MicroRNA-125b regulates proliferation and radioresistance of oral squamous cell carcinoma.

BACKGROUND: MicroRNAs (miRNAs) are involved in essential biological activities, and have been reported to exhibit differential expression profiles in various cancers. Our previous study demonstrated that intercellular adhesion molecule-2 (ICAM2) inhibition induces radiosensitisation in oral squamous cell carcinoma (OSCC) cells. Thus, we hypothesised that certain miRNAs play crucial roles in radioresistance in OSCC by regulating ICAM2 expression. METHODS: Because predicted target gene analyses revealed that microRNA-125b (miR-125b) potentially regulates ICAM2 mRNA expression, we examined the association between miR-125b and radioresistance. The expression of miR-125b was investigated by real-time quantitative reverse transcriptase-PCR. For a functional analysis, miR-125b was transfected to OSCC-derived cells. RESULTS: A downregulated expression of miR-125b was found in OSCC-derived cell lines and OSCC samples. The miR-125b-transfected cells showed a decreased proliferation rate, enhanced radiosensitivity to X-ray irradiation and diminished ICAM2 mRNA expression. Moreover, miR-125b expression correlated with OSCC tumour staging and survival. CONCLUSION: These findings suggested that the downregulated miR-125b expression was associated with proliferation and radioresistance mechanisms, probably through ICAM2 signalling. Thus, controlling the expression or activity of miR-125b might contribute to suppressing proliferation and overcoming radioresistance in OSCC.

Oncolytic activity of Sindbis virus in human oral squamous carcinoma cells.

BACKGROUND: Sindbis virus (SIN) infection causes no or only mild symptoms (fever, rash, and arthralgia) in humans. However, SIN has a strong cytopathic effect (CPE) on various cancer cells. This study focuses on the oncolytic activity of SIN AR399 on oral cancer cells compared with reovirus, a well-known oncolytic virus that targets cancer cells. METHODS: We analysed the cytotoxicity and growth of SIN in 13 oral squamous cell carcinoma (OSCC) cell lines (HSC-2, HSC-3, HSC-4, Ca9-22, H-1, Sa-3, KON, KOSC-2, OK-92, HO-1-N1, SCC-4, SAT, SKN-3) and normal human oral keratinocytes (NHOKs). RESULTS: Sindbis virus infection induced CPE in 12 OSCC cell lines at a low multiplicity of infection (MOI) of 0.01, but not in the OSCC cell line, HSC-4 or NHOKs. Sindbis viral growth was not observed in NHOKs, whereas high SIN growth was observed in all OSCC cell lines, including HCS-4. The cytotoxicity and growth of SIN was the same as reovirus at an MOI of 20 in 12 OSCC cell lines. The CPE was shown, by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling assays, to be apoptotic cell death. Furthermore, quantitative RT-PCR of mRNA in HSC-3 and HSC-4 cells after SIN infection showed that activation of caspases, cytochrome c, and IkappaBalpha was associated with SIN-induced apoptosis. CONCLUSION: As a replication-competent oncolytic virus, SIN may be a useful therapeutic modality for oral cancers.

Role of adenosine in insulin-stimulated release of leptin from isolated white adipocytes of Wistar rats.

Leptin, the ob gene product that can decrease caloric intake and increase energy expenditure, is functionally released by insulin from adipose tissue. Adenosine is thought to be an important regulator of the action of insulin in adipose tissue. The present study investigated the role of adenosine in the release of leptin by insulin in isolated rat white adipocytes. Release of leptin, measured by radioimmunoassay, from insulin-stimulated samples was seen after 30 min. Adenosine deaminase, at concentrations sufficient to metabolize endogenous adenosine, decreased insulin-stimulated leptin release. Also, the insulin-stimulated leptin release was completely blocked by the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). Mediation of endogenous adenosine in this action of insulin was further supported by the assay of adenosine released into the medium from adipocytes stimulated with insulin. In addition, activation of adenosine A1 receptors by N6-cyclopentyladenosine (CPA) induced an increase in leptin release in a concentration-dependent manner that could be blocked by antagonists, either DPCPX or 8-(p-sulfophenyl)theophylline (8-SPT). In the presence of U73312, a specific inhibitor of phospholipase C (PLC), CPA-stimulated leptin secretion from adipocytes was reduced in a concentration-dependent manner, but it was not affected by U73343, the negative control for U73312. Moreover, chelerythrine and GF 109203X diminished the CPA-stimulated leptin secretion at concentrations sufficient to inhibit protein kinase C (PKC). These results suggest that, in isolated white adipocytes, the released adenosine acts as a helper and/or a positive regulator for insulin in the release of leptin via an activation of adenosine A1 receptors that involves the PLC-PKC pathway.

