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*

BACKGROUND: Adenosine 5'-tetraphosphate(ATPP), an endogenous nucleotide, is stored in cells and released into the extracellular space upon stimulation. Some of the biological responses to ATPP were reported, but characteristics of its receptor were not well known. Present study was conducted to investigate the effects of ATPP on mechanical contractility, resting membrane potential and action potential of rat left atrium. METHODS: Left atrium was isolated from Sprague-Dawley rat. Mechanical contraction induced by electrical field stimulation(EFS) was recorded on polygraph using force transducer. With glass microelectrodes(10 MOmega), potential difference across the membrane was measured and recorded on an oscilloscope and a polygraph. RESULTS: ATPP reduced the left atrial contractility with concentration-dependent manner. ATPP also hyperpolarized the resting membrane potential and decreased the action potential duration of the left atrial cell. Nucleotides other than ATPP, such as ATP, ADP, AMP and adenosine, have the same effect as ATPP. However, there is no difference among the nucleotides. Prior treatment of DPCPX, a P1-purinoceptor blocker, inhibited the ATPP-induced negative inotropism and changes of the membrane potential. But suramin, a nonselective P2-purinoceptor blocker, did not alter the effects of ATPP. alpha, beta methylene ADP and adenosine deaminase, which attenuates hydrolysis of adenine nucleotides and inactivates adenosine respectively, did not influence the effects of adenine nucleotides except for adenosine. CONCLUSION: ATPP reduced the mechanical contractility, hyperpolarized the resting membrance potential and decreased duration of action potential of rat left atrium. These effects were induced by ATPP directly, not by adenosine from hydrolyzed ATPP.
Action Potentials ; Adenine Nucleotides ; Adenosine Deaminase ; Adenosine Diphosphate ; Adenosine Triphosphate ; Adenosine* ; Animals ; Extracellular Space ; Glass ; Heart Atria ; Hydrolysis ; Membrane Potentials ; Membranes ; Muscle Contraction ; Nucleotides ; Rats ; Rats, Sprague-Dawley ; Receptors, Purinergic ; Suramin ; Transducers

BACKGROUND: One of the most common side effects of antidepressant medication is orthostatic hypotension, which can be caused by impaired vasoconstriction. This study was designed to compare the inhibitory effects of antidepressants, including selective serotonin reuptake inhibitors (SSRIs), on the contractile responses to alpha1-adrenergic receptor activation and depolarization in isolated rat aorta. METHODS: Vascular rings were suspended for the measurement of isometric tension in a water-jacketed bath filled with Tyrode solution. After pretreatment with antidepressant for 20 min, vasoconstriction induced by norepinephrine (NE) or 35 mM K+ was measured and compared to the control response. RESULTS: Whereas trazodone and tricyclic antidepressants (TCAs) selectively inhibited NE-induced vasoconstriction, SSRIs inhibited depolarization-induced vasoconstriction more potently. The IC50 value of fluoxetine on depolarization- induced vasoconstriction was 3.29 microM, which is consistent with the previous results on L-type Ca2+ currents of cardiac myocyte. Moclobemide, a monoamine oxidase inhibitor, had no effect on vasoconstriction induced by either alpha- adrenergic receptor activation, or depolarization. CONCLUSION: These results suggest that SSRIs, different from TCAs and trazodone, have potent inhibitory actions to depolarization-induced contraction that may be due to blocking Ca2+ entry through L-type Ca2+ channel.
Animals ; Antidepressive Agents ; Antidepressive Agents, Tricyclic ; Aorta* ; Baths ; Fluoxetine ; Hypotension, Orthostatic ; Inhibitory Concentration 50 ; Moclobemide ; Monoamine Oxidase Inhibitors ; Myocytes, Cardiac ; Norepinephrine ; Rats* ; Receptors, Adrenergic ; Serotonin Uptake Inhibitors* ; Trazodone ; Vasoconstriction*

