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.     

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.     

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.     

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.     

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.     

Prostaglandin E2, a postulated astrocyte-derived neurovascular coupling agent,constricts rather than dilates parenchymal arterioles

It has been proposed that prostaglandin E₂ (PGE₂) is released from astrocytic endfeet to dilate parenchymal arterioles through activation of prostanoid (EP₄) receptors during neurovascular coupling. However, the direct effects of PGE₂ on isolated parenchymal arterioles have not been tested. Here, we examined the effects of PGE₂ on the diameter of isolated pressurized parenchymal arterioles from rat and mouse brain. Contrary to the prevailing assumption, we found that PGE₂ (0.1, 1, and 5 μmol/L) constricted rather than dilated parenchymal arterioles. Vasoconstriction to PGE₂ was prevented by inhibitors of EP₁ receptors. These results strongly argue against a direct role of PGE₂ on arterioles during neurovascular coupling.     

Involvement of tachykinin NK1 receptors in nociceptin-induced hyperalgesia in mice

Intrathecal (i.t.) injection of nociceptin at small doses (3.0 and 30.0 fmol) produced a significant hyperalgesic response as assayed by the tail-flick test. This hyperalgesic effect peaked at 15 min following i.t. administration of nociceptin (3.0 fmol) and returned to control level within 30 min. Hyperalgesia elicited by nociceptin was inhibited dose-dependently by i.t. co-administration of tachykinin NK₁ receptor antagonists, CP-99,994 and sendide. A significant antagonistic effect of [ -Phe⁷, -His⁹] substance P (6–11), a selective antagonist for substance P, was observed against the nociceptin-induced hyperalgesia. Pretreatment with i.t. substance P antiserum and i.t. capsaicin resulted in a complete block of the reduced threshold produced by nociceptin. The NK₂ receptor antagonist, MEN-10,376 and pretreatment with neurokinin A antiserum did not alter the behavioural effect of nociceptin. The N-methyl- -aspartate (NMDA) receptor antagonists, dizocilpine (MK-801) and (−)-2-amino-5-phosphonovaleric acid ( -APV), and -N^G-nitro arginine methyl ester ( -NAME), a nitric oxide synthase inhibitor, failed to inhibit nociceptin-induced hyperalgesia. The results obtained suggest that the hyperalgesic effect of nociceptin may be mediated through tachykinin NK₁ receptors in the spinal cord.     

Prostaglandin E2 protects cultured cortical neurons against receptor-mediated glutamate cytotoxicity

The effects of prostaglandin (PG) E₂ on glutamate-induced cytotoxicity were examined using primary cultures of rat cortical neurons. The cell viability was significantly reduced when cultures were briefly exposed to either glutamate or (NMDA) then incubated with normal medium for 1 h. Similar cytotoxicity was observed with the brief application of ionomycin, a calcium ionophore, and S-nitrosocysteine, a nitric oxide (NO)-generating agent. PGE₂ at concentrations of 0.01–1 μM dose-dependently ameliorated the glutamate-induced cytotoxicity. PGE₁, butaprost, an EP₂ receptor agonist, and 8-bromo-cAMP were also effective in protecting cultures against glutamate cytotoxicity. By contrast, neither 17-phenyl-ω-trinor-PGE₂, an EP₁ receptor agonist, nor M & B 28767, an EP₃ receptor agonist, affected glutamate-induced cytotoxicity. NMDA-induced cytotoxicity was ameliorated by PGE₂, butaprost, MK-801, , a NO synthase inhibitor, and hemoglobin, which binds NO. These agents excluding MK-801 ameliorated the ionomycin-induced cytotoxicity. The cytotoxicity induced by S-nitrosocysteine was prevented only by hemoglobin but not by the other agents including PGE₂. These findings indicate that PGE₂ protects cultured cortical neurons against NMDA receptor-mediated glutamate neurotoxicity via EP₂ receptors. EP₂ receptor stimulation may suppress a step in NO formation triggered by Ca²⁺-influx through NMDA receptors.     