Synthesis of phosphorothioate analogues of oligodeoxyribonucleotides and their antiviral activity against human immunodeficiency virus (HIV).

Nuclease-resistant phosphorothioate analogues of oligodeoxynucleotides (oligos) were synthesized by sulfurization of either internucleoside phosphite linkages, in a repetitive manner during chain extension, or internucleoside hydrogen phosphonate linkages, in a single step following chain assembly. These analogues were tested as antiviral agents against human immunodeficiency virus (HIV). In a cytopathic effect inhibition assay using HIV-uninfected susceptible T cells (tetanus toxoid-specific normal T cells) co-cultured with irradiated chronically HIV-infected cells, phosphorothioate oligomers inhibited the cytopathic effect and replication of several isolates of HIV-1 and HIV-2. Thus phosphorothioate analogues of oligos could inhibit cell-to-cell transmission of the virus as well as the infection by cell-free virus particles and also could inhibit a variety of isolates of human retroviruses.

Phosphorothioate and normal oligodeoxyribonucleotides with 5'-linked acridine: characterization and preliminary kinetics of cellular uptake.

Certain phosphorothioate oligodeoxynucleotide (S-oligo) analogs, unlike their normal congeners, have been found to exhibit significant anti-HIV activity [Matsukura et al., Proc. Natl. Acad. Sci. USA 84 (1987) 7706-7710]. Here we report melting temperatures (Tm) of a series of S-oligos compared with those of the corresponding normal oligomers. The Tm's for AT base pairs of S-oligos are significantly depressed relative to normal oligos, while GC-containing S-oligos show much less Tm depression. The Tm's of S-dT oligomers with poly(rA) are reduced relative to the duplexes with normal dA oligomers. These results provide a rational basis for the S-d(CG) sequences as anti-message inhibitors of gene expression. We also describe an automated synthesis of 5'-acridine linked oligothymidylates using phosphoramidite-linked acridine. During this synthesis we noted the replacement of thiophenol for the 6-chloro substituent on acridine. We have measured the Tm's of the compounds with 3 and 5 methylene groups linked to normal and phosphorothioate dTn (with n = 3-40) on duplex formation with the equivalent dAn, and have found small increases of Tm for the 5-methylene-linked acridine derivative. We have monitored the uptake of these fluorescently labeled oligos into HL60 cells, and found that the shorter oligos are more rapidly taken up than the longer, and the normal oligos faster than the S-oligos. The temperature dependence of the cellular uptake suggests an energy-dependent process, and a possible membrane receptor for oligos. These results have significance for the potential use of such compounds as inhibitors of gene expression.

Adenosine and dipyridamole: actions and interactions on the contractile response of guinea-pig ileum to high frequency electrical field stimulation.

The action of adenosine on the myenteric plexus-longitudinal muscle strips from guinea-pig ileum to high frequency electrical field stimulation (10 Hz) was investigated. Electrically induced contractions were reduced markedly by tetrodotoxin (0.2 microM) and atropine (1 microM), and partially by noradrenaline (3 microM) and morphine (3 microM). Adenosine, adenosine 5'-monophosphate (AMP) and adenosine triphosphate (ATP) produced a concentration-dependent inhibition of the high frequency contractions over the range of 0.1-100 microM, the most potent being adenosine. The concentration-response curve for adenosine was significantly shifted to the left by dipyridamole (10 nM), while dipyridamole at higher concentrations (30 nM-10 microM), depressed the contraction markedly by itself. Dipyridamole decreased [3H]-adenosine uptake into strips of ileum in a concentration-dependent manner. There was a significant correlationship between the reduction of adenosine uptake and the inhibition of the contraction induced by dipyridamole (r = 0.970). In strips desensitized to adenosine or treated with adenosine deaminase, the inhibitory effect of dipyridamole was significantly reduced. The present investigation revealed that adenosine depressed responses of guinea-pig ileum to high frequency electrical stimulation and suggested that the inhibitory effect of dipyridamole may be closely associated with the behaviour of endogenous adenosine or related compounds.

