We cultured the rabbit inner medullary collecting duct (IMCD) cells as monolayers on collagen-coated membrane filters, and investigated distribution of the P2Y receptors by analyzing nucleotide-induced short circuit current (Isc) responses. Exposure to different nucleotides of either the apical or basolateral surface of cell monolayers stimulated Isc. Dose-response relationship and cross-desensitization studies suggested that at least 3 distinct P2Y receptors are expressed asymmetrically on the apical and basolateral membranes. A P2Y2-like receptor, which responds to UTP and ATP, is expressed on both the apical and basolateral membranes. In addition, a uracil nucleotide receptor, which responds to UDP and UTP, but not ATP, is expressed predominantly on the apical membrane. In contrast, a P2Y1-like receptor, which responds to ADP and 2-methylthio-ATP, is expressed predominantly on the basolateral membrane. These nucleotides stimulated intracellular cAMP production with an asymmetrical profile, which was comparable to that in the stimulation of Isc. Our results suggest that the adenine and uracil nucleotides can interact with different P2Y nucleotide receptors that are expressed asymmetrically on the apical and basolateral membranes of the rabbit IMCD cells, and that both cAMP- and Ca2+-dependent signaling mechanisms underlie the stimulation of Isc.
Adenine ; Adenosine Diphosphate ; Adenosine Triphosphate ; Membranes ; Nucleotides ; Uracil ; Uracil Nucleotides ; Uridine Diphosphate ; Uridine Triphosphate

The energy metabolism of the brain has been measured in cat model using high performance liquid chromatography(HPLC). The experimental groups were divided into three according to the duration of ischemia. In 1- and 3-hour ischemia groups, recirculation had increased the ATP, UTP and GTP significantly to 39-49%, 53-57% and 39-62% of the sham control value respectively. Also in these groups, recirculation had increased adenylate energy charge(E.C.) to 75-82% of sham control value. Whereas there were slight increase in adenylate E.C. after recirculation in 5-hr ischemia group, with the remainders not increasing significantly. The Na+, K+-ATPase activities were not significant statistically among the groups. These results suggest that in order to prevent from the irreversible ischemic brain damage, restoration of blood flow must be accomplished within 3 hours from the onset of the acute focal ischemia in cat.
Adenosine Triphosphate ; Animals ; Brain ; Cats ; Chromatography, High Pressure Liquid ; Energy Metabolism* ; Guanosine Triphosphate ; Ischemia ; Uridine Triphosphate

BACKGROUND: Uracil nucleotides are stored in platelets and all other cells, and are released into the extracellular space upon stimulation. They show various biological responses but their actions and mechanism are not well understood. This study was conducted to investigate the effects of uridine 5'-triphosphate(UTP) on vascular tone and to identify the characteristics of their receptors. METHODS: Aortic ring preparation were made from the rat descending thoracic aorta. Endo-thelial cells were preserved or removed by gentle rubbing, The basal tension of aortic ring was lgm and isometric contraction were recorded on polygraph using force transducer. RESULTS: In aortic ring Precontracted by 100nM norepinephrine, UTP induced dual effect with various concentrations. UTP elicited endothelium-dependent relaxation at low concentrations(100nM-10microM), and endothelium-independent contraction at high concentrations(more than 30microM). Among uracil nucleotides, UDP was as much effective as UTP in vascular tone, but UMP and uridine were not. UTP(pA50 6.15) was more potent than ATP(5.17), ITP(4.75) and other nucleotides(TTP, GTP, CTP). At basal tension, UTP induced relaxation at low concentrations and contraction at hige concentrations in endothelium-intact ring. But in endothelium-removed ring, UTP elicited only contraction. Prior treatment of aortic ring with suramin, a non-selective P2-purinoceptor blocker, inhibited UTP-Induced relaxation and contraction. Reactive blue-2, a P2gamma purinoceptor blocker, inhibited relaxation only, but alpha, beta-methylene ATP, a P2x Purinoceptor blocker, enhanced contractile response. ATP inhibited the UPT-induced relaxation, but 2-methylthio ATP did not alter the effects of UTP. It means that UTP and ATP act at the same receptor but 2-methylthio ATP does not. CONCLUSION: These results suggest that UTP-induced relaxation is mediated by nucleotide receptors on endothelium and the contraction is mediated by pyrimidinoceptors on vascular smooth muscle.
Adenosine Triphosphate ; Animals ; Aorta ; Aorta, Thoracic* ; Endothelium ; Extracellular Space ; Guanosine Triphosphate ; Isometric Contraction ; Muscle, Smooth, Vascular ; Norepinephrine ; Rats* ; Receptors, Purinergic ; Receptors, Purinergic P2X ; Relaxation ; Suramin ; Transducers ; Uracil Nucleotides ; Uridine Diphosphate ; Uridine Monophosphate ; Uridine Triphosphate ; Uridine*

