Inhibition of interleukin-1β and prostaglandin E2 thermogenesis by glycyl-glutamine, a pro-opiomelanocortin-derived peptide

Interleukin-1β (IL-1β) and other cytokines produce fever by stimulating prostaglandin E₂ (PGE₂) synthesis in thermoregulatory regions of the preoptic area and anterior hypothalamus (POA/AH). Prostaglandin E₂ is thought to raise body temperature, at least in part, by stimulating β-endorphin release from pro-opiomelanocortin neurons that innervate the POA/AH. In this study, we investigated whether glycyl-glutamine (β-endorphin_30–31), an inhibitory dipeptide synthesized from β-endorphin post-translationally, inhibits IL-1β and PGE₂-induced hyperthermia. Hyperthermic sites were identified by microinjecting PGE₂ (3 fmol/1 μl) into the medial preoptic area (mPOA) of conscious, unrestrained rats. Interleukin-1β (1 U) injection into the same PGE₂ responsive thermogenic sites in the mPOA elicited a prolonged rise in colonic temperature (T_c) (+1.02±0.06°C) that persisted for at least 2 h. Glycyl-glutamine (3 nmol) co-injection into the mPOA inhibited IL-1β thermogenesis completely (T_c=−0.18±0.22°C). Glycyl-glutamine had no effect on body temperature when given alone to normothermic rats. Co-injection of individual amino acids, glycine and glutamine (3 nmol each amino acid), failed to influence IL-1β-induced thermogenesis, which indicates that Gly-Gln hydrolysis does not explain its inhibitory activity. Glycyl-glutamine (3 nmol) also prevented the rise in body temperature produced by PGE₂ (PGE₂=0.89±0.05°C; PGE₂ plus Gly-Gln=−0.16±0.14°C), consistent with evidence that PGE₂ mediates IL-1β-induced fever. These findings demonstrate that Gly-Gln inhibits the thermogenic response to endogenous pyrogens.     

Intracerebroventricular injection of prostaglandin E2 induces thermal hyperalgesia in rats: the possible involvement of EP3 receptors

To determine what types of prostanoid receptors are involved in the central effect of prostaglandin E₂ (PGE₂) on nociception, we administered PGE₂ and its agonists, i.e., 17-phenyl-ω-trinor PGE₂ (an EP₁ receptor agonist), butaprost (an EP₂ receptor agonists), 11-deoxy PGE₁ (an EP₂/EP₃ receptor agonist, EP₂ EP₃) and M&B28767 (an EP₃ receptor agonist) into the lateral cerebroventricle (LCV) of rats and observed the changes of paw-withdrawal latency on a hot plate. The LCV injection of PGE₂ (10 pg/kg-10 ng/kg), 11-deoxy PGE₁ (100 pg/kg-10 ng/kg) and M&B28767 (1 pg/kg-100 pg/kg) produced a significant reduction in the paw-withdrawal latency. The maximal reduction was observed 15 min after the LCV injection of these drugs. Neither 17-phenyl-ω-trinor PGE₂ (1 pg/kg-1 μg/kg) nor butaprost (1 pg/kg-100 μg/kg) induced any significant changes in the paw-withdrawal latency. The LCV injection of PGE₂ (1 μg/kg) and 17-phenyl-ω-trinor PGE₂ (50 μg/kg) increased the latency only 5 min after LCV injection. These findings indicate that the LCV injection of PGE₂ induces thermal hyperalgesia through EP₃ receptors and analgesia through EP₁ receptors by its central action in rats.     

Antibodies to macrophage inflammatory protein-1β in preoptic area of rats fail to suppress PGE2 hyperthermia

This study determined whether macrophage inflammatory protein-1β (MIP-1β) plays a role in the hyperthermia caused by prostaglandin E₂ (PGE₂) given intracerebroventricularly (i.c.v.) in the rat. In these experiments, anti-murine MIP-1β antibody (anti-MIP-1β) was micro-injected in the anterior hypothalamic, preoptic area (AH/POA) just before i.c.v. PGE₂. The results showed that anti-MIP-1β failed to alter the PGE₂ hyperthermia. However, immunocytochemical studies revealed MIP-1β immunoreactivity detectable in both the organum vasculosum laminae terminalis (OVLT) and AH/POA in the febrile rat. These data thus demonstrate that MIP-1β is sequestered in diencephalic structures underlying thermoregulation even though it is not involved in PGE₂ hyperthermia. This dissociation supports the viewpoint that at least two distinct systems exist in the brain which underlie a febrile response: MIP-1β underlies one component whereas PGE₂ comprises the other.     

