Ca2+ signalling by P2Y receptors in cultured rat aortic smooth muscle cells

Background and purpose:

P2Y receptors evoke Ca²⁺ signals in vascular smooth muscle cells and regulate contraction and proliferation, but the roles of the different P2Y receptor subtypes are incompletely resolved.

Experimental approach:

Quantitative PCR was used to define expression of mRNA encoding P2Y receptor subtypes in freshly isolated and cultured rat aortic smooth muscle cells (ASMC). Fluorescent indicators in combination with selective ligands were used to measure the changes in cytosolic free [Ca²⁺] in cultured ASMC evoked by each P2Y receptor subtype.

Key results:

The mRNA for all rat P2Y receptor subtypes are expressed at various levels in cultured ASMC. Four P2Y receptor subtypes (P2Y1, P2Y2, P2Y4 and P2Y6) evoke Ca²⁺ signals that require activation of phospholipase C and comprise both release of Ca²⁺ from stores and Ca²⁺ entry across the plasma membrane.

Conclusions and implications:

Combining analysis of P2Y receptor expression with functional analyses using selective agonists and antagonists, we isolated the Ca²⁺ signals evoked in ASMC by activation of P2Y1, P2Y2, P2Y4 and P2Y6 receptors.     

Two subtypes of G protein-coupled nucleotide receptors, P2Y(1) and P2Y(2) are involved in calcium signalling in glioma C6 cells

1. In glioma C6 cells, the stimulation of P2Y receptors by ADP, ATP and UTP initiated an increase in the intracellular Ca2+ concentration, in a process that involved the release of Ca2+ from InsP(3)-sensitive store and the capacitative, extracellular Ca2+ entry. The presence of external Ca2+ was not necessary to elevate Ca(2+). 2. The rank order of potencies of nucleotide analogues in stimulating [Ca2+](i) was: 2MeSADP > ADP > 2MeSATP = 2ClATP > ATP > UTP. alpha,beta-Methylene ATP, adenosine and AMP were ineffective. 3. ADP and UTP effects were additive, while actions of ATP and UTP were not additive on [Ca2+](i) increase. Similarly, cross-desensitization between ATP and UTP but not between ADP and UTP occurred. 4. Suramin, a non-specific nucleotide receptors inhibitor, antagonized ATP-, UTP- and ADP-evoked Ca2+ responses. PPADS, a selective antagonist of the P2Y(1) receptor-generated InsP(3) accumulation, decreased ADP-initiated Ca2+ response with no effect on ATP and UTP. 5. Pertussis toxin (PTX) reduced ADP- and ATP-induced Ca2+ increases. Short-term treatment with TPA, inhibited both ATP and ADP stimulatory effects on [Ca2+](i). 6. ADP inhibited isoproterenol-induced cyclic AMP accumulation. PTX blocked this effect, but PPADS did not. 7. RT - PCR analysis revealed the molecular identity of P2Y receptors expressed by glioma C6 cells to be both P2Y(1) and P2Y(2). 8. It is concluded that both P2Y(1) and P2Y(2) receptors co-exist in glioma C6 cells. ADP acts as agonist of the first, and ATP and UTP of the second one. Both receptors are linked to phospholipase C (PLC).     

P2Y receptor-mediated Ca(2+) signaling increases human vascular endothelial cell permeability

We investigated the effects of P2-receptor agonists on cell size, intracellular calcium levels ([Ca(2+)](i)), and permeation of FITC-labeled dextran (FD-4) as well as the relationship between these effects in human umbilical vein endothelial cells (HUVEC). FD-4 concentration, cell size, and [Ca(2+)](i) were analyzed by HPLC with fluorescence, phase contrast microscopic imaging, and fluorescent confocal microscopic imaging, respectively. The P2Y(1)-receptor agonists 2-methylthio ATP (2meS-ATP) and ADP decreased cell size and increased [Ca(2+)](i) in HUVEC. The P2Y(2)-receptor agonist UTP increased [Ca(2+)](i), but did not influence cell size. The P2X-receptor agonist alpha,beta-methylene ATP did not induce either response. The decrease in size and increase in [Ca(2+)](i) by 2meS-ATP were blocked by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, P2Y(1)-antagonist), thapsigargin (Ca(2+)-pump inhibitor), and U73122 (phospholipase C inhibitor). Furthermore, 2meS-ATP (P2Y(1)-receptor agonist) enhanced permeation of FD-4 through the endothelial cell monolayer. The 2meS-ATP-induced enhancement of the permeation was also prevented by PPADS, thapsigargin, and U73122. These results indicate that activation of P2Y receptors induces a decrease in cell size, an increase in [Ca(2+)](i), and may participate in facilitating macromolecular permeability in HUVEC.     

