Involvement of glutamate receptors in allodynia induced by prostaglandins E2 and F2 alpha injected into conscious mice

In order to investigate the involvement of glutamate receptor systems in allodynia induced by prostaglandin (PG) E2 or F2 alpha, we co-administered antagonists for N-methyl-D-aspartate (NMDA), non-NMDA, or metabotropic glutamate receptors intrathecally with PGE2 or PGF2 alpha and examined their effects on the allodynia evoked in conscious mice by non-noxious brushing of the flanks. MK-801, a non-competitive NMDA receptor channel blocker, and D-AP-5, a selective NMDA receptor antagonist, dose-dependently blocked PGE2-induced allodynia with an IC50 of 1.60 and 0.52 microgram/mouse, respectively. A glycine binding-site antagonist for the NMDA receptor, 7-Cl-KYNA, did not influence it. None of these NMDA receptor antagonists inhibited PGF2 alpha-evoked allodynia. Non-NMDA receptor antagonists GAMS and CNQX inhibited both PGE2- and PGF2 alpha-induced allodynia. On the other hand, L-AP-3 and L-AP-4, putative metabotropic glutamate receptor antagonists, dose-dependently antagonized the allodynia induced by PGF2 alpha with an IC50 of 0.92 and 3.26 ng/mouse, respectively, but not that induced by PGE2. Intrathecal administration of L-glutamate produced allodynia over a wide range of low doses from 0.1 pg to 0.1 microgram/mouse, and the maximal effect was observed at 1 ng. Similar to allodynia induced by prostaglandins, the response lasted over a 50-min experimental period. These results demonstrate that both PGE2- and PGF2 alpha-evoked allodynia are mediated through a pathway that includes the glutamate receptor system but that subtypes of glutamate receptors involved and sites of action in the spinal cord may be different between them.     

Inhibition of nociceptin-induced allodynia in conscious mice by prostaglandin D2

1. We recently showed that intrathecal administration of nociceptin induced allodynia by innocuous tactile stimuli and hyperalgesia by noxious thermal stimuli in conscious mice. In the present study, we examined the effect of prostaglandins on nociceptin-induced allodynia and hyperalgesia. 2. Prostaglandin D2 (PGD2) blocked the allodynia induced by nociceptin in a dose-dependent manner with an IC50 of 26 ng kg(-1), but did not affect the nociceptin-induced hyperalgesia at doses up to 500 ng kg(-1). BW 245C (an agonist for PGD (DP) receptor) blocked the allodynia with an IC50 of 83 ng kg(-1). 3. The blockade of nociceptin-induced allodynia by PGD2 was reversed by the potent and selective DP-receptor antagonist BW A868C in a dose-dependent manner with an ED50 of 42.8 ng kg(-1). 4. Glycine (500 ng kg[-1]) almost completely blocked the nociceptin-induced allodynia. A synergistic effect on the inhibition of nociceptin-evoked allodynia was observed between glycine and PGD2 at below effective doses. 5. Dibutyryl cyclic AMP, but not dibutyryl cyclic GMP, blocked the nociceptin-induced allodynia with an IC50 of 2.9 microg kg(-1). 6. PGE2, PGF2alpha, butaprost (an EP2 agonist) and cicaprost (a PGI receptor agonist) did not affect the nociceptin-induced allodynia. 7. These results demonstrate that PGD2 inhibits the nociceptin-evoked allodynia through DP receptors in the spinal cord and that glycine may be involved in this inhibition.     

Characterization of the inhibitory prostanoid receptors on human neutrophils

1. We have evaluated the effects of various prostanoid agonists on the release of leukotriene B4 (LTB4) and superoxide anions (O2-) from human neutrophils stimulated with opsonized zymosan (OZ) and formyl-methionyl-leucyl-phenylalanine (FMLP), respectively. 2. Prostaglandin E2 (PGE2) and PGD2 inhibited both OZ-induced LTB4 release (EC50 0.72 microM and 0.91 microM respectively), and FMLP-induced O2- release (EC50 0.42 microM and 0.50 microM respectively). PGF2 alpha, the TP-receptor agonist, U46619, and the IP-receptor agonist, iloprost, were also active, but were all at least an order of magnitude less potent than PGE2 and PGD2. 3. The EP2/EP3-receptor agonist, misoprostol, and the selective EP2-agonist, AH13205, were both effective inhibitors of LTB4 release, being approximately equipotent with and 16-times less potent than PGE2, respectively. In contrast, the EP1/EP3-receptor agonist, sulprostone, had no inhibitory activity at concentrations of up to 10 microM. 4. The selective DP-receptor agonist, BW245C, inhibited LTB4 release, (EC50 0.006 microM) being approximately 50 times more potent than PGD2. BW245C also inhibited O2- release, and this inhibition was antagonized competitively by the DP-receptor blocking drug, AH6809 (pA2 6.6). 5. These data indicate the presence of both inhibitory EP- and DP-receptors on the human neutrophil. The rank order of potency of EP-receptor agonists suggest that the EP-receptors are of the EP2-subtype.     