OBJECTIVE: It has been well known that alcohol can modulate several ligand-gated ion channel and voltage-gated ion channels. But the roles of alcohol in the autonomic neurons still remain unclear. In this study, thus we characterized the neuronal acetylcholine receptor (nnAChRs) and investigated the modulation of nnAChRs by ethanol (EtOH). METHODS: We used whole-cells which were acutely dissociated male rat major pelvic ganglion (MPG) neurons, and used gramicidin perforated patch clamp techniques. RESULTS: MPG neurons can be classified on the basis of the response of the soma membrane to depolarizing current pulses ; either tonic or phasic neurons. Sympathetic neurons expressing T-type Ca(2+) channels showed tonic firing pattern, while parasympathetic neurons lacking T-type Ca(2+) channels phasic firing to depolarizing current pulses. When hyperpolarizing currents were injected, sympathetic neurons produced post-anodal rebound spikes, while parasympathetic neurons were silent. Under current clamp mode, Acetylcholine (ACh) evoked significant membrane depolarization and produced subsequently marked membrane hyperporization. Under whole-cell mode, application of ACh-induced inward currents held at holding potentials below 0 mV and reversal potential was close to 0 mV, an equilibrium potential of nonselective cation channel. The ACh-activated current was blocked by methyllycaconitine (MLA ; 10 micrometer), hexamethonium (100 micrometer) and alpha-bungarotoxin (alpha-BuTx ; 100 nM), nAChRs antagonists. EtOH (40 mM) potentiated ACh-induced depolarization and hyperpolarization. EtOH also increased both alpha-BuTx-sensitive and -insensitive ACh-activated currents. Futhermore, EtOH potentiated 5-HT-activated current but had a little effect on GABA-activated current. CONCLUSION: These results suggest that EtOH modulates nnAChRs and 5-HT receptors in MPG neurons.
Acetylcholine ; Animals ; Bungarotoxins ; Carisoprodol ; Ethanol ; Fires ; Ganglia, Autonomic ; Ganglion Cysts ; Gramicidin ; Hexamethonium ; Humans ; Ion Channels ; Male ; Membranes ; Neurons ; Patch-Clamp Techniques ; Rats ; Receptors, Nicotinic* ; Receptors, Serotonin

PURPOSE: The present study aimed to identify the characteristics and physiological function of the P2Y11 receptor, a receptor likely expressed in human retinoblastoma cells. METHODS: We measured possible P2Y11 signaling in WERI-Rb-1 cells using a Ca2+ imaging technique and RT-PCR. RESULTS: 1) 10 micro M ATP elicited a strong but transient increase in Ca2+ in the WERI-Rb-1 cells, and this Ca2+ rise was well maintained after external Ca2+-depletion. 2) ATP-induced Ca2+ response arose entirely through Ca2+ mobilization. 3) P2Y11 agonist (BzATP, 100 micro M) increased Ca2+ by 31.2+/-3.7 % of ATP effect. 4) mRNA for P2Y11 subtype was identified using RT-PCR. CONCLUSIONS: P2Y11 purinergic activation can increase the intracellular calcium level through calcium mobilization in undifferentiated retinoblastoma cells, which may play an important role in cell proliferation, differentiation, and even pathologic processes.
Adenosine Triphosphate ; Calcium ; Cell Proliferation ; Humans* ; Pathologic Processes ; Retinoblastoma* ; RNA, Messenger

BACKGROUND: Extracellular ATP, released from platelets and nerve endings, plays significant roles in the regulation of circulation. The effects of ATP depend on the location of the vessels and the species of experimental animals. Until now, studies were limited to arteries, so we compared the effects of ATP in rat vena cava with those in the aorta and attempted to identify the characteristics of their receptors. METHODS: Vascular rings were isolated from the rat inferior vena cava and descending thoracic aorta. Endothelial cells were preserved or removed by gentle rubbing. The isometric contractions were recorded on polygraph using a force transducer. RESULTS: In the vena cava ring precontracted by 100 nM norepinephrine (NE), ATP elicited relaxations in a dose-dependent manner. These effects were abolished by removal of the endothelium or pretreatment with a nitric oxide synthase inhibitor. Relaxations to ATP in the vena cava (EC50 :9.9 microM) were less potent than those in the aorta (1.7 microM). The relative order of potencies was ADP>ATP>AMP>adenosine, but the maximal relaxation to ADP was smaller than to ATP. ATP-induced vasorelaxation was blocked by suramin, a nonselective antagonist for P2 purinoceptor and reactive blue-2, a P2Y blocker. At basal tension, ATP contracted the vena cava dose-dependently and these effects were potentiated by endothelium-removal. Contractions in the vena cava were also less potent than in the aorta, and the order of potencies was alpha, beta-MeATP>UTP>ATP>ADP>AMP=adenosine. ATP-induced vasoconstriction was blocked by suramin and alpha, beta-MeATP, a desensitizing antagonist of P2X purinoceptor, and potentiated by pretreatment with UTP. CONCLUSION: These results suggest that ADP and ATP acts on P2Y1- and P2Y2-purinoceptor in the endothelium, respectively and induces vasorelaxation of the vena cava, which is mediated by nitric oxide. Since ATP and UTP induced vasoconstriction in endothelium-denuded condition, it may be mediated by the activation of the P2X and P2Y4, 6 purinoceptor on smooth muscles, respectively.
Adenosine Diphosphate ; Adenosine Triphosphate ; Animals ; Aorta ; Aorta, Thoracic ; Arteries ; Endothelial Cells ; Endothelium ; Isometric Contraction ; Muscle, Smooth ; Nerve Endings ; Nitric Oxide ; Nitric Oxide Synthase ; Norepinephrine ; Rats* ; Receptors, Purinergic P2 ; Receptors, Purinergic P2X ; Receptors, Purinergic* ; Relaxation ; Suramin ; Transducers ; Uridine Triphosphate ; Vasoconstriction ; Vasodilation ; Vena Cava, Inferior*

