Enhancement of acid-sensing ion channel activity by metabotropic P2Y UTP receptors in primary sensory neurons.

Peripheral purinergic signaling plays an important role in nociception. Increasing evidence suggests that metabotropic P2Y receptors are also involved, but little is known about the underlying mechanism. Herein, we report that selective P2Y receptor agonist uridine 5'-triphosphate (UTP) can exert an enhancing effect on the functional activity of acid-sensing ion channels (ASICs), key sensors for extracellular protons, in rat dorsal root ganglia (DRG) neurons. First, UTP dose-dependently increased the amplitude of ASIC currents. UTP also shifted the concentration-response curve for proton upwards, with a 56.6 ± 6.4% increase of the maximal current response to proton. Second, UTP potentiation of proton-gated currents can be mimicked by adenosine 5'-triphosphate (ATP), but not by P2Y1 receptor agonist ADP. Potentiation of UTP was blocked by P2Y receptor antagonist suramin and by inhibition of intracellular G protein, phospholipase C (PLC), protein kinase C (PKC), or protein interacting with C-kinase 1 (PICK1) signaling. Third, UTP altered acidosis-evoked membrane excitability of DRG neurons and caused a significant increase in the amplitude of the depolarization and the number of spikes induced by acid stimuli. Finally, UTP dose-dependently exacerbated nociceptive responses to injection of acetic acid in rats. These results suggest that UTP enhanced ASIC-mediated currents and nociceptive responses, which reveal a novel peripheral mechanism underlying UTP-sensitive P2Y2 receptor involvement in hyperalgesia by sensitizing ASICs in primary sensory neurons.

Effects of Zidovudine Treatment on Heart mRNA Expression and Mitochondrial DNA Copy Number Associated with Alterations in Deoxynucleoside Triphosphate Composition in a Neonatal Rat Model.

The prevention of mother-to-child transmission (MTCT) of HIV is a crucial component in HIV therapy. Nucleoside reverse transcriptase inhibitors (NRTIs), primarily 3'-azido-3'-thymidine (AZT [zidovudine]), have been used to treat both mothers and neonates. While AZT is being replaced with less toxic drugs in treating mothers in MTCT prevention, it is still commonly used to treat neonates. Problems related to mitochondrial toxicity and potential mutagenesis associated with AZT treatment have been reported in treated cohorts. Yet little is known concerning the metabolism and potential toxicity of AZT on embryonic and neonatal tissues, especially considering that the enzymes of nucleoside metabolism change dramatically as many tissues convert from hyperplastic to hypertrophic growth during this period. AZT is known to inhibit thymidine phosphorylation and potentially alter deoxynucleoside triphosphate (dNTP) pools in adults. This study examines the effects of AZT on dNTP pools, mRNA expression of deoxynucleoside/deoxynucleotide metabolic enzymes, and mitochondrial DNA levels in a neonatal rat model. Results show that AZT treatment dramatically altered dNTP pools in the first 7 days of life after birth, which normalized to age-matched controls in the second and third weeks. Additionally, AZT treatment dramatically increased the mRNA levels of many enzymes involved in deoxynucleotide synthesis and mitochondrial biogenesis during the first week of life, which normalized to age-matched controls by the third week. These results were correlated with depletion of mitochondrial DNA noted in the second week. Taken together, results demonstrated that AZT treatment has a powerful effect on the deoxynucleotide synthesis pathways that may be associated with toxicity and mutagenesis.

CysLT1 leukotriene receptor antagonists inhibit the effects of nucleotides acting at P2Y receptors.