Angiotensin AT1 receptors in the preoptic area negatively modulate the cardiovascular and ACTH responses induced in rats by intrapreoptic injection of prostaglandin E2

We previously reported that brain angiotensin II type 2 (AT₂) receptors contribute to the hyperthermia induced by intrahypothalamic (intrapreoptic (i.p.o.)) administration of prostaglandin E₂ (PGE₂) in rats. The present study was carried out to investigate the role of angiotensin II (ANG II) receptors in the cardiovascular and adrenocorticotropic hormone (ACTH) responses induced in rats by i.p.o. injection of PGE₂. PGE₂ (100 ng) produced marked increases in blood pressure, heart rate, and plasma ACTH concentration. These changes were significantly enhanced by i.p.o. treatment with an AT₁-receptor antagonist, losartan, while an AT₂-receptor antagonist, CGP 42112A, had no effect. In contrast, losartan, but not CGP 42112A, reduced the pressor and ACTH responses to i.p.o. injection of a large dose of “exogenous” ANG II (25 ng). These results suggest that while “endogenous” ANG II exerts inhibitory effects on both the cardiovascular and the ACTH responses to i.p.o. PGE₂ by way of preoptic AT₁-receptors, a large dose of exogenous ANG II produces effects opposite to those induced by the endogenous ANG II that is released locally and in small amounts by i.p.o. PGE₂.     

Effects of perfusion flow rate, prostaglandin F2α, phenylephrine, and serotonin on isolated, perfused brains of spontaneously hypertensive rats

Effects of perfusion flow rate and three vasoconstrictors, phenylephrine, prostaglandin F_2α (PGF_2α) and serotonin, on isolated, perfused brain preparations of spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto rats (WKY) were investigated. The basal perfusion pressure of the cerebral vascular beds at a flow rate of 2.5 ml/min was 48 ± 3mm Hg(n = 11) in SHR and 32 ± 2mm Hg(n = 12)in WKY(P < 0.005). The perfusion pressures at all flow rates tested (2.5−6.5 ml/min) in SHR were significantly greater than those in WKY. Concentration-perfusion pressure curves for the vasoconstrictors showed that the brain vascular bed was much more reactive to sertonin compared with phenylephrine and PGF_2α. EC₅₀ values (−logM) for serotonin in the perfused brains of SHR and WKY were 7.0 ± 0.06 (n = 10)and6.5 ± 0.06 (n = 11), respective (P < 0.01). There were no differences in EC₅₀ values for phenylephrine or PGF_2α between SHR and WKY. Exogenous serotonin and phenulephrine caused significantly greater maximal vasoconstrictor responses in SHR compared with WKY, while the pressor response to PGF_2α was very weak and no significant difference between SHR and WKY preparations was observed. These results indicate that cerebral vascular beds in SHR exhibit higher cerebrovascular resistance than those in WKY, and that reactivity and sensitivity to serotonin and reactivity to phenylephrine in SHR rats are enhanced to a greater extent compared to WKY.     

Prostaglandin E1 modifies porcine cerebral arterial tone through cyclooxygenase related eicosanoid(s) release

The effects of prostaglandin E₁ (PGE₁) on porcine cerebral arteries were studied in the absence and presence of cyclooxygenase inhibitors, 5×10⁻⁶ M indomethacin or 10⁻⁴ M aspirin. In preparations placed at a resting tension, 3×10⁻⁹ to 10⁻⁶ M PGE₁ caused dose-dependent contractions. In prostaglandin F_2α (PGF_2α)- contracted preparations, low concentrations of 10⁻⁹ and 3 × 10⁻⁹ M PGE₁ did not modify arterial tone and higher concentrations of 10⁻⁸ to 10⁻⁶ M PGE₁ further increased the arterial tone in all preparations tested. In contrast, in the presence of the cyclooxygenase inhibitors, low concentration of 10⁻⁹ and 3×10⁻⁹ M PGE₁ markedly decreased the arterial tone induced by PGF_2α and higher dose of 10⁻⁸ to 10⁻⁶ M PGE₁ increased the arterial tone in 15 of 18 preparations (83%). These findings suggest that PGE₁ modifies porcine cerebral vascular tone at least partly through cyclooxygenase-related eicosanoid(s) production.     