Prostaglandin E2 suppression of acetylcholine release from parasympathetic nerves innervating guinea-pig trachea by interacting with prostanoid receptors of the EP3-subtype

1. We have demonstrated recently that exogenous prostaglandin E2 (PGE2) inhibits electrical field stimulation (EFS)-induced acetylcholine (ACh) release from parasympathetic nerve terminals innervating guinea-pig trachea. In the present study, we have attempted to characterize the pre-junctional prostanoid receptor(s) responsible for the inhibitory action of PGE2 and to assess whether other prostanoids modulate, at a prejunctional level, cholinergic neurotransmission in guinea-pig trachea. To this end, we have investigated the effect of a range of both natural and synthetic prostanoid agonists and antagonists on EFS-evoked [3H]-ACh release. 2. In epithelium-denuded tracheal strips pretreated with indomethacin (10 microM), PGE2 (0.1 nM-1 microM) inhibited EFS-evoked [3H]-ACh release in a concentration-dependent manner with an EC50 and maximal effect of 7.62 nM and 74% inhibition, respectively. Cicaprost, an IP-receptor agonist, PGF2alpha and the stable thromboxane mimetic, U46619 (each at 1 microM), also inhibited [3H]-ACh release by 48%, 41% and 35%, respectively. PGD2 (1 microM) had no significant effect on [3H]-ACh release. 3. The selective TP-receptor antagonist, ICI 192,605 (0.1 microM), completely reversed the inhibition of cholinergic neurotransmission induced by U-46619, but had no significant effect on similar responses effected by PGE2 and PGF2alpha. 4. A number of EP-receptor agonists mimicked the ability of PGE2 to inhibit [3H]-ACh release with a rank order of potency: GR63799X (EP3-selective) > PGE2 > M&B 28,767 (EP3 selective) > 17-phenyl-omega-trinor PGE2 (EP1-selective). The EP2-selective agonist, AH 13205 (1 microM), did not affect EFS-induced [3H]-ACh release. 5. AH6809 (10 microM), at a concentration 10 to 100 times greater than its pA2 at DP-, EP1- and EP2-receptors, failed to reverse the inhibitory effect of PGE2 or 17-phenyl-omega-trinor PGE2 on [3H]-ACh release. 6. These results suggest that PGE2 inhibits [3H]-ACh release from parasympathetic nerves supplying guinea-pig trachea via an interaction with prejunctional prostanoid receptors of the EP3-receptor subtype. Evidence for inhibitory prejunctional TP- and, possibly, IP-receptors was also obtained although these receptors may play only a minor role in suppressing [3H]-ACh release when compared to receptors of the EP3-subtype. However, the relative importance of the different receptors will depend not only on the sensitivity of guinea-pig trachea to prostanoids but on the nature of the endogenous ligands released locally that have activity on parasympathetic nerves.     

Human endometrial stromal cells generate uncombined alpha-subunit from human chorionic gonadotropin, which can synergize with progesterone to induce decidualization