The development of tachyphylaxis to electrical stimulation in guinea-pig ileal longitudinal muscles and the possible participation of adenosine and adenine nucleotides.

1 Electrically (30 Hz) induced contractions of guinea-pig isolated ileal longitudinal muscles were reduced by tetrodotoxin (1 micron), adenosine (30 micron) and morphine (10 micron). 2 When stimulated with 10 or 30 Hz for 10 s at 1 min intervals, a progressive decline of amplitude of the contraction was seen (development of tachyphylaxis). At this time, the contractile response to 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) (10 micron) was also greatly reduced. 3 The smaller responses to electrical stimulation and DMPP during tachyphylaxis were restored to their initial amplitude by the addition of theophylline (10 micron). The appearance of tachyphylaxis was prevented by pretreatment with theophylline (1 to 10 micron) and was greatly accelerated by pretreatment with dipyridamole (0.1 1 micron). 4 In [14C]-choline or [3H]-adenosine preloaded muscle strips, electrical stimulation (30 Hz) increased the 14C- or 3H-output, the effect being sensitive to tetrodotoxin blockade. The tachyphylaxis to electrical stimulation was accompanied by a considerable and sustained increase in 3H-output, an effect that was accelerated by dipyridamole (1 micron). The 14C-output initially increased but fell off gradually with the development of tachyphylaxis at which time theophylline (30 micron) reversed the fall. 5 There was a marked increase in the proportion of released [3H]-adenosine to its derivatives during the development of tachyphylaxis. Approximately 60% of the released total radioactivity after tachyphylaxis was found to be [3H]-adenosine. 6 These results suggest that the development of tachyphylaxis may be closely associated with the release of endogenous adenosine derivatives (mostly adenosine) which have presynaptic inhibitory actions on the cholinergic elements in guinea-pig ileum.

In vitro studies of release of adenine nucleotides and adenosine from rat vascular endothelium in response to alpha 1-adrenoceptor stimulation.

1. Noradrenaline-induced release of endogenous adenine nucleotides (ATP, ADP, AMP) and adenosine from both rat caudal artery and thoracic aorta was characterized, using high-performance liquid chromatography with fluorescence detection. 2. Noradrenaline, in a concentration-dependent manner, increased the overflow of ATP and its metabolites from the caudal artery. The noradrenaline-induced release of adenine nucleotides and adenosine from the caudal artery was abolished by bunazosin, an alpha 1-adrenoceptor antagonist, but not by idazoxan, an alpha 2-adrenoceptor antagonist. Clonidine, an alpha 2-adrenoceptor agonist, contracted caudal artery smooth muscle but did not induce release of adenine nucleotides or adenosine. 3. Noradrenaline also significantly increased the overflow of ATP and its metabolites from the thoracic aorta in the rat; however, the amount of adenine nucleotides and adenosine released from the aorta was considerably less than that released from the caudal artery. 4. Noradrenaline significantly increased the overflow of ATP and its metabolites from cultured endothelial cells from the thoracic aorta and caudal artery. The amount released from the cultured endothelial cells from the thoracic aorta and caudal artery. The amount released from the cultured endothelial cells from the aorta was also much less than that from cultured endothelial cells from the caudal artery. In cultured smooth muscle cells from the caudal artery, a significant release of ATP or its metabolites was not observed. 5. These results suggest that there are vascular endothelial cells that are able to release ATP by an alpha 1-adrenoceptor-mediated mechanism, but that these cells are not homogeneously distributed in the vasculature.

Role of 5-HT(2) receptor subtypes in depletion of 5-HT induced by p-chloroamphetamine in the mouse frontal cortex.

A serotonin (5-hydroxytryptamine, 5-HT)-releasing drug, p-chloroamphetamine elicited decreases in 5-HT levels in the mouse frontal cortex. 5-HT reduction elicited by p-chloroamphetamine was inhibited by the 5-HT(2A/2B/2C) receptor antagonist, LY 53857 and the 5-HT(2A) receptor antagonist, ketanserin. However, the 5-HT(2B/2C) receptor antagonist, SB 206553, enhanced it. LY 53857 and ketanserin can inhibit hyperthermia elicited by p-chloroamphetamine, although SB 206553 enhances it. The effects of the 5-HT(2) receptor antagonists on neurotoxicity are very similar to those on hyperthermia. Since hyperthermia facilitates neurotoxicity induced by amphetamine analogue, these 5-HT(2) receptor antagonists may modify 5-HT depletion induced by p-chloroamphetamine through responses to body temperature.