Biphasic modulation in the trigeminal nociceptive neuronal responses by the intracerebroventricular prostaglandin E2 may be mediated through different EP receptors subtypes in rats

To determine which prostaglandin E₂ (PGE₂) receptor subtypes are involved in the brain-derived PGE₂-induced changes in nociception, we injected synthetic EP₁, EP₂ and EP₃ receptor agonists (0.01 fmol to 10 nmol) into the lateral cerebroventricle (LCV) of urethane-anesthetized rats and observed the changes in the responses of the wide dynamic range (WDR) neurons in the trigeminal nucleus caudalis to noxious pinching of facial skin. The enhancement and suppression of the nociceptive responses of the WDR neurons were observed after the LCV injection of MB28767 (an EP₃ receptor agonist) at a low dose range (1–100 fmol) and 17-phenyl-ω-trinor PGE₂ (an EP₁ receptor agonist) at high doses (1–10 nmol), respectively. Furthermore, the suppression of nociceptive neuronal responses after the LCV injection of PGE₂ (1 nmol) was completely blocked by SC19220 (an EP₁ receptor antagonist, 300 nmol). On the other hand, butaprost (an EP₂ receptor agonist) at any doses tested (0.1 fmol to 1 nmol) had no effect on the nociceptive responses. The LCV injection of MB28767 (10 fmol) and 17-phenyl-ω-trinor PGE₂ (1 nmol), which respectively enhanced and suppressed the nociceptive neuronal responses, did not affect the responses of the low threshold mechanoreceptive neurons to innocuous tactile stimuli. These results provide electrophysiological evidence that brain-derived PGE₂ induces mechanical hyperalgesia and hypoalgesia through EP₃ and EP₁ receptors, respectively, in the rat.     

Prostaglandin E2 mediates the stimulatory effect of methoxamine on in vivo luteinizing hormone-releasing hormone (LH-RH) release in the ovariectomized female rhesus monkey

Previously, we found that noradrenergic input throughα₁-receptors modulates pulsatile release of luteinizing hormone-releasing hormone (LH-RH) in ovariectomized rhesus monkeys in the absence of estrogen. In the present study, the role of prostaglandin E₂ (PGE₂) in mediating α-adrenergic stimulation of LH-RH release is investigated. In the first experiment the effects of theα₁-adrenergic agonists methoxamine (MTX) on LH-RH and PGE₂ release were examined. Push-pull perfusion of the stalk-median eminence (S-ME) was performed in conscious, ovariectomized monkeys, and perfusate samples were collected on ice. MTX (10⁻⁵ M) was infused into the S-ME through the push cannula for 10 min at 90-min intervals, and LH-RH and PGE₂ in aliquots of the same perfusate samples were measured by radioimmunoassay. Infusion of MTX significantly stimulated LH-RH release (n = 12; P < 0.01) and PGE₂ release (P < 0.05). In the second experiment, the effect of PGE₂ infusion on LH-RH release was tested. PGE₂ (10⁻⁷ M) was infused using the same protocol as above, and LH-RH was measured in the perfusates. Infusion of PGE₂ through the push cannula significantly stimulated LH-RH release (n = 23; P < 0.05). These results suggest that the stimulatory effect of MTX on LH-RH release is at least partly mediated by PGE₂, since MTX stimulated not only LH-RH but also PGE₂ release, and since PGE₂ itself stimulated LH-RH release. Therefore, PGE₂ may be an important endogenous mediator ofα₁-adrenergic input stimulating pulsatile PH-RH release. Moreover, the stimulatory effects of MTX and PGE₂ can be observed in the absence of estrogen in the rhesus monkey, unlike in rhodents. Our results also demonstrate the usefulness of the push-pull perfusion technique for studies of cellular mechanisms in neuroendocrine research.     