Prostaglandin E2 (PGE2) is an endogenous hormone of adrenal zona glomerulosa cells and is released in response to stimulation by agonists such as angiotensin II (Ang II). It stimulates the release of aldosterone from cultured bovine adrenal zona glomerulosa cells. These studies were designed to determine whether this steroidogenic effect of PGE2 was mediated by an EP1, EP2, or EP3 receptor. Prostaglandin E2 and 11-deoxy PGE1, an EP2-selective agonist, stimulated aldosterone release in a concentration-related manner with an ED50 of 300 nmol/L for PGE2 and 2 micromol/L for 11-deoxy PGE1. The maximal effect of PGE2 was less than that of angiotensin II. 17-Phenyl trinor PGE2, an EP1-selective agonist, required concentrations of 100 micromol/L to stimulate aldosterone release and sulprostone, an EP3/EP1-selective agonist, failed to alter aldosterone release. The EP1-selective antagonist SC19220 failed to alter basal or PGE2-stimulated aldosterone release over a range of concentrations. PGE2 and 11-deoxy PGE1 also stimulated an increase in both intracellular and extracellular cAMP. This increase was time- and concentration-related. The ED50 for PGE2 was 9.8 micromol/L. 17-Phenyl trinor PGE2 and sulprostone were without effect. Using fura-2 loaded cells, PGE2 (2 micromol/L), dibutyryl cAMP (2 mmol/L), and Ang 11 (2 micromol/L) increased intracellular calcium over basal concentrations by 5.5-fold, 3-fold, and 6.2-fold, respectively. Like PGE2, dibutyryl cAMP also stimulated aldosterone release. PGE2- and dibutyryl cAMP-induced aldosterone release were blocked by the calcium channel inhibitor diltiazem. These studies indicate that PGE2 is a potent stimulus for aldosterone release and that the effect is mediated by EP2 receptors. Both cAMP and calcium appear to mediate the steroidogenic effect of PGE2 and calcium seems to be distal to cAMP.     

Lack of tactile pain (allodynia) in lipocalin-type prostaglandin D synthase-deficient mice

Prostaglandin (PG) D2 is the most abundant prostanoid produced in the central nervous system of mammals and has been implicated in the modulation of neural functions such as sleep induction, nociception, regulation of body temperature, and odor responses. We generated gene-knockout mice for lipocalin-type PGD2 synthase (L-PGDS) and found that the intrathecal administration of PGE2, an endogenous pain-producing substance, failed to elicit allodynia (touch-evoked pain), which is one typical phenomenon of neuropathic pain, whereas it evoked thermal hyperalgesia, in L-PGDS-/- mice. We also found that the allodynic response induced by the gamma-aminobutyric acid (GABA)A receptor antagonist bicuculline was selectively abolished in the L-PGDS-/- mice, among excitatory and inhibitory agents that induced allodynia in wild-type mice. Interestingly, simultaneous injection of a femtogram amount of PGD2 with PGE2 or bicuculline induced allodynia in L-PGDS-/- mice to the same extent as in wild-type mice. The PGE2- or bicuculline-evoked allodynia in wild-type and in PGD2-supplemented L-PGDS-/- mice was blocked by a PGD2 receptor antagonist given in a femtogram amount. These results reveal that endogenous PGD2 is essential for both PGE2- and bicuculline-induced allodynia.     

Modulation of noradrenaline release from the sympathetic nerves of the human saphenous vein and pulmonary artery by presynaptic EP3- and DP-receptors

1. Spirally cut strips of the human saphenous vein and pulmonary artery were used to determine the pharmacological properties of the presynaptic prostanoid receptors involved in the modulation of sympathetic [3H]-noradrenaline release. Strips preincubated with [3H]-noradenaline were superfused with physiological salt solution containing inhibitors of uptake1 and uptake2 and rauwolscine to eliminate involvement of presynaptic alpha 2-adrenoceptors. Tritium overflow was evoked by transmural electrical stimulation (standard frequency: 2 Hz). 2. In the saphenous vein, prostaglandin E2 (PGE2) inhibited the electrically-evoked tritium overflow; at the highest concentration investigated, tritium overflow was inhibited by more than 75% and the pEC50 value was 7.00. These effects were mimicked by prostaglandin E1, the EP1/EP3 receptor agonist, sulprostone and the EP2/EP3 receptor agonist, misoprostol with the rank order (pEC50): sulprostone (8.60) > PGE1 (7.25) > misoprostol (6.96). This rank order of potency suggests that the inhibitory effect of the drugs is mediated by presynaptic EP3-receptors. In contrast, PGF2 alpha did not inhibit evoked tritium overflow; the IP/EP1 receptor agonist iloprost and the stable thromboxane A2 analogue U 46619 (9, 11-dideoxy-11 alpha,9 alpha-epoxy-methanoprostaglandin F2 alpha) produced inhibition only at concentrations above 1 microM. 3. The EP1-receptor antagonist, AH 6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid) had no effect on the evoked tritium overflow nor did it modify the inhibitory effect of PGE2, further excluding involvement of inhibitory presynaptic EP1-receptors. 4. PGD2 caused a facilitation of evoked tritium overflow in the saphenous vein; this facilitation is probably mediated by presynaptic DP-receptors, since it was abolished by the selective DP-receptor antagonist, BW A868C (3-benzyl-5-(6-carboxyhexyl)-1-(2-cyclohexyl-2-hydroxyethylamino)hydantoin).5. In the pulmonary artery, sulprostone (pECm value 8.35), misoprostol (7.70) and PGE2 (6.80)inhibited electrically-evoked tritium overflow. This rank order of potency is consistent with the involvement of inhibitory presynaptic EP3-receptors.6. These results suggest that the sympathetic nerve fibres of both human saphenous vein and pulmonary artery are endowed with presynaptic inhibitory EP3 receptors. The EP3-receptors do not interact with the alpha 2-autoreceptors. In addition, the human saphenous vein seems to be endowed with presynaptic facilitatory DP-receptors.     