Effects of adenosine on 45Ca uptake and [3H]acetylcholine release in synaptosomal preparation from guinea-pig ileum myenteric plexus.

The effects of adenosine on acetylcholine (ACh) release and calcium uptake were examined in a synaptosomal fraction prepared from guinea-pig ileum myenteric plexus-longitudinal muscle. A high concentration of potassium (40 mM) and electrical pulses (ES:10Hz) caused a marked increase in the output of [3H]ACh from [3H]choline-preloaded crude synaptosomes. This [3H]ACh output was calcium- and temperature-dependent. Adenosine reduced the high potassium-induced release significantly, and the electrically stimulated release completely. When the preparation was depolarized by high potassium or electrical pulses, the 45Ca uptake by synaptosomes was significantly enhanced. The uptake of 45Ca induced by high potassium was significantly reduced and that induced by electrical stimulation was completely abolished by adenosine. From these results, it may be suggested that adenosine inhibits neurotransmitter release by suppressing the presynaptic influx of calcium ion during depolarization of the cholinergic nerve terminals in guinea-pig ileum.

Sites of actions of adenosine in intrinsic cholinergic nerves of ileal longitudinal muscle from guinea pig.

The sites of presynaptic actions of adenosine on electrically stimulated longitudinal muscle strip of guinea pig ileum were studied with a special dual organ bath. Local stimulation (0.1 Hz) of the anal part of the strip induced a twitch response in the stimulated part and in the non-stimulated oral part; both of these twitch responses were depressed by an application of adenosine to the anal, stimulated part of the strip. The present results suggested that electrical stimulation applied to the anal part of the strip travelled along the network of Auerbach's plexus in the oral direction, and adenosine might depress such conduction in cholinergic nerve fibers in addition to nerve terminals.

Effect of ascorbate on the contractile response induced by DMPP in guinea-pig ileal longitudinal muscle strip.

The study concerned the effect of ascorbate on the contractile response induced by DMPP in the guinea-pig ileal longitudinal muscle. At 0.5-10 mM, sodium ascorbate shifted the dose-response curve for DMPP to the left and enhanced the maximum contraction. On the other hand, in the presence of ascorbate, the high potassium (40 mM)-induced contraction was not altered, and the contraction by acetylcholine and histamine in concentrations higher than 1 microM and 0.3 microM, respectively, was slightly reduced. Thus, ascorbate enhanced the contractile response caused by DMPP which induced acetylcholine release from nerve terminals indirectly by stimulating the ganglia of the Auerbach's plexus elements. This potentiating effect of ascorbate was studied with a dual organ bath which was partitioned into two compartments. When the oral half of the muscle strip was directly stimulated by DMPP applied to the oral compartment, a contraction of the unstimulated anal part was observed. This contraction was blocked by atropine or adenosine applied to the unstimulated part. Tetrodotoxin, applied to the stimulated part, abolished the contraction of the unstimulated part. The contraction of the unstimulated part was enhanced by ascorbate applied to the stimulated part but not to the unstimulated part. These results indicate that the contraction observed in the unstimulated part may have been caused by acetylcholine release from cholinergic nerves as a result of conduction of the oral part excitation by DMPP along cholinergic nerve fibers; ascorbate may have affected the ganglion cells in Auerbach's plexus to potentiate the action of DMPP.

Role of nitric oxide from the endothelium on the neurogenic contractile responses of rabbit pulmonary artery.

The effects of L-NG-nitro arginine (L-NO2Arg), a stereospecific inhibitor of nitric oxide formation, on the responsiveness of rabbit pulmonary artery to transmural electrical stimulation were studied. The contractile response evoked by electrical stimulation at 4 Hz was abolished by tetrodotoxin (10(-7) M) and depressed to approximately 10% by bunazosin (10(-6) M), an alpha 1-antagonist. Pretreatment with L-NO2Arg (10(-5) M) significantly potentiated the response to electrical stimulation without changing the resting tension. D-NO2Arg (10(-5) M) did not show such a potentiating action. In endothelium-denuded arteries, L-NO2Arg did not potentiate the response to electrical stimulation. The effect of L-NO2Arg on endogenous noradrenaline release in response to electrical stimulation was also examined by HPLC with electrochemical detection; L-NO2Arg did not affect noradrenaline release. The contractions induced by exogenous noradrenaline (10(-6)-10(-5) M) were enhanced by L-NO2Arg, but not by D-NO2Arg. These results suggest that the vasoconstriction induced by sympathetic nerve stimulation in the rabbit pulmonary artery is modulated by endogenous nitric oxide or nitric oxide-like substances released from endothelial cells.