Prostaglandin E1 and dibutyryl cyclic AMP enhance platelet resistance to deformation

The effect of prostaglandin E₁ (PGE₁) on platelets is mediated through the PGE₁ receptor and the consequent maintenance of the platelet's discoid shape. The effects of PGE₁ and dibutyryl cAMP (dbcAMP) on the deformability of human platelets were studied. Deformability tests based upon the micropipette aspiration on the platelets were performed by using pipettes with radii (Rp) of 0.26-0.36 gm. The time course of the extension length (Dp, in μg) of the platelets in response to aspiration with a negative pressure (ΔP) of 5 cm H₂ O (ΔP × Rp = 0.15 dynes/cm) was analyzed. PGE₁ treatment (0.1 μM) resulted in a decrease of platelet deformability as compared with results obtained for apparently non-activated, control platelets. The deformation index, i.e., Dp/Rp (PGE₁ -treated) / Dp/Rp (control), was significantly reduced to 0.90 ± 0.04. DbcAMP treatment also significantly decreased the deformability of platelets and this decrease was dbcAMP dose dependent. In contrast, colchicine- or cytochalasin D-treated platelets increased deformability. PGE₁ -treated platelets had a higher [cAMP]_i than controls. Platelets treated with PGE₁ or dbcAMP showed a reduced [Ca²⁺]i increment induced by thrombin as compared to non-treated controls. These results indicate that PGE₁ and dbcAMP treatment of platelets is accompanied by an enhancement of platelet resistance to deformation. The increased [cAMP]_i and low [Ca²⁺]_i after PGE₁ treatment may limit the rearrangement of cytoskeleton and thus enhance platelet resistance to deformation.     

The involvement of the sympathetic nervous system in the centrogenic pressor and tachycardiac effects of prostaglandins E2 and F2 alpha in anaesthetised cats

The intracerebroventricular (i.c.v.) administration of prostaglandin E2 (PGE2, 1 micrograms) and prostaglandin F2 alpha (PGF2 alpha, 10 micrograms) produced prolonged pressor and tachycardiac responses in chloralose-anaesthetised cats. Phenoxybenzamine-pretreatment completely prevented the pressor response without altering the tachycardiac response, whereas propranolol intervention completely inhibited the tachycardiac response and also attenuated the pressor response. The pretreatment with pentolinium completely antagonised both the pressor and tachycardiac responses to i.c.v. PGE2 and PGF2 alpha. The results suggest that the centrally administered PGE2 and PGF2 alpha augment sympathetic outflow to the heart and vascular system and thereby cause excitatory cardiovascular responses in anaesthetised cats.     

The formation and regional distribution of prostaglandins D2 and F2α in the brain of spontaneously convulsing gerbils

The distribution of the two major cycloxygenase products prostaglandin D₂ (PGD₂) and prostaglandin F_2α (PGF_2α) in 7 different regions of the brain (medulla, cerebellum, hypothalamus, striatum, midbrain, hippocampus and cerebral cortex) was studied. Basal levels were highest in hypothalamus and cortex. Following convulsions elicited by environmental stress prostaglandin concentrations increased in all areas, with largest increases (10–20-fold) in hippocampus and cortex, reaching 70 ng/g PGD₂ in hippocampus and 115 ng/g PGD₂ in cortex. These results demonstrate that, during spontaneous seizures, there is a greater increase in prostanoid production in those areas involved in the convulsive process.     

Prostaglandin E2 may induce hyperthermia through EP1 receptor in the anterior wall of the third ventricle and neighboring preoptic regions

We have previously reported that intracerebroventricular injection of prostaglandin E₂ (PGE₂) induces hyperthermia possibly through EP₁ receptors in the rat. In the present study, to determine the sites in the brain where PGE₂ induces hyperthermia through EP₁ receptors, we microinjected an EP₁ receptor agonist, 17-phenyl-ω-trinor PGE₂ (17-Ph-PGE₂, 100 ng) into different sites in the rat brain and observed the colonic temperature (T_co) for 2 h in a 23±1°C environment. Responsive sites where 17-Ph-PGE₂ (100 ng) produced a rise in the T_co of more than 1.1°C within 60 min after injection were found in the medial preoptic area, the subchiasmatic portion of the median preoptic nucleus, the anterior wall of the third ventricle (A3V) and the ventral portion of the diagonal band of Broca. Among these sites, the A3V was the most responsive. In contrast, microinjection of neither butaprost (an EP₂ agonist, 100 ng) nor M&B28767 (an EP₃ agonist, 100 ng) into these four sites had any effect on the T_co. Intracerebroventricular pretreatment with SC-19220 (an EP₁ antagonist, 100 μg) inhibited the rise in the T_co which was induced by microinjection of PGE₂ (50 ng) into the A3V. These results thus suggest that PGE₂ induces hyperthermia by stimulating EP₁ receptors in the A3V and the neighboring preoptic region.     