Ethanol administration delays recovery from acute pancreatitis induced by exocrine hyperstimulation

A cholera toxin-sensitive, prostaglandin E2 (PGE2) specific receptor has been identified in the plasma membrane fraction of tick salivary glands. In the present study, we report that stimulation of dispersed salivary glands of the lone star tick Amblyomma americanum (L.) with 1 nM to 10 microM PGE2 increased the intracellular concentration of inositol trisphosphate (IP3) in a dose-dependent manner. Incubation of dispersed tissue with 1 nM to 10 microM PGE2 also stimulated release of 45Ca2+ from preloaded tissue. PGE2 (10 microM) did not stimulate an influx of 45Ca2+. Therefore, the PGE2 receptor in the salivary glands appears to activate a phosphoinositide phospholipase C signalling pathway to increase formation of intracellular IP3 and, thus, mobilize Ca2+ from intracellular stores. Incubation of dispersed salivary glands with 1 nM to 1 microM PGE2 stimulated secretion of anticoagulant protein, but not at < 1 nM or > 1 microM PGE2. In addition, the mammalian PGE2 EP1 receptor antagonist AH-6809 affected secretion of anticoagulant by dispersed salivary gland tissue at a low concentration supporting the hypothesis that the PGE2 receptor in tick salivary glands is EP1-like. We propose that a major function for PGE2 in tick salivary glands is to mobilize Ca2+ and stimulate secretion (exocytosis) of bioactive proteins into the tick's saliva during feeding.     

Interactions between excitatory amino acids and tachykinins in the rat spinal dorsal horn

Whole-cell patch-clamp technique of freshly isolated rat spinal dorsal horn (DH) neurons, intracellular recording from DH neurons in a slice preparation, and high performance liquid chromatography with fluorimetric detection of release of endogenous glutamate and aspartate from spinal cord slice following activation of primary afferent fibers were employed to investigate interactions between excitatory amino acids (EAA) and tachykinins [substance P (SP) and neurokinin A (NKA)]. Potentiation of N-methyl-D-aspartate (NMDA)-, quisqualate (QA)- and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-, but not kainate-induced currents by SP and NKA was found. Spantide II, a claimed novel nonselective tachykinin antagonist, effectively blocked the SP (2 nM)-induced potentiation of the responses of DH neurons to NMDA. In the presence of glycine (0.1 microM), the SP-evoked increase of the NMDA-induced current was prevented. However, 7-chlorokynurenic acid (2 microM), a competitive antagonist at the glycine allosteric site of the NMDA receptor, led to the reestablishment of the SP effect. Brief high frequency electrical stimulation of primary afferent fibers produced a long-lasting potentiation of presumed monosynaptic and polysynaptic excitatory postsynaptic potentials and sustained enhanced release of endogenous glutamate (218.3 +/- 66.1%) and aspartate (286.3 +/- 58.0%). Possible functional implications of the observed phenomena are discussed in relation to transmission and integration of sensory information, including pain.     

Cyclic-AMP-dependent Ca2+ influx elicited by prostaglandin D2 in freshly isolated nonchromaffin cells from bovine adrenal medulla