Montelukast and pranlukast are orally active leukotriene receptor antagonists selective for the CysLT1 receptor. Conversely, the hP2Y(1,2,4,6,11,12,13,14) receptors represent a large family of GPCRs responding to either adenine or uracil nucleotides, or to sugar-nucleotides. Montelukast and pranlukast were found to inhibit nucleotide-induced calcium mobilization in a human monocyte-macrophage like cell line, DMSO-differentiated U937 (dU937). Montelukast and pranlukast inhibited the effects of UTP with IC50 values of 7.7 and 4.3 microM, respectively, and inhibited the effects of UDP with IC50 values of 4.5 and 1.6 microM, respectively, in an insurmountable manner. Furthermore, ligand binding studies using [3H]LTD4 excluded the possibility of orthosteric nucleotide binding to the CysLT1 receptor. dU937 cells were shown to express P2Y2, P2Y4, P2Y6, P2Y11, P2Y13 and P2Y14 receptors. Therefore, these antagonists were studied functionally in a heterologous expression system for the human P2Y receptors. In 1321N1 astrocytoma cells stably expressing human P2Y(1,2,4,6) receptors, CysLT1 antagonists inhibited both the P2Y agonist-induced activation of phospholipase C and intracellular Ca2+ mobilization. IC50 values at P2Y1 and P2Y6 receptors were <1 microM. In control astrocytoma cells expressing an endogenous M3 muscarinic receptor, 10 microM montelukast had no effect on the carbachol-induced rise in intracellular Ca2+. These data demonstrated that CysLT1 receptor antagonists interact functionally with signaling pathways of P2Y receptors, and this should foster the study of possible implications for the clinical use of these compounds in asthma or in other inflammatory conditions.

Evaluation of reactive blue 2 derivatives as selective antagonists for P2Y receptors.

P2Y receptor pharmacology is hampered by a lack of subtype selective antagonists. However, a recent study evaluated series of compounds, structurally related to the dye reactive blue 2, for their antagonist selectivity at P2X vs. P2Y receptors. Acid blue 129, acid blue 80, acid blue 25 and acid violet 34 were found to be the most potent of the antagonists studied, at P2Y receptors [Naunyn Schmiedeberg's Arch. Pharmacol. 357 (1998) 111]. In this study, we have determined the ability of these four agents to selectively antagonize inositol phosphate turnover mediated by P2Y1 and P2Y2 receptors that are natively expressed in bovine aortic endothelial (BAE) cells. Acid blue 129, acid blue 80, and acid violet 34 shifted the dose-response curve of the P2Y1 agonist 2-methylthio adenosine trisphosphate (2MeSATP) to the right. Acid blue 129 and acid blue 80 were also very weak antagonists of the P2Y2 agonist uridine 5'-triphosphate (UTP). At 30 and 100 microM, acid violet 34 failed to have any significant effect on the dose-response to UTP. However, at 10 microM, acid violet 34 enhanced the UTP responses. Acid blue 80, acid blue 129 and acid violet 34 are P2Y vs. P2X selective, but show poor selectivity between P2Y1 and P2Y2 receptors and are therefore of limited use in the field of P2Y receptor pharmacology. Furthermore, contrary to previous reports, acid blue 25 is not a P2Y-selective antagonist.

Methyl orange antagonizes uridine 5' triphosphate and not alpha,beta-methylene-adenosine 5' triphosphate-evoked depolarization of rat superior cervical ganglia.

1. Compared with the effects of adenosine 5' triphosphate (ATP) on the nervous system, the actions of pyrimidine nucleosides and their 5'-nucleotides, such as uridine 5' triphosphate (UTP), have received less attention. In part, this is because there is a need for a selective antagonist for responses mediated by UTP-activated receptors. The objective of this study was to discover such an antagonist. 2. Superior cervical ganglia isolated from male rats were superfused with a physiological salt solution. Responses to alpha,beta-methylene-ATP (alpha,beta-Me-ATP), potassium, adenosine and UTP were determined before and in the presence of 1-300 microM methyl orange. 3. Methyl orange at 1-100 microM did not alter resting potential or depolarizing responses to alpha,beta-Me-ATP, potassium, or adenosine-evoked hyperpolarizations, but at 10 and 100 microM methyl orange significantly antagonized UTP-evoked depolarizations (P < 0.05). 4. Although the antagonistic effects of methyl orange were not dramatic, this is the first report of a putative pyrimidinoceptor antagonist. These observations also support the idea of distinct receptors for UTP and ATP on rat superior cervical ganglia.

Interplay between the NO pathway and elevated [Ca2+]i enhances ciliary activity in rabbit trachea.