Nitric oxide and prostaglandin E2 formation parallels blood–brain barrier disruption in an experimental rat model of bacterial meningitis

During meningitis, the host produces a plethora of signaling agents as part of a coordinated defense mechanism against invading pathogens. Nitric oxide (NO) and prostaglandin E₂ (PGE₂) are two such inflammatory mediators produced in response to bacterial endotoxins. Disruption of the blood–brain barrier (BBB) is one of many pathophysiological consequences of meningitis. The present objective was to examine the time-course of NO and PGE₂ production in relationship to BBB permeability alterations during experimentally-induced meningitis. Meningeal inflammation was elicited by intracisternal administration of the bacterial endotoxin, lipopolysaccharides (LPS; 200 μg), and NO, PGE₂, and BBB integrity were monitored over the next 24 h. Meningeal NO production was assessed by headspace chemiluminescence; cerebrospinal fluid PGE₂ was determined by enzyme-linked immunosorbent assay (ELISA) immunoassay; and BBB integrity was determined by the brain accumulation of ¹⁴C-sucrose. Similar time-course profiles for NO and PGE₂ were observed, with a peak effect for both inflammatory mediators observed within 6–8 h after intracisternal LPS dosing. Statistically significant (p < 0.05) disruption of the BBB was observed in various brain regions. Strikingly similar temporal relationships were observed for NO and PGE₂ production and BBB disruption. These results suggest the hypothesis that NO and PGE₂ may act in conjunction to disrupt the BBB during experimental meningitis.     

NSAIDS inhibit the IL-1β-induced IL-6 release from human post-mortem astrocytes: the involvement of prostaglandin E2

Epidemiological studies have shown that steroidal as well as non-steroidal anti-inflammatory drugs lower the risk of developing Alzheimer's Disease (AD). A suppressive effect of these anti-inflammatory drugs on local inflammatory events in AD brains has been suggested, however the mechanisms responsible are still unknown. In this study we investigated at cellular level the influence of two anti-inflammatory drugs—dexamethasone and indomethacin—and an experimental specific cyclooxygenase-2 inhibitor, BF389, on the production of the pro-inflammatory cytokine IL-6 and the inflammatory mediator PGE₂ by human astrocytes. Two human post-mortem astrocyte cultures (A157 and A295) and astroglioma cell lines (U251 and U373 MG) were found to secrete considerable amounts of IL-6 upon stimulation with IL-1β. The glucocorticoid dexamethasone inhibited the IL-1β-activated release of IL-6 from the postmortem astrocyte cultures A157 and A295 and from the astroglioma cell lines. The non-specific cyclooxygenase inhibitor indomethacin and BF389 only suppressed the IL-6 release by post-mortem astrocyte culture A157. This post-mortem astrocyte culture was found to produce large amounts of PGE₂ upon stimulation with IL-1β, whereas in the supernatants of the postmortem astrocyte culture A295 and the astroglioma cell lines, low PGE₂ concentrations were detected. Addition of exogenous PGE₂ prevented the inhibitory effect of indomethacin and BF389 on the IL-1β-activated IL-6 release from A157 astrocytes and largely potentiated the IL-1-induced release of IL-6 from all astrocytes/astroglioma cells tested. Dexamethasone also inhibited the PGE₂ release from the astrocytes and astroglioma cells, however the inhibitory effect of dexamethasone on the IL-1β-activated IL-6 release could not be prevented by the addition of PGE₂. The observed reduction of IL-6 and/or PGE₂ from astrocytes may be involved in the mechanism underlying the beneficial effects of these drugs in AD.     