We previously reported that prostaglandin D2 (PGD2) specifically elevates intracellular cyclic AMP in nonchromaffin cells isolated from bovine adrenal medulla (Biochim. Biophys. Acta (1989) 1011, 75-80). Here we again found that PGD2 increased intracellular Ca2+ concentration ([Ca2+]i) in freshly isolated nonchromaffin cells and investigated the cellular mechanisms of PGD2-induced [Ca2+]i increase using the Ca2+ indicator fura-2 and a fluorescence microscopic imaging system. Treatment of the cells with PGD2 receptor agonists BW245C and ZK110841 resulted in both marked stimulation of cyclic AMP formation and an increase in [Ca2+]i. The [Ca2+]i response was also induced by bypassing of the receptor with forskolin, a direct activator of adenylate cyclase, but not by PGE2 or PGF2 alpha both of which are devoid of the ability to generate cyclic AMP in the cells. These cyclic AMP and [Ca2+]i responses induced by PGD2 were completely blocked by the PGD2 receptor antagonist BWA868C. The time-course of cyclic AMP production stimulated by PGD2 coincided with that of the [Ca2+]i increase. While the Ca(2+)-mobilizing hormone bradykinin stimulated a rapid inositol phosphate accumulation in nonchromaffin cells, PGD2 did not stimulate it significantly. Removal of extracellular Ca2+ markedly reduced the Ca2+ response to PGD2 in magnitude and duration, but did not alter the peak [Ca2+]i response to bradykinin. These results demonstrate that PGD2 receptor activation induces the increase in [Ca2+]i via cyclic AMP mainly by increasing the Ca2+ influx from the outside, unlike inositol trisphosphate which causes release of Ca2+ from internal stores.     

M3 muscarinic receptor-mediated enhancement of NMDA-evoked adenosine release in rat cortical slices in vitro

Acetylcholine plays an important role in cortical arousal. Adenosine is released during increased metabolism and has been suggested to be a sleep-promoting factor. To understand the interaction of acetylcholine and adenosine in regulating cortical excitability, we examined the effect of carbachol on NMDA-evoked adenosine release and identified the muscarinic receptor subtype that mediated this effect in adult rat cortical slices in vitro. Carbachol (to 300 microM) alone did not affect the basal release of adenosine. However, carbachol (100 microM) induced a 253% increase in NMDA (20 microM)-evoked adenosine release in the presence of Mg2+. In the absence of Mg2+, carbachol's potentiating effect was less (60% increase). The nonselective muscarinic antagonist atropine (1.5 microM) blocked the facilitatory effect of carbachol on NMDA-evoked adenosine release, and this was mimicked by the M3-selective antagonist 4-diphenylacetoxy-N-methylpiperidine (1 microM). Neither an M1-selective dose of pirenzepine (50 nM) nor the M2-selective antagonist methoctramine (1 microM) affected carbachol's action on NMDA-evoked adenosine release. Carbachol had no effect on adenosine release evoked by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA). These results suggest that acetylcholine does not affect basal adenosine release but enhances NMDA receptor-mediated evoked adenosine release by acting at M3 receptors in the cortex. This interaction may have a role in regulating cortical neuronal excitability on a long-term basis.     

The type 1 angiotensin-II receptor mediates intracellular calcium mobilization in rat luteal cells

The intracellular cytosolic calcium concentration ([Ca]i) was determined in cultured rat luteal cells using the calcium-chelating dye fura-2 and microspectrofluorimetry. Angiotensin-II (Ang-II) induced a dose-dependent transient increase in [Ca]i (ED50, 9.0 +/- 6.5 nM). After the initial peak in [Ca]i, cytosolic calcium returned to a secondary elevated basal level that was dependent upon the presence of extracellular calcium. Pretreatment of rat luteal cells with Ang-II (100 nM) desensitized a subsequent response to a higher concentration (1 microM), but did not desensitize a prostaglandin F2 alpha (PGF2 alpha)-induced calcium flux. Although the peak increases in [Ca]i induced by Ang-II (1 microM) and PGF2 alpha (10 microM) were not significantly different, the plateau phase stimulated by PGF2 alpha was significantly higher (P < 0.05) than that stimulated by Ang-II (1 microM). Pretreatment of luteal cells with the type 2 Ang-II receptor antagonist PD 123319 (10 microM) did not inhibit calcium mobilization; however, Ang-II (1 microM)-induced calcium mobilization was dose dependently blocked by the type 1 Ang-II receptor antagonist Losartan (DuP 753). The ID50 for Losartan was 5.2 +/- 1.8 nM. Pretreatment of the luteal cells with the endoplasmic reticulum calcium ATPase inhibitor thapsigargin (1 microM) also blocked Ang-II-induced calcium mobilization. These data demonstrate the presence of the type 1 Ang-II receptor in rat luteal cells, through which Ang-II dose dependently mobilizes calcium from an intracellular source, probably the endoplasmic reticulum.     