Molecular genetics of Ca(2+) stores and intracellular Ca(2+) signalling.

An increasing number of studies based on recombinant cells and on mouse models that express an altered repertoire of some of the key components of the intracellular Ca(2+) release stores are becoming available as a result of molecular genetics techniques. Information from these studies, together with results from studies of human diseases caused by mutations in genes that encode proteins of the intracellular Ca(2+) stores, are providing a significant advancement in understanding the interactive nature of the molecular machinery that underlies intracellular Ca(2+) signalling and how the different components of the Ca(2+) stores contribute to the regulation of cellular functions.     

P2Y receptor-mediated enhancement of permeation requires Ca2+ signalling in vascular endothelial cells

1. We investigated the effects of 2-methylthioATP (2meS-ATP; a P2Y receptor agonist) on the permeation of fluorescein isothiocyanate (FITC)-labelled dextran, transendothelial electrical resistance (TEER) and intracellular calcium levels ([Ca2+]i) in cultured endothelial cells isolated from the rat caudal artery. 2. The cellular transport of FITC-labelled dextran was enhanced and TEER of the endothelial monolayer was reduced by 2meS-ATP. Both these effects were prevented by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, a P2Y receptor antagonist, which also inhibited the increase in [Ca2+]i induced by 2meS-ATP in endothelial cells. 3. The increase in [Ca2+]i induced by 2meS-ATP was inhibited by thapsigargin (a Ca2+ pump inhibitor) and by U-73122 (a phospholipase C inhibitor). 4. These findings suggested that activation of the P2Y receptor enhances the passage of material in the endothelium, which is associated with Ca2+ signalling in endothelial cells.     

P2Y receptor-mediated Ca2+ signalling in cultured rat aortic smooth muscle cells.

1. ATP, UTP, ADP and ADP-beta-S elicited Ca2+ -signals in cultured aortic smooth muscle cells although ADP, UDP and ADP-beta-S gave approximately 40% of the maximal response seen with ATP and UTP. Adenosine, AMP or alpha,beta-methylene-ATP had no effect. These responses were attributed to P2Y2/4 and P2Y1 receptors, which we assumed could be selectively activated by UTP and ADP-beta-S respectively. 2. The response to UTP was reduced (approximately 50%) by pertussis toxin, whilst this toxin had no effect upon the response to ADP-beta-S. This suggests P2Y2/4 receptors simultaneously couple to pertussis toxin-sensitive and -resistant G proteins whilst P2Y1 receptors couple to only the toxin-resistant proteins. 3. Repeated stimulation with UTP or ADP-beta-S caused desensitization which was potentiated by 12-O-tetradecanoyl phorbol-13-acetate (TPA) and attenuated by staurosporine. 4. TPA completely abolished sensitivity to ADP-beta-S but the response to UTP had a TPA-resistant component. In pertussis toxin-treated cells, however, TPA could completely abolish sensitivity to UTP and so the TPA-resistant part of this response seems to be mediated by pertussis toxin-sensitive G proteins. 5. Loss of sensitivity to UTP did not occur when pertussis toxin-treated cells were repeatedly stimulated with this nucleotide, suggesting that pertussis toxin-sensitive G proteins mediate this effect. The toxin did not, however affect desensitization to ADP-beta-S.     