1. Average intracellular calcium concentration ([Ca2+]i) and ciliary beat frequency (CBF) were simultaneously measured in rabbit airway ciliated cells in order to elucidate the molecular events that lead to ciliary activation by purinergic stimulation. 2. Extracellular ATP and extracellular UTP caused a rapid increase in both [Ca2+]i and CBF. These effects were practically abolished by a phospholipase C inhibitor (U-73122) or by suramin. 3. The effects of extracellular ATP were not altered: when protein kinase C (PKC) was inhibited by either GF 109203X or chelerythrine chloride, or when protein kinase A (PKA) was inhibited by RP-adenosine 3', 5'-cyclic monophosphothioate triethylamine (Rp-cAMPS). 4. Activation of PKC by phorbol 12-myristate, 13-acetate (TPA) had little effect on CBF or on [Ca2+]i, while activation of PKA by forskolin or by dibutyryl-cAMP led to a small rise in CBF without affecting [Ca2+]i. 5. Direct activation of protein kinase G (PKG) with dibutyryl-cGMP had a negligible effect on CBF when [Ca2+]i was at basal level. However, dibutyryl-cGMP strongly elevated CBF when [Ca2+]i was elevated either by extracellular ATP or by ionomycin. 6. The findings suggest that the initial rise in [Ca2+]i induced by extracellular ATP activates the NO pathway, thus leading to PKG activation. In the continuous presence of elevated [Ca2+]i the stimulated PKG then induces a robust enhancement in CBF. In parallel, activated PKG plays a central role in Ca2+ influx via a still unidentified mechanism, and thus, through positive feedback, maintains CBF close to its maximal level in the continuous presence of ATP.

La3+ inhibits the UTP-induced Ca2+ mobilization in MDCK cells.

We have studied the effects of La3+ on UTP-induced rises in intracellular calcium levels ([Ca2+]i) measured by fura-2 fluorimetry in Madin Darby canine kidney (MDCK) cells. UTP induced [Ca2+]i rises dose-dependently with an EC50 of 1 mM. The Ca2+ signal was triggered by a Ca2+ release from the inositol 1,4,5-trisphosphate (IP3)-sensitive pool because the signal was completely blocked by pretreatment of the endoplasmic reticulum (ER) Ca2+ pump inhibitor thapsigargin (TG) or the phospholipase C (PLC) inhibitor U73,122. Both the peak height and under-curve area of 10 microM UTP-induced Ca2+ signal was reduced by approximately 40% by extracellular Ca2+ removal, suggesting that UTP induced capacitative Ca2+ entry. La3+ inhibited the UTP-induced Ca2+ signal dose-dependently when added before or after UTP. Pretreatment of 0.1 mM La3+ inhibited the UTP response more than Ca2+ removal did. The mechanisms underlying the La3+ inhibition appear to involve not only block of capacitative Ca2+ entry.

Priming effects of lipopolysaccharide on UTP-induced arachidonic acid release in RAW 264.7 macrophages.

Stimulation of mouse RAW 264.7 macrophages with UTP activates both the inositol phosphate signal transduction pathway and the phospholipase A2 pathway. In the present study, we investigated the interactions between bacterial lipopolysaccharide and UTP in these two systems and the underlying mechanisms involved. While the UTP-induced release of arachidonic acid was only 2.9-fold that in controls, priming the cells with 1 microgram/ml lipopolysaccharide for 1 h before UTP treatment resulted in 9.2-fold arachidonic acid release upon stimulation with UTP. Lipopolysaccharide priming was both concentration- and time-dependent with a peak effect after 1 h treatment at a concentration of 1 microgram/ml. Lipopolysaccharide treatment affect neither the basal nor the UTP-stimulated inositol phosphate formation and [Ca2+]i rise. Pretreatment of the cells with staurosporine, calphostin, N-(2-aminoethyl)-5-isoquinolinesulfonamide H-7), genistein or K-252a led marked inhibition of the priming effect, suggesting that both protein kinase C and tyrosine kinase are involved in the lipopolysaccharide effect. Buffering intracellular Ca2+ levels using [1,2-bis-(o-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester] (BAPTA/AM) or pretreatment with either N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide (H-89), 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD098059) or {1-N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl] -4-phenyl-piperazine (KN-62) did not affect the lipopolysaccharide-induced priming effect. Primed UTP stimulation was inhibited by actinomycin D and cycloheximide, indicating a requirement for both gene expression and protein translation. To further examine whether the stimulatory effects of lipopolysaccharide on phospholipase A2 activity were independent of [Ca2+]i levels but dependent on protein phosphorylation, a fixed Ca2+ concentration and inhibitors of protein phosphatases were used in primed permeabilized cells. Arachidonic acid release from permeabilized cells containing 100 nM Ca2+ was high in lipopolysaccharide-primed cells and potentiated by addition of microcystin, orthovanadate or FK 506. These results that the Ser/Thr and tyrosine phosphorylation cascades induced by protein kinase C and tyrosine kinase, respectively, are required for the arachidonic acid potentiation effect of lipopolysaccharide, which was independent of modulation of the upper stream signaling pathways of UTP.