Oligomeric interaction of the PapB transcriptional regulator with the upstream activating region of pili adhesin gene promoters in Escherichia coli

1. Prostanoid receptor-mediated sensitization, or excitation, of sensory nerve fibres contributes to the generation of hyperalgesia. To characterize the prostanoid receptors present on sensory neurones, biochemical assays were performed on primary cultures of adult rat dorsal root ganglia (DRG) and the F-11 (embryonic rat DRG x neuroblastoma hybrid) cell line. 2. In DRG cultures, the IP receptor agonists, cicaprost and carbaprostacyclin (cPGI2) stimulated cyclic AMP accumulation. Prostaglandin E2 (PGE2) also increased cyclic AMP levels, but to a lesser extent, while carbocyclic thromboxane A2 (cTxA2), PGD2 and PGF2alpha had negligible effects. The rank order of agonist potency was cicaprost>PGE2=BMY45778=cPGI2=PGI2. In the F-11 cells, the rank order of agonist potency for the stimulation of cyclic AMP accumulation was: cicaprost>iloprost=cPGI2=PGI2=BMY45778>PGE2=cTXA2++ +. In DRG cultures, cicaprost induced significantly more accumulation of inositol phosphates than PGE2. 3. To examine the effects of prostanoids on C-fibre activity, extracellular recordings of d.c. potentials from the rat isolated vagus nerve were made with the 'grease-gap' technique. PGI2 (0.1 nM-10 microM) produced the largest depolarizations of the nerve. The rank order of agonist potency was: PGI2=cPGI2=PGE1>cTXA2>PGE2=PGD2=TXB2>PGF2alpha. 4. Prior depolarization of nerves with either forskolin (10 microM) or phorbol dibutyrate (1 microM) alone significantly reduced the response to PGI2 (10 microM), while simultaneous application of both forskolin and phorbol dibutyrate attenuated PGI2 responses almost completely. 5. Putative EP1 and/or TP receptor-selective antagonists had no effect on the responses to PGI2, cPGI2 or PGE2 in the three preparations studied. 6. Collectively, these data are consistent with a positive coupling of IP receptors to both adenylyl cyclase and phospholipase C in sensory neurones. These findings suggest that IP receptors play a major role in the sensitization of rat sensory neurones.     

NMDA receptor-mediated control of presynaptic calcium and neurotransmitter release

Before action potential-evoked Ca2+ transients, basal presynaptic Ca2+ concentration may profoundly affect the amplitude of subsequent neurotransmitter release. Reticulospinal axons of the lamprey spinal cord receive glutamatergic synaptic input. We have investigated the effect of this input on presynaptic Ca2+ concentrations and evoked release of neurotransmitter. Paired recordings were made between reticulospinal axons and the neurons that make axo-axonic synapses onto those axons. Both excitatory and inhibitory paired-cell responses were recorded in the axons. Excitatory synaptic inputs were blocked by the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM) and by the NMDA receptor antagonist 2-amino-5-phosphonopentanoate (AP-5; 50 microM). Application of NMDA evoked an increase in presynaptic Ca2+ in reticulospinal axons. Extracellular stimulation evoked Ca2+ transients in axons when applied either directly over the axon or lateral to the axons. Transients evoked by the two types of stimulation differed in magnitude and sensitivity to AP-5. Simultaneous microelectrode recordings from the axons during Ca2+ imaging revealed that stimulation of synaptic inputs directed to the axons evoked Ca2+ entry. By the use of paired-cell recordings between reticulospinal axons and their postsynaptic targets, NMDA receptor activation was shown to enhance evoked release of transmitter from the axons that received axoaxonic inputs. When the synaptic input to the axon was stimulated before eliciting an action potential in the axon, transmitter release from the axon was enhanced. We conclude that NMDA receptor-mediated input to reticulospinal axons increases basal Ca2+ within the axons and that this Ca2+ is sufficient to enhance release from the axons.     

Rate of vasoconstrictor prostanoids released by endothelial cells depends on cyclooxygenase-2 expression and prostaglandin I synthase activity