Decreased cardiac contractility occurs in endotoxicosis, but little is known about the ionic mechanism responsible for myocardial dysfunction. In this study, we examined the changes in Ca2+ and K+ currents in cardiac myocytes from endotoxin-treated rat. Ventricular myocytes were isolated from normal and endotoxemic rats (ex vivo), that were treated for 10 hours with Salmonella enteritidis lipopolysaccharides (LPS; 1.5 mg/kg) intravenously. Normal cardiac myocytes were also incubated for 6 hours with 200 ng/ml LPS (in vitro). L-type Ca2+ current (ICa,L) and transient outward K+ current (Ito) were measured using whole cell patch clamp techniques. Peak ICa,L was reduced in endotoxemic myocytes (ex vivo; 6.00.4 pA/pF, P<0.01) compared to normal myocytes (control; 10.90.6 pA/pF). Exposure to endotoxin in vitro also attenuated ICa,L (8.40.4 pA/pF, P<0.01). The amplitude of Ito on depolarization to 60 mV was reduced in endotoxin treated myocytes (16.51.5 pA/pF, P<0.01, ex vivo; 20.00.9 pA/pF, P<0.01, in vitro) compared to normal myocytes (control; 24.71.0 pA/pF). There was no voltage shift in steady-state inactivation of ICa,L and Ito between groups. These results suggest that endotoxin reduces Ca2+ and K+ currents of rat cardiac myocytes, which may lead to cardiac dysfunction.
Animals ; Depression* ; Lipopolysaccharides ; Muscle Cells ; Myocytes, Cardiac* ; Patch-Clamp Techniques ; Rats* ; Salmonella enteritidis

No abstract available.
Wolf-Hirschhorn Syndrome*

Uridine 5'-triphosphate (UTP) is stored in the granules of cells such as platelets and is released into the extracellular space upon cell stimulation. Extracellular UTP is known to influence many biological processes. We investigated the hemodynamic effects of UTP on the perfused rat liver and characterized its receptors. Liver perfusions were performed in a recirculation system under constant pressure (28 cmH2O). The perfusion flow and oxygen consumption rate were measured at 30 second intervals. UTP decreased the perfusion flow and the oxygen consumption rate, dose-dependently. UTP-induced changes were transient and disappeared in about 10 minutes. Suramin (P2-purinergic antagonist, 100 uM) and indomethacin (cyclooxygenase inhibitor, 20 uM) blocked UTP-induced hemodynamic changes significantly. The effects of UTP were also inhibited when Kupffer cells were damaged with treatment of gadolinium chloride (10 mg/kg iv). L-NAME (1 mM), a potent inhibitor of nitric oxide synthase, markedly enhanced and prolonged the contractile response of UTP in the hepatic vessel. These results suggest that UTP acts mainly on suramin-sensitive UTP receptors on the Kupffer cell through prostanoid synthesis. The nitric oxide systems in the endothelium seem to counteract the vasoconstrictile action of UTP in the hepatic circulation.
Animal ; Extracellular Space/*metabolism ; Hemodynamics ; Liver/*metabolism ; *Liver Circulation ; Perfusion ; Rats ; Rats, Sprague-Dawley ; Support, Non-U.S. Gov't ; Uridine Triphosphate/*metabolism

OBJECTIVE: The purpose of this study was to investigate the effects of haloperidol on [Ca2+]i in hamster insulinoma cells (HIT T-15). METHODS: [Ca2+]i levels were measured by calcium imaging techniques, and membrane potential ionic currents were recorded using conventional patch-clamp methods. RESULTS: Haloperidol induced a transient [Ca2+]i increase, which was abolished by the removal of extracellular Ca2+ or pretreatment with Ca2+ channel blockers (nimodipine and mibefradil). Haloperidol depolarized the membrane potential and inhibited the ATP-sensitive K+ (KATP) channels. Sigma receptor agonists, (+)-SKF10047 and ifenprodil, induced a transient [Ca2+]i increase similar to haloperidol. BD1047, a sigma receptor antagonist, completely blocked the [Ca2+]i increase induced by haloperidol. Haloperidol inhibited the KCl-induced [Ca2+]i increase and voltage-dependent Ca2+ currents. Sigma receptor agonists [(+)-SKF10047, ifenprodil] also inhibited the KCl-induced [Ca2+]i increase. CONCLUSION: Our results suggest that haloperidol induces depolarization, which increases [Ca2+]i by voltage-gated Ca2+ currents via the closing of KATP channels. Haloperidol also inhibits KCl-induced [Ca2+]i increases in the same manner. These effects of haloperidol seemed to be mediated by sigma receptors, which might be linked to the pathogenesis of haloperidol-induced diabetes mellitus.
Animals ; Calcium ; Cricetinae ; Diabetes Mellitus ; Haloperidol* ; Insulinoma* ; KATP Channels ; Membrane Potentials ; Receptors, sigma