOS activity and NO production in neuropathic mice were blocked by pretreatment of oral JTC-801. Although intraperitoneal injection of the nonselective NOS inhibitor NG.-nitro-L-arginine methyl ester transiently, but significantly, attenuated neuropathic hyperalgesia, inducible NOS-deficient mice showed neuropathic pain after L5 spinal nerve transection. These results suggest that N/OFQ is involved in the maintenance of neuropathic pain and that the analgesic effect of JTC-801 on neuropathic pain is mediated by inhibition of NO production by neuronal NOS.