Prostaglandin E2 excites neurons of the nucleus tractus solitarius by activating cation channels

Nucleus tractus solitarius (NTS) has a high density of prostaglandin E₂ (PGE₂)-binding sites. Action of PGE₂ (10⁻⁹–10⁻⁶ M) was tested on neurons in a NTS slice with patch-clamp recording under synaptic blockade. PGE₂ raised the firing rate in approximately half of the neurons in cell-attached patch mode. In whole-cell current clamp, PGE₂ depolarized membrane potential accompanied by an increase in firing rate. In whole-cell voltage clamp (−58 mV), PGE₂ induced the inward current with an increase in conductance. The current was linearly related to voltage from −100 mV to −10 mV and suppressed between −10 mV and 20 mV. The current-voltage curve remained similar under low external Cl⁻ or high internal Cl⁻ conditions and after external Na⁺ was replaced by Cs⁺. It is concluded that PGE₂ excites NTS neurons by activating cation conductance.     

Prostanoids in the preoptic hypothalamus mediate systemic lipopolysaccharide-induced hyperalgesia in rats

The systemic administration of lipopolysaccharide (LPS), an experimental model of systemic bacterial infection is known to modulate nociception. It increases the prostaglandin E₂ (PGE₂) levels in the preoptic area of the hypothalamus (POA) and the microinjection of PGE₂ into the POA and the neighboring basal forebrain induces hyperalgesia. We, therefore, hypothesized that the PGE₂ synthesized in these regions mediates intravenous (i.v.) LPS-induced hyperalgesia. To test this hypothesis, we microinjected cyclooxygenase (COX) inhibitors into several sites in the rat hypothalamus and observed their effects on the LPS (0.1–100 μg/kg, i.v.)-induced changes in nociceptive behavior as assessed by a plantar test. LPS (10 and 100 μg/kg, i.v.) reduced the paw-withdrawal latency at 90 min and 45–60 min after injection, respectively, both thus indicating a hyperalgesic effect. This hyperalgesia was observed only in the period before the development of fever which started 120–135 min after the LPS injection. The LPS (100 μg/kg, i.v.)-induced hyperalgesia was completely abolished by pretreatment with the microinjection of diclofenac (an inhibitor of COX-1 and 2) at 1.0 ng into the bilateral POA. Furthermore, it was also blocked by the microinjection of NS-398 (a selective COX-2 inhibitor) at 1.0 ng into the bilateral POA and the horizontal limb of the diagonal band of Broca (DBB), but not the lateral hypothalamic area, the paraventricular hypothalamic nucleus, and the ventromedial hypothalamic nucleus. These findings suggest that LPS (i.v.)-induced hyperalgesia is mediated predominantly through a COX-2 induced prostanoids in the POA and the DBB in rats.     

Cardiovascular effects of central administration of clonidine in conscious cats

The effects on arterial blood pressure and heart rate after an intracerebroventricular (i.c.v.) administration of clonidine were investigated using conscious normotensive cats. Injection of clonidine (5–10 μg; 5 μl; i.c.v.) elicited a decrease in mean arterial pressure (MAP) and heart rate (HR) in a dose-dependent manner. The highest dose of 10 μg of clonidine decreased MAP and HR by 39 ± 3 mmHg and 74 ± 5 b.p.m., respectively (n = 7). Pretreatment with yohimbine, the α₂-adrenoceptor antagonist (8 μg; 5 μl; i.c.v.) blocked the cardiovascular responses to a subsequent i.c.v. injection of 10 μg clonidine (n = 7). Furthermore, preadministration of cimetidine (100 μg; 5 μl; i.c.v.), the H₂ histamine receptor antagonist with imidazoline receptor activating properties, prevented the decreases in MAP and HR to a subsequent i.c.v. injection of 10 μg clonidine (n = 7). By contrast, pretreatment with the specific I₁ imidazoline receptor blocker, efaroxan (100–500 μg; 5 μl; i.c.v.), failed to inhibit the cardiovascular effects of an i.c.v. administration of 10 μg clonidine (n = 7). These results suggest that the effects of centrally administered clonidine on MAP and HR are probably not mediated through activation of the I₁ subtype of imidazoline receptors in conscious cats. However, the cardiovascular effects elicited by i.c.v. administration of clonidine appear to result from stimulation of central α₂-adrenergic or the H₂ histaminergic-like receptors.     