Participation by purines in the modulation of norepinephrine release by methoxamine.

The alpha 1-receptor agonist methoxamine reduced, by a prazosin sensitive mechanism, the nerve stimulation evoked release of norepinephrine in the rat caudal artery. The effect of methoxamine was also antagonized by the purinoceptor antagonist 8-(p-sulfophenyl)theophylline suggesting an involvement of endogenous purines in this process. Indeed, methoxamine caused the release of adenine nucleotides and adenosine, an action which was blocked by prazosin. These results suggest that methoxamine releases ATP or a related purine which in turn decreases transmitter release by acting on prejunctional purinoceptors.

Methoxamine-induced release of endogenous ATP from rabbit pulmonary artery.

Methoxamine, an alpha 1-adrenoceptor agonist, significantly increased the overflow of ATP, ADP and AMP, but not adenosine, by a prazosin-sensitive mechanism in the rabbit pulmonary artery. Among the adenine nucleotides released, the amount of ATP was larger than those of the other two. Such release of adenine nucleotides was not induced by clonidine, an alpha 2-adrenoceptor agonist, and isoproterenol, a beta-adrenoceptor agonist. Methoxamine-induced release was observed in the absence of extracellular calcium, but was not observed at a low temperature, 27 degrees C. This suggests an extracellular calcium-independent and temperature-dependent ATP-releasing mechanism coupled with alpha 1-adrenoceptors in rabbit pulmonary artery.

Dietary cholesterol enhances impaired endothelium-dependent relaxations in aortas of salt-induced hypertensive Dahl rats.

We investigated the effect of hypercholesterolemia on the vascular reactivity of thoracic aortas isolated from hypertensive Dahl salt-sensitive (DS) rats. DS rats were fed on a low-sodium diet (control group), a low-sodium plus high-cholesterol diet (CHOL group), a high-sodium diet (NaCl group) or a high-sodium plus high-cholesterol diet (NaCl + CHOL group) for 8 weeks. Hypercholesterolemia developed in the CHOL and NaCl + CHOL groups, while hypertension developed in the NaCl and NaCl + CHOL groups, with these changes being greatest in the NaCl + CHOL group. Aortic cholesteryl ester accumulation was attenuated in the aortic rings from the NaCl and NaCl + CHOL groups, compared to the control group. The degree of attenuation in the NaCl + CHOL group was significantly greater than that in the NaCl group. Endothelium-dependent relaxations induced by the calcium ionophore A23187 were attenuated only in the NaCl + CHOL group. Endothelium-independent relaxations in response to sodium nitroprusside were slightly but significantly attenuated in the NaCl + CHOL group. The relaxations in the CHOL group were comparable to those in the control group. These findings indicate that cholesterol feeding strikingly enhances the impaired endothelium-dependent relaxations and the slightly impaired endothelium-independent relaxations in the aorta of DS rats with salt-induced hypertension, parallel to the development of hypertension, hypercholesterolemia and cholesterol deposition.

Release of endogenous ATP from the caudal artery in rats with arteriosclerosis.

Noradrenaline significantly increased (by a prazosin-sensitive mechanism) the overflow of ATP and its metabolites from the caudal arteries of rats treated with excess vitamin D2 and a high-cholesterol diet (arteriosclerotic rats), although the amount of the overflow was smaller than that in the normal rats. The arteries from the arteriosclerotic rats showed a marked increase in the calcium content and there was a significant negative correlation between the noradrenaline-induced overflow of ATP and the arterial calcium content. These findings indicate that ATP release from arteriosclerotic rat caudal arteries mediated by alpha 1-adrenoceptors is impaired in proportion to the extent of arterial calcification.

Persistent release of noradrenaline caused by anticancer drug 4'-epidoxorubicin in rat tail artery in vitro.