Ca2+ signalling by endothelin receptors in rat and human cultured airway smooth muscle cells

1. The aim of the current study was to characterize the ET receptor subtypes in cultured airway smooth muscle cells derived from rat trachea and human bronchus using radioligand binding techniques and to investigate the coupling of ET receptors to intracellular calcium signalling mechanisms using endothelin receptor-selective agonists (sarafotoxin S6c) and antagonists (BQ-123, BQ-788) and digital image fluorescence microscopy.
2. Confluent rat airway smooth muscle cells in culture possessed a mixed ET receptor population (30% ET_A : 70% ET_B), with a density of approximately 3400±280 ET_A and 8000±610 ET_B receptors/cell (n=3 experiments). The density of ET_B, but not ET_A receptors increased substantially in serum-containing medium. However, a 2-day period of serum deprivation, which inhibited cellular growth, substantially reduced ET_B receptor density such that the ET receptor subtype proportions were approximately equal (55% ET_A; 45% ET_B) and similar to those previously observed in intact rat tracheal smooth muscle.
3. Challenge of rat airway smooth muscle cells in culture with endothelin-1 elicited a concentration-dependent biphasic increase in [Ca²⁺]_i (EC₅₀: 16 nM), that comprised an initial transient peak [Ca²⁺]_i increase (typically 350 nM) followed by a modest sustained component. The endothelin-1-induced biphasic [Ca²⁺]_i increase was primarily due to ET_A receptor activation, although a modest and inconsistent ET_B response was observed. The ET_A-mediated [Ca²⁺]_i increase was due primarily to the mobilization of IP₃-sensitive and to a lesser extent ryanodine-sensitive intracellular calcium stores. In contrast, ET_B receptor activation was exclusively coupled to extracellular calcium influx.
4. Somewhat surprisingly, human airway smooth muscle cells in culture contained a homogeneous population of ET_A receptors at a density of 6100±800 receptors cell⁻¹ (n=3 experiments). Serum deprivation was without effect on either ET receptor subtype proportion or ET_A receptor density. Challenge of human airway smooth muscle cells with endothelin-1 provoked a concentration-dependent increase in [Ca²⁺]_i (EC₅₀: 15 nM), with a peak [Ca²⁺]_i increase to greater than 700 nM. Furthermore, the ET_A-mediated calcium response in these human airway smooth muscle cells in culture was entirely dependent upon the mobilization of calcium from intracellular stores.
5. In summary, rat cultured tracheal airway smooth muscle cells contained both ET_A and ET_B receptors. ET_A receptors, the numbers of which remained constant during cell growth, were linked to the release of Ca²⁺ from intracellular stores and a strong rise in [Ca²⁺]_i in the majority of airway smooth muscle cells. In stark contrast, the numbers of ET_B receptors increased significantly during cell growth, an effect that was diminished substantially by incubation in serum-free medium. Moreover, despite the greater number of ET_B receptors, their activation in a small number of airway smooth muscle cells produced only a weak rise in [Ca²⁺]_i, which appeared to be attributable to the influx of extracellular Ca²⁺. In contrast, the populations of ET receptors and their linkage to [Ca²⁺]_i were markedly different in the human cultured airway smooth muscle cells used in the current study compared to that previously observed in intact human isolated bronchial smooth muscle.

     

Opposite diastereoselective activation of P2Y1 and P2Y11 nucleotide receptors by adenosine 5'-O-(alpha-boranotriphosphate) analogues

BACKGROUND AND PURPOSE: We explored the stereoselective activation of the P2Y11 receptor, stably expressed and tagged with GFP, in 1321N1 cells, in comparison to its closest homologue, the P2Y1 receptor. EXPERIMENTAL APPROACH: The potency of several chiral ATP analogues was determined by measuring increases in intracellular calcium concentration ([Ca2+]i). In a series of ATP-alpha-B and ATP-alpha-S analogues, a non-bridging oxygen atom of Palpha was substituted by BH3 or sulphur, respectively, introducing a chiral center at Palpha. The pairs of diastereoisomers (A and B isomers) were each applied as purified compounds. KEY RESULTS: The (B) isomers (ATP-alpha-B Sp isomers and ATP-alpha-S Rp isomers) of all derivatives tested were more potent at the P2Y11 receptor than the corresponding (A) isomers (ATP-alpha-B Rp isomers and ATP-alpha-S Sp isomers) and the parent compounds. This characteristic of the P2Y11 receptor is opposite to the behaviour of the same diastereoisomers at the P2Y1 receptor, at which the (A) isomers are more active. CONCLUSIONS AND IMPLICATIONS: The distinctly opposite diastereoselective activity of ATP derivatives at the P2Y11 and the P2Y1 receptor will allow the deciphering of structural differences of the ligand recognition sites between these receptor subtypes and may aid in the development of subtype-selective agonists. Moreover, ATP-alpha-B diastereoisomers are not active at the P2Y2 receptor. Thus, they are compounds suitable for distinguishing the functional contribution of the two ATP-activated P2Y receptors, the P2Y2 and P2Y11 receptor, in physiological or pathophysiological responses of cells.     