N-Monomethyl-l-arginine co-injection attenuates the thermogenic and hyperthermic effects of E2 prostaglandin microinjection into the anterior hypothalamic preoptic area in rats

Prostaglandin E₂ (PGE₂) microinjection (25 ng, 250 nl) into the preoptic area of the anterior hypothalamus (POAH) stimulated heat production in brown adipose tissue (BAT) and increased core temperature in urethane-anesthetized rats. These thermogenic and hyperthermic effects were attenuated by co-injection of N^G-monomethyl-l-arginine (NMMA, 25 μg), a competitive inhibitor of nitric oxide (NO) production froml-arginine. Inclusion ofl-arginine (50 μg), though notd-arginine (50μg) reversed the inhibitory effect of NMMA (25μg) on intra-POAH PGE₂-induced increases in interscapular BAT (IBAT) and core temperatures. Intra-POAH injection of NMMA (25 μg) orl-arginine (50 μg) alone had no effect on IBAT and core temperatures. The results suggest that the effect on thermoregulation induced by action of PGE₂ in the POAH is modulated by a locall-arginine-dependent and NMMA-sensitive NO-generating system.     

Arachidonic acid and prostaglandin D2 cooperatively accelerate desensitization of nicotinic acetylcholine receptor channel in mouse skeletal muscles

To clarify the effects of arachidonic acid (AA) and its metabolites on desensitization of nicotinic acetylcholine (ACh) receptor channel in mouse skeletal muscle cells, we investigated the time-dependent decrease in the channel opening frequency of ACh (1 μM)-activated channel currents by the cell-attached patch clamp technique. AA (30–100 μM) applied to a patched membrane or to non-patched membrane accelerated the decrease in the channel opening frequency. A cyclooxygenase inhibitor, indomethacin (10 μM), prevented the acceleration elicited by 30 μM AA, but not by 100 μM AA. A lipoxygenase inhibitor, nordihydroguaiaretic acid (10 μM), and a cytochrome P-450 inhibitor, ketoconazole (3 μM), did not affect the acceleration by 30 μM AA. Prostaglandin (PG) D₂ at 10 μM alone and at 25 nM in combination with 10 μM AA accelerated the decrease in the channel opening frequency. No acceleration was observed with PGE₂ at 10 μM alone and at 25 nM in combination with 10 μM AA. Pretreatment with a protein kinase (PK) C inhibitor, staurosporine (10 nM), but not with a PKA inhibitor, H-89 (3 μM), prevented the acceleration elicited by AA+PGD₂. These results suggest that AA, and PGD₂ of its metabolites, cooperatively accelerate desensitization of nicotinic ACh receptor channel. The activation of PKC by AA and PGD₂ may be involved in the mechanism of the cooperative acceleration of desensitization.     

Characterization of the glutamatergic system for induction and maintenance of allodynia

Glutamate is the main excitatory neurotransmitter in the central nervous system and has been shown to be involved in spinal nociceptive processing. We previously demonstrated that intrathecal (i.t.) administration of prostaglandin (PG) E₂ and PGF_2α induced touch-evoked pain (allodynia) through the glutamatergic system by different mechanisms. In the present study, we characterized glutamate receptor subtypes and glutamate transporters involved in induction and maintenance of PGE₂- and PGF_2α-evoked allodynia. In addition to PGE₂ and PGF_2α, N-methyl- -aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), but not kainate, induced allodynia. PGE₂- and NMDA-induced allodynia were observed in NMDA receptor 4 (NR2D) subunit knockout (GluR4(−/−)) mice, but not in 1 (NR2A) subunit knockout (GluR1(−/−)) mice. Conversely, PGF_2α- and AMPA-induced allodynia were observed in GluR1(−/−) mice, but not in GluR4(−/−) mice. The induction of allodynia by PGE₂ and NMDA was abolished by the NMDA receptor 2 (NR2B) antagonist CP-101,606 and neonatal capsaicin treatment. PGF_2α- and AMPA-induced allodynia were not affected by CP-101,606 and by neonatal capsaicin treatment. On the other hand, the glutamate transporter blocker -threo-β-benzyloxyaspartate ( -TBOA) blocked all the allodynia induced by PGE₂, PGF_2α, NMDA, and AMPA. These results demonstrate that there are two pathways for induction of allodynia mediated by the glutamatergic system and suggest that the glutamate transporter is essential for the induction and maintenance of allodynia.