Hyperalgesia onset latency suggests a hierarchy of action

Hyperalgesia onset latencies of inflammatory mediators were quantified by measuring the threshold of the nociceptive flexion reflex in the rat at 1 min intervals after intradermal injection. Prostaglandin E2 and 8(R), 15(S)-dihydroxyicosa-(5E,9,11,13Z)-tetraenoic acid induced hyperalgesia with short onset latencies, compatible with a direct action on primary afferent nociceptors. Bradykinin, norepinephrine and leukotriene B4 induced hyperalgesia with a significant delay in onset, compatible with their known indirect mechanisms of producing hyperalgesia. We propose that use of this approach, rapid frequent measurement of nociceptive threshold, can be used to determine the hierarchy of action of mediators in hyperalgesic mechanisms.     

Injection of muscimol in the posterior hypothalamus reduces the pge,-hyperthermia in the rat

The firing rate of the nerves innervating interscapular brown adipose tissue PBAT), MAT and colonic temperatures (T_IBAT and T_c, heart rate, and oxygen (O₂) consumption were monitored in urethane-anesthetized male Sprague-Dawley rats. These variables were measured for 40 min before (baseline values) and 40 min after a 56 ng muscimol injection in the posterior hypothalamus and an intracerebroventricular administration of 500 ng prostaglandin E, (PGE₁). The same variables were monitored in other rats with muscimol injection or PEE₁ administration alone. No drug was Injected in control rats. The results show that muscirnol injection reduces the increases in firing rate, T_IBAT, T_c, heart rate, 02 consumption induced by PGE₁. These findings suggest that GABAergic tone in the posterior hypothalamus is important in the control of thermogenic changes induced by PGE₁.     

Locomotor effects of amantadine in the mouse are not those of a typical glutamate antagonist

Summary Amantadine has been shown to displace [³H]MK 801 from its binding site on the NMDA receptor. We have therefore studied the motor effects of amantadine in normal and 24h reserpine-treated mice to determine whether the behavioural profile of this drug resembles that of other NMDA receptor antagonists (e.g. MK 801). In common with the latter, amantadine (5–40 mg/kg IP) produced a modest dose-dependent sedation in dopamineintact mice, with a reduction in locomotion and other species-typical behaviours (e.g. rearing and grooming), but with no signs of the hyperactivity, stereotypy, ataxia or loss of muscle tone commonly seen with MK 801. Amantadine (5–80 mg/kg IP) effected a small incrase in motility in akinetic reserpine-treated mice by itself, but this response was highly variable and not statistically significant. As with MK 801, amantadine significantly inhibited the locomotion induced by the selective D₂ agonist RU 24213 (5 mg/kg SC) and the mixed D₁/D₂ agonist apomorphine (0.5 mg/kg SC) in monominedepleted mice, without altering the animals' responsiveness to threshold doses of these drugs. However, amantadine did not facilitate the locomotion induced by threshold (3 mg/kg IP) or fully active doses (30 mg/kg IP) of the selective D₁ agonist SKF 38393, which distinguishes amantadine from other NMDA receptor blockers. Since the potentiation of dopamine D₁-dependent locomotion may be a major factor in the antiparkinson activity of MK 801 and other glutamate receptor antagonists the inability of amantadine to potentiate SKF 38393 in this study suggests the mechanism of its anti-akinetic activity differs, from that of conventional glutamate blocking drugs.     

The effect of α-trinositol (d-myo-Inositol 1,2,6-trisphosphate) on formalin-evoked spinal amino acid and prostaglandin E2 levels

The effect of the inositol trisphosphate analog α-trinositol on noxious-evoked behavior, amino acid and prostaglandin E₂ (PGE₂) release was examined in unanesthetized rats using intrathecal microdialysis probes. Subcutaneous injection of 50 μl 5% formalin solution produced two phases of pain-like behavior and significant elevation of glutamate, aspartate, glycine, taurine and serine during phase 1. PGE₂ concentrations were increased during both phases 1 and 2. Intraperitoneal delivery of 300 mg/kg α-trinositol significantly suppressed both phases 1 and 2 of formalin-induced behavior and the associated elevation of amino acids and PGE₂. These data demonstrate that the antinociceptive effect of α-trinositol corresponds to suppression of noxious-evoked release of amino acids and PGE₂ from the spinal cord.     

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.     

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.     