This study was undertaken to investigate the enzymatic regulation of the biosynthesis of vasoconstrictor prostanoids by resting and interleukin (IL)-1(beta)stimulated human umbilical vein endothelial cells (HUVECs). Biosynthesis of eicosanoids in response to IL-1beta, exogenous labeled arachidonic acid (AA), or histamine, as well as their spontaneous release, was evaluated by means of HPLC and RIA. HUVECs exposed to IL-1beta produced prostaglandin (PG) I2 for no longer than 30 seconds after the substrate was added irrespective of the cyclooxygenase (COX) activity, whereas the time course of PGE2 and PGD2 formation was parallel to the COX activity. The ratio of PGE2 to PGD2 produced by HUVECs was similar to that obtained by purified COX-1 and COX-2. Production of PGF2alpha from exogenous AA was limited and similar in both resting and IL-1beta-treated cells. PGF2alpha was the main prostanoid released into the medium during exposure to IL-1beta, whereas when HUVECs treated with IL-1beta were stimulated with histamine or exogenous AA, PGE2 was released in a higher quantity than PGF2alpha. PGF2alpha released into the medium during treatment with IL-1beta and the biosynthesis of PGE2 and PGD2 in response to exogenous AA or histamine increased with COX-2 expression, whereas this did not occur in the case of PGI2. We observed that PGI synthase (PGIS) mRNA levels were not modified by the exposure to IL-1beta, but the enzyme was partially inactivated. When SnCl2 was added to the incubation medium, the transformation of exogenous AA-derived PGH2 into PGE2 and PGD2 was totally diverted toward PGF2alpha. Overall, these results support the conclusions that PGE2 and PGD2 (and also probably PGF2alpha) were nonenzymatically derived from PGH2 in HUVECs. The concept that a high ratio of PGH2 was released by the IL-1beta-treated HUVECs and isomerized outside the cell into PGE2 and PGD2 was supported by the biosynthesis of thromboxane B2 by COX-inactivated platelets, indicating the uptake by platelets of HUVEC-derived PGH2. The IL-1beta-induced increase in the release of PGH2 by HUVECs was suppressed by the COX-2-selective inhibitor SC-58125 and correlated with both COX-2 expression and PGIS inactivation. An approach to the mechanism of inactivation of PGIS by the exposure to IL-1beta was performed by using labeled endoperoxides as substrate. The involvement of HO. in the PGIS inactivation was supported by the fact that deferoxamine, pyrrolidinedithiocarbamate, DMSO, mannitol, and captopril antagonized the effect of IL-1beta on PGIS to different degrees. The NO synthase inhibitor NG-monomethyl-L-arginine also antagonized the PGIS inhibitory effect of IL-1beta, indicating that NO. was also involved. NO. reacts with O2-. to form peroxynitrite, which has been reported to inactivate PGIS. Homolytic fission of the O-O bond of peroxynitrite yields NO2. and HO.. The fact that 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), which reacts with NO. to form NO2., dramatically potentiated the IL-1beta effect suggests that NO2. could be a species implicated in the inactivation of PGIS. Cooperation of HO. was supported by the fact that DMSO partially antagonized the effect of carboxy-PTIO. Although our results on the exact mechanism of the inactivation of PGIS caused by IL-1beta were not conclusive, they strongly suggest that both NO. and HO. were involved.     

Cognitive changes during growth hormone replacement in adult men

Prostaglandins (PGs) lower intraocular pressure by increasing uveoscleral outflow, presumably via a receptor-mediated mechanism coupled to a second messenger pathway in the ciliary muscle. In the present study, we examined the effect of prostanoids on cyclic AMP production in cultured human ciliary muscle cells. Cells were identified based on their expression of smooth muscle specific alpha-actin and monoclonal antibody against desmin. Cyclic AMP production in confluent cells incubated with buffer solution containing various concentrations of prostanoids was analyzed by radioimmunoassay. PGE2 caused a time-dependent increase in cyclic AMP concentrations which reached a maximum after 10 mins. With the exception of PGD2, all prostanoids produced a concentration-dependent increase in cyclic AMP levels with the following rank order of activity: PGE2 > 11-deoxy-PGE1 > 16,16-dimethyl PGE2 > sulprostone > PGF2alpha. PGE2-induced increase on cyclic AMP levels was unaffected by AH6809, an antagonist at both PGD2 (DP) and E2 (EP1) receptors. Flurbiprofen decreased basal cyclic AMP concentrations suggesting that intramurally-generated PGs stimulate the formation of the nucleotide in ciliary smooth muscle cells. PGE2-induced increases in cyclic AMP production was synergistic with those induced by the diterpene activator of adenylyl cyclase, forskolin. We conclude that prostanoids active at EP2-receptors can stimulate cyclic AMP production in cultured human ciliary muscle cells.     