Tumor necrosis factor alpha-induced apoptosis in astrocytes is prevented by the activation of P2Y6, but not P2Y4 nucleotide receptors

The physiological role of the uracil nucleotide-preferring P2Y(6) and P2Y(4) receptors is still unclear, although they are widely distributed in various tissues. In an effort to identify their biological functions, we found that activation by UDP of the rat P2Y(6) receptor expressed in 1321N1 human astrocytes significantly reduced cell death induced by tumor necrosis factor alpha (TNF alpha). This effect of UDP was not observed in non-transfected 1321N1 cells. Activation of the human P2Y(4) receptor expressed in 1321N1 cells by UTP did not elicit this protective effect, although both receptors were coupled to phospholipase C. The activation of P2Y(6) receptors prevented the activation of both caspase-3 and caspase-8 resulting from TNF alpha exposure. Even a brief (10-min) incubation with UDP protected the cells against TNF alpha-induced apoptosis. Interestingly, UDP did not protect the P2Y(6)-1321N1 cells from death induced by other methods, i.e. oxidative stress induced by hydrogen peroxide and chemical ischemia. Therefore, it is suggested that P2Y(6) receptors interact rapidly with the TNF alpha-related intracellular signals to prevent apoptotic cell death. This is the first study to describe the cellular protective role of P2Y(6) nucleotide receptor activation.     

Physiological evidence for a P2Y receptor responsive to diadenosine polyphosphates in human lung via Ca(2+) release studies in bronchial epithelial cells

P2Y(2) receptors that are activated by the extracellular nucleotides ATP or UTP mediate Cl(-) secretion via an increase in [Ca(2+)](i) (intracellular calcium concentration). Therefore, in the lung of patients suffering from cystic fibrosis, inhalation of aerosolized UTP offers a way to circumvent the defect in Cl(-) secretion by the cystic fibrosis transmembrane conductance regulator. A possible alternative for the relatively unstable UTP in inhalation therapy is the more resistant diadenosine tetraphosphate (Ap(4)A). In human and rat lung membranes, Ap(4)A binds to P2 receptor sites coupled to G proteins. Here, we showed that Ap(4)A caused an increase in [Ca(2+)](i) with an EC(50) of 17 microM in human bronchial epithelial cells (HBE1). The [Ca(2+)](i) rise evoked by ATP and UTP was completely, but that induced by Ap(4)A only partially, caused by release of Ca(2+) from internal stores. Moreover, the potency of Ap(4)A to mobilize Ca(2+) was lower than that of ATP and UTP (EC(50) 1.5 and 1.8 microM, respectively), and the maximal increase in [Ca(2+)](i) was considerably smaller than that after ATP or UTP. In accordance with our previous results providing evidence for a common binding site for various diadenosine polyphosphates in lung membranes, all Ap(n)A analogues tested (n = 3 to 6) caused a comparable [Ca(2+)](i) increase. Homologous or heterologous prestimulation largely diminished the increase in [Ca(2+)](i) found after a second pulse of either UTP or Ap(4)A. Although specific binding characteristics and functional responses of Ap(4)A on lung cells are in favor of a distinct receptor for Ap(4)A, the cross-talk between UTP and Ap(4)A in HBE1 cells and the only slight differences in Ca(2+) mobilization by ATP or UTP and Ap(4)A render it impossible at this point to state unequivocally whether there exists a distinct P2Y receptor specific for diadenosine polyphosphates in lung epithelia or whether Ap(4)A activates one of the nucleotide receptors already described.     