Diverging effects of cortistatin and somatostatin on the production and release of prostanoids from rat cortical microglia and astrocytes

Here we compared the effects of cortistatin and somatostatin on the production of prostanoids from primary cultures of rat cortical microglia and astrocytes. We found that both cortistatin and somatostatin do not modify basal PGE₂ release from cultured astrocytes in 24-h experiments. Somatostatin further enhanced the increase in PGE₂ release induced by IL-1β, whereas cortistatin inhibited such increase. Experiments on microglia showed that somatostatin has no effect on basal and IL-1β-stimulated PGE₂ release, whereas cortistatin reduced baseline prostanoids production and abolished stimulation elicited by IL-1β. The latter effect was associated to the inhibition of COX-2 gene over-expression induced by the cytokine.     

Nociceptive effects induced by intrathecal administration of prostaglandin D2, E2, or F2 alpha to conscious mice

The effects of intrathecal administration of prostaglandins on pain responses in conscious mice were evaluated by using hot plate and acetic acid writhing tests. Prostaglandin D2 (0.5-3 ng/mouse) had a hyperalgesic action on the response to a hot plate during a 3-60 min period after injection. Prostaglandin E2 showed a hyperalgesic effect at doses of 1 pg to 10 ng/mouse, but the effect lasted shorter (3-30 min) than that of prostaglandin D2. Similar results were obtained by acetic acid writhing tests. The hyperalgesic effect of prostaglandin D2 was blocked by simultaneous injection of a substance P antagonist (greater than or equal to 100 ng) but not by AH6809, a prostanoid EP1-receptor antagonist. Conversely, prostaglandin E2-induced hyperalgesia was blocked by AH6809 (greater than or equal to 500 ng) but not by the substance P antagonist. Prostaglandin F2 alpha had little effect on pain responses. These results demonstrate that both prostaglandin D2 and prostaglandin E2 exert hyperalgesia in the spinal cord, but in different ways.     

Diencephalic sites responsive to prostaglandin E2 facilitation of sexual receptivity in estrogen-primed ovariectomized rats

Crystalline prostaglandin E₂ (PGE₂) induced high levels of sexual receptivity when stereotaxically implanted into the anterior-basal hypothalamic and preoptic regions of estrogen-primed, ovariectomized rats. Anterior- as well as medial basal hypothalamic implants of PGE₂ induced a significant elevation of rectal body temperature, but had no effect on open-field activity scores. The possibility that PGE₂ exerts its facilitatory effects upon sexual receptivity via an LHRH mechanism is discussed.     

Characterization of the EP receptor types that mediate longitudinal smooth muscle contraction of human colon,mouse colon and mouse ileum

Background Prostaglandin E₂ (PGE₂) is an inflammatory mediator implicated in several gastrointestinal pathologies that affect normal intestinal transit. The aim was to establish the contribution of the four EP receptor types (EP_1–4), in human colon, that mediate PGE₂‐induced longitudinal smooth muscle contraction. Methods Changes in isometric muscle tension of human colon, mouse colon and mouse ileum were measured in organ baths in response to receptor‐specific agonists and antagonists. In addition, lidocaine was used to block neurogenic activity to investigate whether EP receptors were pre‐ or post‐junctional. Key Results PGE₂ contracted longitudinal muscle from human and mouse colon and mouse ileum. These contractions were inhibited by the EP₁ receptor antagonist, EP₁A in human colon, whereas a combination of EP₁A and the EP₃ antagonist, L798106 inhibited agonist responses in both mouse preparations. The EP₃ agonist, sulprostone also increased muscle tension in both mouse tissues, and these responses were inhibited by lidocaine in the colon but not in the ileum. Although PGE₂ consistently contracted all three muscle preparations, butaprost decreased tension by activating smooth muscle EP₂ receptors in both colonic tissues. Alternatively, in mouse ileum, butaprost responses were lidocaine‐sensitive, suggesting that it was activating prejunctional EP₂ receptors on inhibitory motor neurons. Conversely, EP₄ receptors were not functional in all the intestinal muscle preparations tested. Conclusions & Inferences PGE₂‐induced contraction of longitudinal smooth muscle is mediated by EP₁ receptors in human colon and by a combination of EP₁ and EP₃ receptors in mouse intestine, whereas EP₂ receptors modulate relaxation in all three preparations.     

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.     

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.