The study was designed to examine the effects of pretreatment with mannitol, methyl prednisolone and nimodipine on the acute focal cerebral ischemia in the cats of occlusion of the proximal part of the middle cerebral artery via the postorbital approach. The energy metabolisms of the brain was measured utilizing the high liquid performance chromatography in the brain tissues of cats. The experimental animals were seperated into 3 groups. group I: the sham control group. group II: the recirculation group. group III: the treatment group. There were significant increase in the ATP, GTP, UTP and E.C. levels in focal ischemic cerebral tissues of the treatment group when compared with the recirculation group. It is suggested that pretreatment with the combination of these drugs may prevent the ischemic damage from the acute focal cerebral ischemia by the maintenance of high energy metabolites. However further studies should determine the synergistic pharmacologic mechanisms in this therapeutic strategy.
Adenosine Triphosphate ; Animals ; Brain ; Brain Ischemia* ; Cats ; Chromatography ; Energy Metabolism ; Guanosine Triphosphate ; Mannitol ; Middle Cerebral Artery ; Nimodipine ; Prednisolone ; Uridine Triphosphate

OBJECTIVETo test the different contrctile responses of extracellular nucleotides, such as ATP, UTP and nucleotide uridine adenosine tetraphosphate (Up4A) in gastric longitudinal muscle (LM) and circular muscle (CM). Examined the effect of P2X and P2Y receptor antagonists (in this study, we used IP5I and suramin) and cyclooxygenase inhibitor (indomethacin) on Up4A induced contractile responses in LM and CM.

METHODSThe rats were sacrificed and the stomachs were opened to gain LM and CM. Using organ bath system to assess contrctile responses of smooth muscle.

RESULTSUp4A could induce contractile responses in both CM and LM, which were similar with ATP and UTP. IP5 did not attenuate Up4A could induce contractions in both LM and CM, but suramin and indomethacin significantly inhibited Up4A contraction in CM, but not in LM.

CONCLUSIONOur results suggest that extracellular nucleosides and their inhibitors induce different responses between LM and CM.

Adenosine Triphosphate ; pharmacology ; Animals ; Dinucleoside Phosphates ; pharmacology ; Indomethacin ; Muscle Contraction ; Muscle, Smooth ; physiology ; Nucleotides ; pharmacology ; Rats ; Suramin ; Uridine Triphosphate ; pharmacology