Pharmacological characterization of nucleotide P2Y receptors on endothelial cells of the mouse aorta

Nucleotides regulate various effects including vascular tone. This study was aimed to characterize P2Y receptors on endothelial cells of the aorta of C57BL6 mice. Five adjacent segments (width 2 mm) of the thoracic aorta were mounted in organ baths to measure isometric force development. Nucleotides evoked complete (adenosine 5' triphosphate (ATP), uridine 5' triphosphate (UTP), uridine 5' diphosphate (UDP); >90%) or partial (adenosine 5' diphosphate (ADP)) relaxation of phenylephrine precontracted thoracic aortic rings of C57BL6 mice. Relaxation was abolished by removal of the endothelium and was strongly suppressed (>90%) by inhibitors of nitric oxide synthesis. The rank order of potency was: UDP approximately UTP approximately ADP>adenosine 5'-[gamma-thio] triphosphate (ATPgammaS)>ATP, with respective pD2 values of 6.31, 6.24, 6.22, 5.82 and 5.40. These results are compatible with the presence of P2Y1 (ADP>ATP), P2Y2 or P2Y4 (ATP and UTP) and P2Y6 (UDP) receptors. P2Y4 receptors were not involved, since P2Y4-deficient mice displayed unaltered responses to ATP and UTP.The purinergic receptor antagonist suramin exerted surmountable antagonism for all agonists. Its apparent pKb for ATP (4.53+/-0.07) was compatible with literature, but the pKb for UTP (5.19+/-0.03) was significantly higher. This discrepancy suggests that UTP activates supplementary non-P2Y2 receptor subtype(s). Further, pyridoxal-phosphate-6-azophenyl-2'-4'-disulphonic acid (PPADS) showed surmountable (UTP, UDP), nonsurmountable (ADP) or no antagonism (ATP). Finally, 2'-deoxy-N6-methyladenosine3',5'-bisphosphate (MRS2179) inhibited ADP-evoked relaxation only. Taken together, these results point to the presence of functional P2Y1 (ADP), P2Y2 (ATP, UTP) and P2Y6 (UDP) receptors on murine aorta endothelial cells. The identity of the receptor(s) mediating the action of UTP is not fully clear and other P2Y subtypes might be involved in UTP-evoked vasodilatation.     

Synthesis and structure-activity relationships of uracil nucleotide derivatives and analogues as agonists at human P2Y2, P2Y4, and P2Y6 receptors

A series of UTP, UDP, and UMP derivatives and analogues were synthesized and evaluated at the human pyrimidinergic P2Y receptor subtypes P2Y2, P2Y4, and P2Y6 stably expressed in 1321N1 astrocytoma cells. Substituents at N3 of UTP were poorly tolerated by P2Y2 and P2Y4 receptors. In contrast, a large phenacyl substituent at N3 of UDP was well tolerated by the P2Y6 receptor, yielding a potent and selective P2Y6 receptor agonist (3-phenacyl-UDP, EC50=70 nM, >500-fold selective). The most potent and selective P2Y2 receptor agonist of the present series was 2-thio-UTP (EC50=50 nM, >or=30-fold selective vs P2Y4 and P2Y6). All modifications at the uracil base of UTP led to a decrease in potency at the P2Y4 receptor. A beta,gamma-dichloromethylene modification in the triphosphate chain of 5-bromo-UTP was tolerated by all three receptor subtypes, thus opening up a new strategy to obtain ectonucleotide diphosphohydrolase- and phosphatase-resistant P2Y2, P2Y4, and P2Y6 receptor agonists.     

Distinct Ca(2+) signalling mechanisms induced by ATP and sphingosylphosphorylcholine in porcine aortic smooth muscle cells