The effect of PPADS as an antagonist of inositol (1,4,5)trisphosphate induced intracellular calcium mobilization.

1. Brain capillary endothelial cells responded to uridine 5'-triphosphate (UTP) and adenosine 5'-triphosphate (ATP) by activation of phospholipase C and by large changes in [Ca2+]i. These cells expressed mRNA sequences identical to the sequence of the P2Y2-purinoceptor of rat pituitaries. 2. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) at 100 microM did not prevent UTP and ATP induced accumulations of total [3H]-inositol (poly)phosphates. It inhibited UTP and ATP induced intracellular Ca2+ mobilization (IC50 = 30 microM) by non competitive mechanism. 3. PPADS (100 microM) inhibited endothelin-1 induced accumulation of total [3H]-inositol (poly)phosphates by less than 20% and prevented most of endothelin-1 induced intracellular Ca2+ mobilization (IC50 = 30 microM). 4. PPADS (100 microM) had no action on ionomycin induced intracellular Ca2+ mobilization. 5. Microinjection of inositol (1,4,5)trisphosphate (InsP3) into Xenopus oocytes induced large Ca2+ activated Cl- currents that were prevented by heparin and by PPADS. 6. It is concluded that PPADS does not recognize rat P2Y2-purinoceptors and prevents UTP and ATP induced intracellular Ca2+ mobilization by a non-specific mechanism that could involve the inhibition of InsP3 channels.

Ca2+ responses to ATP via purinoceptors in the early embryonic chick retina.

1. The action of adenosine triphosphate on cytoplasmic Ca2+ concentration ([Ca2+]i) was studied in the retinal cell of early embryonic chicks with fura-2 fluorescence measurements. The fluorescence was measured from the whole neural retina dissected from chick embryos at embryonic day three (E3). 2. Bath application of ATP (> or = 30 microM; EC50, 128 microM) raised [Ca2+]i by the release of Ca2+ from intracellular Ca2+ stores, since the Ca2+ response to ATP occurred even in a Ca(2+)-free medium. 3. The Ca2+ response to ATP was mediated by P2U purinoceptors. An agonist for P2U purinoceptors, uridine triphosphate (UTP), evoked Ca2+ rises more potently (> or = 3 microM; EC50, 24 microM) than ATP. Agonists for P2X purinoceptors, alpha, beta-methylene ATP and beta, gamma-methylene ATP, or an agonist for P2Y purinoceptors, 2-methylthio ATP (500 microM each), caused no Ca2+ response. Suramin (100 microM) and Reactive Blue 2 (50 microM) almost completely blocked the Ca2+, responses to 500 microM ATP and 200 microM UTP. 4. The developmental profile of the Ca2+ response to ATP was studied from E3 to E13. The Ca2+ response to ATP was largest at E3, drastically declined towards E8 and decreased further until E11-13. 5. These results suggest that the Ca2+ mobilization by ATP via P2U purinoceptors is characteristic of early embryonic retinal cells.

Dual regulation of cerebrovascular tone by UTP: P2U receptor-mediated contraction and endothelium-dependent relaxation.