Anthracycline derivatives including 4'-epidoxorubicin are known to cause cardiovascular side effects. In this study we examined the effects of 4'-epidoxorubicin on sympathetic nerves of rat tail artery in vitro. Treatment with 4'-epidoxorubicin at concentrations higher than 10 microM gradually increased the resting tension of the arterial strips, an effect which was greatly enhanced by subsequent addition of 10 microM cocaine. This increase of the resting tension by 4'-epidoxorubicin was prevented by prazosin, suppressed in the arterial strips of reserpine-pretreated rats, and reduced by superoxide dismutase. However, tetrodotoxin and histamine receptor antagonists (diphenhydramine and cimetidine) failed to influence it. The contractile response to electrical sympathetic stimulation was slightly attenuated by 30 microM 4'-epidoxorubicin. 4'-epidoxorubicin did not shift the concentration-response curve for noradrenaline. In the superfusion experiments, the basal release of noradrenaline was increased approximately five-fold by 30 microM 4'-epidoxorubicin, and this increase was not inhibited by 0.1 microM prazosin, 0.5 microM tetrodotoxin, 10 microM cocaine or Ca2+-free medium. Noradrenaline release evoked by electrical stimulation was gradually suppressed by 30 microM 4'-epidoxorubicin treatment. These results suggest that 4'-epidoxorubicin directly acts on the sympathetic nerve to cause persistent release of noradrenaline in rat tail artery.

Nicorandil--induced ATP release in endothelial cells of rat caudal artery is associated with increase in intracellular Ca(2+).

The effect of nicorandil, an ATP-sensitive K(+) channel opener, on the level of intracellular Ca(2+) ([Ca(2+)](i)) and on ATP release in endothelial cells of the rat caudal artery was examined using a fluorescent confocal microscopic imaging system and high-performance liquid chromatography (HPLC) with fluorescent detection, respectively. Nicorandil significantly increased [Ca(2+)](i) and the overflow of ATP and its metabolites. The former reaction was abolished in the absence of extracellular Ca(2+), but it did not change in the presence of thapsigargin or cyclopiazonic acid. The increase in the overflow of ATP and [Ca(2+)](i) induced by nicorandil was markedly suppressed by glibenclamide, an ATP-sensitive K(+) channel blocker. The increase of [Ca(2+)](i) induced by nicorandil was significantly and inversely correlated with the level of intracellular ATP in the endothelial cells, suggesting that activation of ATP-sensitive K(+) channels by nicorandil increases Ca(2+) influx in endothelial cells. The increase of [Ca(2+)](i) might be associated with ATP release.

Prostaglandin E2 induced the cyclic AMP-dependent release of acetylcholine in circular muscles of the isolated guinea pig ileum.

Prostaglandin E2 (PGE2) induced a dose-dependent increase in tone of the circular muscles of guinea pig ileum in vitro. These actions of PGE2 were deleted in the cold-stored preparations and blocked by tetrodotoxin. Atropine reduced the effects of PGE2 and physostigmine potentiated the PGE2-induced contractions. The release of acetylcholine (ACh) by PGE2 was responsible for initiating this contraction. The effect of PGE2 was compared with that of an electrical stimulation which also initiated a non-receptor-mediated release of ACh. Hexamethonium abolished the effect of PGE2 but did not influence the actions of the electrical stimulations. Synaptosomal fractions of the circular muscles were prepared to study the release of [14C]ACh. However, PGE2 failed to evoke a marked increase in the efflux of radioactivity, even at the maximal concentration. Damage and/or removal of the myenteric plexus may be responsible for this result because electrical stimulations that exert a powerful spasmogenic effect on longitudinal muscles also induced an insensitive response. Alloxan and ethacrynic acid, inhibitors of adenylate cyclase, reduced the activity of PGE2 at a concentration insufficient to modify either the actions of ACh or the electrical stimulations. 3-Isobutyl-1-methylxanthine (IBMX) potentiated the responses to PGE2 at a dose sufficient to block the activity of phosphodiesterase (PDE). Imidazole, a stimulator of PDE, decreased the actions of PGE2 in a dose-dependent manner. IBMX, like imidazole, failed to modify the activities of both ACh and the electrical stimulations. These results indicate that PGE2 may function as a releaser of ACh in a cyclic AMP-dependent manner in the circular muscles of guinea pig ileum.