BACKGROUND AND OBJECTIVES: Puringeric receptors and their agonists like uridine-5-triphosphate (UTP) and adenosine triphosphate (ATP), regulate mucin secretion in middle ear epithelial cells. In the present study, we examined the effects of purinergic agonists on Ca2+ influx ([Ca2+]i ) in normal human middle ear epithelial (NHMEE) cells. We also examined the effect of caffeine, an inositol 1, 4, 5-triphosphate (IP3) inhibitor, on UTP induced [Ca2+]i and mucin secretion in NHMEE cells. MATERIALS AND METHOD: NHMEE cells were stimulated with various purinergic agonists, such as UTP, and [Ca2+]i was measured using a miniature double perfusion chamber. UTP-induced mucin secretion was quantitated by immunoblotting assay. RESULTS: The determined order of purinergic agonist potency with respect to [Ca2+]i was ATP=UTP>2-MeSATP>ADP>> adenosine. UTP-induced mucin secretion was inhibited when the intracellular Ca2+ was removed with 2-bis (2-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid-acetoxymethyl ester. Caffeine suppressed UTP-induced [Ca2+]i, and but inhibited UTPinduced and constitutional mucin secretion. CONCLUSION: Our results suggest that caffeine may have a therapeutic effect in mucoid otitis media by suppressing mucin secretion.
Adenosine ; Adenosine Triphosphate ; Caffeine* ; Calcium ; Ear, Middle* ; Epithelial Cells* ; Humans* ; Immunoblotting ; Inositol ; Mucins* ; Otitis Media ; Perfusion ; Purinergic Agonists ; Uridine Triphosphate

BACKGROUND AND OBJECTIVES: Extracellular uridine 5'-triphosphate (UTP) regulates a variety of biological functions in the airway epithelium including chloride and fluid transport, mucociliary clearance, and mucin secretion via P2Y purinergic receptors. This study was undertaken to investigate which P2Y purinergic receptors are expressed in the normal human middle ear epithelial (NHMEE) cells. We also determined the levels of mucin secretion and its mRNA expressions following stimulation with UTP. MATERIALS AND METHOD: The level of P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11 and P2Y12) receptors and mucin gene 5AC (MUC5AC), MUC5B, MUC8 messenger RNA (mRNA)s were measured by reverse transcription (RT)-polymerase chain reaction (PCR). We also determined the levels of mucin secretion following stimulation with UTP by dot-blotting method in NHMEE cells. RESULTS: Middle ear epithelial cells expressed P2Y1, P2Y2, P2Y6, P2Y11 and P2Y12 receptors but not the P2Y4 receptor. Apically applied UTP induced increased the mucin secretion. On the other hand, UTP did not enhance the mucin mRNA expression until 72 h had lapsed after treatment. CONCLUSION: Our study suggests that UTP acts as a secretogogue on mucin secretion in NHMEE cells.
Ear ; Ear, Middle* ; Epithelial Cells* ; Epithelium ; Hand ; Humans* ; Mucins* ; Mucociliary Clearance ; Mucous Membrane ; Receptors, Purinergic ; Reverse Transcription ; RNA, Messenger ; Uridine Triphosphate ; Uridine*

BACKGROUND: The effects of a newly synthesized potassium channel opener, KR-30816((-)(nitro-2-hydroxymethyl-2-methy-2H-1-benzopyran-4-y1)pyridine oxide) on the action potential of papillary muscles of guinea pigs and the ATP-sensitive potassium channel current(IKATP) of single ventricular muscle cells of rats were examined to make clear its action mechanism of the KATPchannel. METHODS: We used the conventional microelectrode and the excised inside-out patch configuration. RESULTS: KR-30816 caused a shortening of the action potential duration in dose-dependent manner, which was inhibited by glibenclamide(3microM). Before run-down of the K+channel, KR-30816 activated the cardiac ATP-sensitive K+ channel only in the presence of ATP and shifted the dose-response relation curve between [ATP]i and the channel activity to the right in parallel. After run-down of the KATP channel, KR-30816 did not after the channel opening either in the absence or in the presence of UDP. CONCLUSION: These results suggest that KR-30816 antagonizes the inhibitory effect of ATP on the KATPchannel in a competitive manner, thereby enhancing the channel openings.
Action Potentials ; Adenosine Triphosphate ; Animals ; Guinea Pigs ; Heart ; Microelectrodes ; Muscle Cells ; Papillary Muscles ; Potassium Channels* ; Potassium* ; Rats ; Uridine Diphosphate