1. The mechanisms of vascular tone regulation by extracellular uridine 5'-triphosphate (UTP) were investigated in bovine middle cerebral arterial strips. Changes in cytosolic Ca2+ concentration ([Ca2+]i) and force were simultaneously monitored by use of front-surface fluorometry of fura-2. 2. In the arterial strips without endothelium, UTP (0.1 microM-1 mM) induced contraction in a concentration-dependent manner. However, when the endothelium was kept intact, cumulative application of UTP (0.1-100 microM) (and only at 1 mM) induced a modest phasic contraction in arterial strips. This endothelium-dependent reduction of the UTP-induced contraction was abolished by 100 microM N omega-nitro-L-arginine (L-NOARG) but not by 10 microM indomethacin. In the presence of intact endothelium, UTP (30 microM) induced a transient relaxation of the strips precontracted with 30 nM U-46619 (a stable analogue of thromboxane A2), which was completely inhibited by pretreatment with L-NOARG but not with indomethacin. 3. In the endothelium-denuded strips, the contractile response to UTP was abolished by desensitization to either ATP gamma S or ATP (P2U receptor agonists), but not by desensitization to alpha, beta-methylene-ATP (P2x receptor agonist) or to 2-methylthio-ATP (P2Y receptor agonist). Desensitization to UTP abolished the contractile response to ATP. 4. In the endothelium-denuded artery, a single dose application of UTP induced an initial transient, and subsequently lower but sustained increase in [Ca2+]i and force. In the absence of extracellular Ca2+, UTP induced only the initial transient increases in [Ca2+]i and force, while the sustained increases in [Ca2+]i and force were abolished. UTP (1 mM) had no effect on the basic [Ca2+]i-force relationship obtained on cumulative application of extracellular Ca2+ at steady state of 118 mM K(+)-depolarization-induced contraction. 5. We conclude that in the presence of an intact endothelium, UTP-induced relaxation of preconstricted middle cerebral artery is mainly mediated indirectly, by the production of an endothelium-derived relaxing factor, but at high doses of UTP, vascular smooth muscle contraction is mediated directly via activation of P2U purinoceptor and [Ca2+]i elevation without Ca(2+)-sensitization of the contractile apparatus. UTP may thus exert a dual regulatory effect upon cerebrovascular tone, but in cases where the endothelium is impaired, it may also act as a significant vasoconstrictor.

Relative contribution of P2U- and P2Y-purinoceptors to endothelium-dependent vasodilatation in the golden hamster isolated mesenteric arterial bed.

1. P2-purinoceptors were characterized pharmacologically in the constantly perfused isolated mesenteric arterial vascular bed of the golden hamster. Vasoconstrictor and vasodilator responses to the nucleotides ATP, ADP, 2 methylthio ATP (2MeSATP), alpha,beta-methylene ATP (alpha,beta-meATP) and uridine 5'-triphosphate (UTP) and a role for ATP in sympathetic constriction were examined. 2. At basal tone nucleotides elicited dose-dependent vasoconstriction with an observed rank order of potency of alpha,beta-meATP >> 2MeSATP > ATP = ADP > UTP (based on the doses required to elicit constrictor responses of 25 mmHg). Adenosine had no vasoconstrictor action at doses up to 5 mumol. After application of a single dose (0.5 mumol) of alpha,beta-meATP preparations were desensitized to constriction by subsequent application of nucleotides. 3. Electrical field stimulation (4-64 Hz, 90 V, 1 ms, 30 s) elicited frequency-dependent constrictions which were abolished by guanethidine (5 microM) and by prazosin (1 microM). 4. The non-selective P2-purinoceptor antagonist suramin (100 microM) did not significantly affect vasoconstrictor responses to ATP. The P2X-selective purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 3 microM), virtually abolished responses to ATP. When the endothelium was removed vasoconstrictor responses to ATP and noradrenaline were augmented. 5. In preparations with tone raised with methoxamine (10-80 microM) nucleotides elicited vasodilatation with an observed potency order of ATP = UTP > ADP >> adenosine. 2MeSATP had relatively minor vasodilator effects and at the highest dose tested (50 nmol) elicited only vasoconstriction. alpha,beta-meATP did not elicit vasodilatation but produced further constriction of the raised tone preparation. At the highest doses of ATP and ADP (0.5 microM) responses were biphasic with vasoconstriction preceding vasodilatation. After removal of the endothelium, with the exception of adenosine, vasodilator responses to purines and to UTP were abolished; vasoconstriction to ATP, ADP, UTP and 2MeSATP was evident at the highest doses. 6. Suramin (100 microM) inhibited vasodilatation to both ATP and UTP and abolished responses to 2MeSATP. PPADS (3 microM) inhibited relaxation to 2MeSATP but did not affect relaxation to ATP, UTP, adenosine and acetylcholine and ADP. 7. Reactive blue 2 (30 microM) blocked vasodilator responses to ATP, UTP, 2MeSATP and acetylcholine; it was without effect when used at 3 microM. 8. The results of this study show that ATP elicits vasoconstriction of mesenteric arteries of the golden hamster via P2X-purinoceptors located on the smooth muscle, and vasodilatation via P2U-receptors which are located on the endothelium. 2MeSATP has marginal vasodilator activity, suggesting that P2Y-purinoceptors contribute minimally to relaxation to ATP in hamster mesenteric arteries.