Gp120 can revert antagonism at the glycine site of NMDA receptors mediating GABA release from cultured hippocampal neurons

The effects of the human immunodeficiency virus type 1 envelope protein gp120 on the release of GABA elicited by N-methyl-D-aspartate (NMDA) from rat hippocampal neurons in primary culture has been investigated. NMDA (1-300 microM) increased in a concentration-dependent manner (EC50 =37.9+/-12 microM) the release of [3H]-GABA. The effect of 100 microM NMDA was prevented by 30 microM of the GABA transport inhibitor N-(4,4-diphenyl-3-butenyl)guvacine (SKF 100330A). Glycine (10 microM) or gp120 (0.01 microM) affected neither the basal nor the NMDA-evoked [3H]-GABA release. The NMDA (100 microM)-evoked release was prevented by 5,7-dichloro-kynurenic acid (5,7-DCKA), a selective antagonist at the glycine site of the NMDA receptor, in a concentration-dependent manner (IC50 approximately 0.3 microM). Glycine (3-10 microM) or gp120 (0.003-0.01 microM) produced reversal of the 5,7-DCKA antagonism in a way that suggested competition at a same site; gp120 was at least 3 orders of magnitude more potent than glycine. It is suggested that gp120 may mimic glycine at NMDA receptors.     

Monoaminergic control of the release of calcitonin gene-related peptide- and substance P-like materials from rat spinal cord slices

The possible control by monoamines of the spinal release of substance P- and calcitonin gene-related peptide-like materials (SPLM and CGRPLM, respectively) was investigated in vitro, using slices of the dorsal half of the rat lumbar enlargement superfused with an artificial cerebrospinal fluid. Whereas the spontaneous outflow of SPLM and CGRPLM was changed by none of the agonists/antagonists of monoamine receptors tested, the overflow of both peptide-like materials due to 30 mM K+ was differentially affected by alpha 2-adrenoreceptor and dopamine D-1 receptor ligands. Noradrenaline (10 microM to 0.1 mM) and clonidine (0.1 mM) significantly reduced the K(+)-evoked overflow of SPLM, and both effects could be prevented by idazoxan (10 microM) and prazosin (10 microM) as expected from their mediation through the stimulation of alpha 2B-adrenoreceptors. In contrast, CGRPLM overflow remained unaffected by alpha 2-adrenoreceptor ligands. Dopamine D-1 receptor stimulation by SKF 82958 (10-100 nM) significantly increased the K(+)-evoked overflow of both SPLM and CGRPLM, and this effect could be prevented by the selective D-1 antagonist SCH 39166 (1 microM). Further studies with selective ligands of other monoamine receptors indicated that neither alpha 1- and beta-adrenergic receptors, dopamine D-2, nor serotonin 5-HT1A and 5-HT3 receptors are apparently involved in some control of the spinal release of CGRPLM and SPLM. These data are discussed in line with the postulated presynaptic control by monoamines of primary afferent fibres conveying nociceptive messages within the dorsal horn of the spinal cord.     

Actions of prostaglandin E2 on rat supraoptic neurones

Prostaglandins (PGs) have been implicated in the regulation of vasopressin (VP) and oxytocin (OT) release in response to various stimuli. To examine the site and mechanism of actions of PGs, we studied effects of PGE2 and PG-receptor agonists on supraoptic nucleus (SON) neurones of rat hypothalamic slice preparations using extracellular recording and whole-cell patch-clamp techniques. PGE2 modulated the electrical activity of more than 80% of the neurones studied. The effects of PGE2 on both phasic and non-phasic neurones were mostly excitatory, and dose-dependent. The effects of PGE2 were mimicked by PGF2alpha or the FP agonist, fluprostenol, whereas PGD2 or the selective EP, IP or TP agonist was less effective or had no effect. The effects of PGE2 were unaffected by the EP1 antagonist, SC-51322, but reduced to 80% of control by the EP1/FP/TP antagonist, ONO-NT-012, which reduced the effects of fluprostenol to 32% of control. Moreover, some neurones responsive to PGE2 did not respond to fluprostenol. Patch-clamp analysis in SON slice preparations revealed that PGE2 at 10(-6) M depolarized the membrane potential by 3.9+/-0.3 mV from the resting membrane potential of -58.4+/-2.2 mV in the current-clamp mode. In the voltage-clamp mode, PGE2 induced inward currents at a holding potential of -70 or -80 mV, while it did not affect spontaneous excitatory postsynaptic currents. PGE2 induced currents also in dissociated SON neurones and the reversal potential of the currents was -35.5+/-0.9 mV, which was similar to that of currents induced by fluprostenol. These results suggest that SON neurones possess at least two types of PG receptors, FP receptors and EP receptors of a subclass different from EP1, EP2, or EP3, and that activation of these receptors leads to the opening of nonselective cation channels, membrane depolarization and increase of the action potential discharge.