We explore the question of whether adenosine 5'-triphosphate (ATP) acts as an excitatory neurotransmitter in guinea-pig gastric smooth muscle. In an organ bath system, isometric force of the circular smooth muscle of guinea-pig gastric antrum was measured in the presence of atropine and guanethidine. Under electrical field stimulation (EFS) at high frequencies (>20 Hz), NO-mediated relaxation during EFS was followed by a strong contraction after the cessation of EFS (a "rebound-contraction"). Exogenous ATP mimicked the rebound-contraction. A known P2Y-purinoceptor antagonist, reactive blue 2 (RB-2), blocked the rebound-contraction while selective desensitization of P2x-purinoceptor with alpha, beta-MeATP did not affect it. ATP and 2-MeSATP induced smooth muscle contraction, which was effectively blocked by RB-2 and suramin, a nonselective P2-purinoceptor antagonist. Particularly, in the presence of RB-2, exogenous ATP and 2-MeSATP inhibited spontaneous phasic contractions, suggestingthe existence of different populations of purinoceptors. Both the rebound-contraction and the agonist-induced contraction were not inhibited by indomethacin. The rank orders of agonists' potency were 2-MeSATP > ATP gtoreq UTP for contraction and alpha, beta-MeATP gtoreq beta, gamma-MeATP for inhibition of the phasic contraction, that accord with the commonly accepted rank order of the classical P2Y-purinoceptor subtypes. Electrical activities of smooth muscles were only slightly influenced by ATP and 2-MeSATP, whereas alpha, beta-MeATP attenuated slow waves with membrane hyperpolarization. From the above results, it is suggested that ATP acts as an excitatory neurotransmitter, which mediates the rebound-contraction via P2Y-purinoceptor in guinea-pig gastric antrum.
Adenosine Triphosphate* ; Adenosine* ; Atropine ; Baths ; Guanethidine ; Indomethacin ; Membranes ; Muscle, Smooth ; Neurotransmitter Agents* ; Pyloric Antrum* ; Receptors, Purinergic ; Relaxation ; Stomach ; Suramin ; Uridine Triphosphate

Prospect Hill virus (PHV) is related antigenically but distinct to Hantaan virus. As a member of genus Hantavirus, PHV has three segmented RNA genome. Among these segments, Small segment(S) encodes nucleocapsid protein (NP) as structural protein and also may do functional nonstructural protein(NSs). We performed in vitro transcription to produce vRNA of PHV S genome. For the first step of in vitro transcription of S genome of PHV, the S RNA segrnent which is 1,675 nucleotides long was amplified by RT-PCR using PCR primers built according to cDNA sequence of PHV S genome. Next, a new PCR primer appended above downstream primer to T7 phage promoter sequence was reconstructed to obtain PCR product containing T7 promoter. Then another PCR was performed. Using this PCR product as the template, in vitro run-off transcription without cloning by transcriptional vector was performed to obtain viral- sense RNA transcript. Thereafter, the size of transcript was assessed by running on formaldehyde agarose gels. Since the transcription reactants contain a-S UTP, the transcript is detectable by autoradiography. The transcript was also detectable by northern hybridization with a-P dCTP- labelled PHV amplicon probe (319 bp) and the initiation start point of run-off transcription was also determined by primer extention analysis. Our data indicate that the in vitro transcript could be produced from the PCR product amplified by PCR primer containing T7 phage promoter without cloning into a phage transcription vector.
Autoradiography ; Bacteriophage T7 ; Bacteriophages ; Clone Cells ; Cloning, Organism ; DNA, Complementary ; Formaldehyde ; Gels ; Genome* ; Hantaan virus ; Hantavirus ; Nucleocapsid Proteins ; Nucleotides ; Polymerase Chain Reaction ; RNA ; Running ; Sepharose ; Uridine Triphosphate ; Viruses*