Effects of potassium channel activators on isolated cerebral arteries of large and small diameter in the cat.

The smooth-muscle relaxant action of adenosine 5'-triphosphate (ATP)-sensitive potassium (KATP) channels in cerebral arteries of large diameter has been confirmed in a number of in vitro studies, but there is still debate about the presence of KATP channels in small cerebral arteries. In the present study, the authors compare the effects of cromakalim and bimakalim, two putative KATP channel activators, in different parts of the feline isolated middle cerebral artery (MCA) designated proximal, intermediate, and distal. The latter corresponds to those small pial arteries that are usually studied in vivo. In ring segments precontracted with 10(-5) M of uridine-5-triphosphate (UTP), both cromakalim and bimakalim induced concentration-related relaxation, with bimakalim being more potent than cromakalim, and no significant differences noted among segments obtained from the different regions of the MCA. In vessels precontracted by adding 30 mM KCl the potency of cromakalim and bimakalim was reduced compared with that obtained after UTP precontraction. In the presence of 10(-6) M glibenclamide, an antagonist of KATP channel activators, the concentration-effect curve to bimakalim was shifted to the right in the proximal and distal MCA, indicating a similar route of action for bimakalim and cromakalim in these arteries. The present study therefore indicates the presence of KATP channels in isolated small cerebral arteries according to results obtained in vivo. Activators of KATP channesl may prove helpful in the treatment of vasospasm, which may occur in large and small cerebral arteries after subarachnoid hemorrhage.

Osteoblast-like cells have a variable mixed population of purino/nucleotide receptors.

Osteoblast-like UMR 106.06 cells respond to extracellular application of nucleotides with a fast intracellular calcium pulse (latency of about 20 s, half-width of about 10 s), as measured with fluo-3 on a confocal laser scanning system. Cross-inhibition experiments at 50 microM show that, on a cell population basis, adenosine triphosphate (ATP) strongly inhibits the effect of uridine triphosphate (UTP) or 2-methylthio-ATP (2-MeSATP) applied within 2 min after the end of the ATP-induced pulse, while prior application of UTP or 2-MeSATP only weakly inhibits the ATP effect, and UTP and 2-MeSATP weakly inhibit each other. Furthermore, there are clear differences in cross-inhibition between individual cells. Our measurements provide strong evidence that these cells have at least two types of purino/nucleotide receptors, probably P2y and P2u, with a proportion that varies between individual cells.

Effect of bilirubin on rabbit cerebral arteries in vivo and in vitro.

The effect of bilirubin on vasoreactivity was examined in the exposed rabbit basilar artery (diameter, 1045 +/- 17 microns; n = 18) and its cortical branches (diameter, 265 +/- 11 microns; n = 43) in vivo. Vasoconstriction induced by uridine triphosphate (UTP; 10(-5) to 10(-3) mol/L) was observed in vivo before and after a 60-minute application of supersaturated bilirubin (10(-4) mol/L). Bilirubin was dissolved in modified artificial cerebrospinal fluid (pH 7.4) or in physiological salt solution (pH 7.6). The latter was spectrophotometrically estimated to contain a higher concentration of free bilirubin because of the formation of less colloid. After treatment with bilirubin in artificial cerebrospinal fluid, the effect was minimal in the basilar arteries (n = 7), whereas the diameter of the branches was reduced by 9.6 +/- 1.5% (n = 23) and UTP-induced vasoconstriction was potentiated. After application of bilirubin in physiological salt solution, the basilar arteries contracted slightly (-2.1 +/- 0.9%; n = 6) and the UTP-induced vasoconstriction in the branches was attenuated (n = 12). After a 60-minute incubation of basilar artery with bilirubin in physiological salt solution in vitro, isometric tension recordings showed a diminution in KCl- and UTP-induced vasoconstrictions. Acetylcholine- and sodium nitroprusside-induced relaxations were also attenuated. It is suggested that bilirubin may exert different effects depending on the size of arteries and the concentration of free bilirubin. The constrictor and potentiating effects of bilirubin could be caused by the impairment of the relaxation mechanism. When the toxic effect of bilirubin becomes severe, the constrictor mechanism is also damaged.