1. The increase in the cytosolic Ca(2+) concentration ([Ca(2+)](i)) following repetitive stimulation with ATP or sphingosylphosphorylcholine (SPC) in single porcine aortic smooth muscle cells was investigated using the Ca(2+) indicator, fura-2. 2. The ATP-induced [Ca(2+)](i) increase resulted from both Ca(2+) release and Ca(2+) influx. The former was stimulated by phospholipase C activation, while the latter occurred predominantly via the receptor-operated Ca(2+) channels (ROC), rather than the store-operated Ca(2+) channels (SOC) or the voltage-operated Ca(2+) channel (VOC). Furthermore, the P2X(5) receptor was shown to be responsible for the ATP-induced Ca(2+) influx. 3. A reproducible [Ca(2+)](i) increase was induced by repetitive ATP stimulation, but was abolished by removal of extracellular Ca(2+) or inhibition of intracellular Ca(2+) release using U-73122 or thapsigargin, and was restored by Ca(2+) readdition in the former case. 4. SPC only caused Ca(2+) release, and the amplitude of the repetitive SPC-induced [Ca(2+)](i) increases declined gradually. However, a reproducible [Ca(2+)](i) increase was seen in cells in which protein kinase C being inhibited, which increased the SPC-induced Ca(2+) influx, rather than IP(3) generation. 5. In conclusion, although the amplitude of the ATP-induced Ca(2+) release, measured when Ca(2+) influx was blocked, or of the Ca(2+) influx when Ca(2+) release was blocked, progressively decreased following repetitive stimulation, the overall [Ca(2+)](i) increase for each stimulation under physiological conditions remained the same, suggesting that the Ca(2+) stores were replenished by an influx of Ca(2+) during stimulation. The SPC-induced [Ca(2+)](i) increase resulted solely from Ca(2+) release and decreased gradually following repetitive stimulation, but the decrease could be prevented by stimulating Ca(2+) influx, further supporting involvement of the intracellular Ca(2+) stores in Ca(2+) signalling.     

Stimulation of sphingosine-1-phosphate formation by the P2Y(2) receptor in HL-60 cells: Ca(2+) requirement and implication in receptor-mediated Ca(2+) mobilization, but not MAP kinase activation

Sphingosine-1-phosphate (SPP), produced by sphingosine kinase, has recently been reported to act as an intracellular second messenger for Ca(2+) and mitogenic responses triggered by membrane receptors and as an extracellular ligand for specific SPP receptors. Here, we investigated the signaling pathway leading to SPP production by the G protein-coupled P2Y(2) receptor and its functional implication in human leukemia (HL-60) cells, which do not respond to extracellular SPP. P2Y(2) receptor activation by UTP or ATP resulted in rapid and transient production of SPP, which was insensitive to pertussis toxin and blocked by the sphingosine kinase inhibitor, DL-threo-dihydrosphingosine. Treatment of HL-60 cells with this inhibitor did not affect activation of mitogen-activated protein kinases, but suppressed Ca(2+) mobilization by the P2Y(2) receptor. However, receptor-induced SPP production apparently required an increase in intracellular Ca(2+) concentration, but not Ca(2+) influx, and was mimicked by exposure of cells to Ca(2+) ionophores. Taken together, activation of the P2Y(2) receptor stimulates SPP production in HL-60 cells, a process apparently not required for mitogen-activated protein kinase activation, but most likely representing an amplification system for receptor-mediated Ca(2+) signaling.     

Pharmacological characterization of small-conductance Ca(2+)-activated K(+) channels stably expressed in HEK 293 cells

Three genes encode the small-conductance Ca(2+)-activated K(+) channels (SK channels). We have stably expressed hSK1 and rSK2 in HEK 293 cells and addressed the pharmacology of these subtypes using whole-cell patch clamp recordings. The bee venom peptide apamin blocked hSK1 as well as rSK2 with IC(50) values of 3.3 nM and 83 pM, respectively. The pharmacological separation between the subtypes was even more prominent when applying the scorpion peptide blocker scyllatoxin, which blocked hSK1 with an IC(50) value of 80 nM and rSK2 at 287 pM. The potent small molecule blockers showed little differentiation between the channel subtypes. The bis-quinolinium cyclophane UCL 1684 blocked hSK1 with an IC(50) value of 762 pM and rSK2 at 364 pM. The antiseptic compound dequalinium chloride blocked hSK1 and rSK2 with IC(50) values of 444 nM and 162 nM, respectively. The nicotinic acetylcholine receptor antagonist d-tubocurarine was found to block hSK1 and rSK2 with IC(50) values of 27 microM and 17 microM when measured at +80 mV. The inhibition by d-tubocurarine was voltage-dependent with increasing affinities at more hyperpolarized potentials. The GABA(A) receptor antagonist bicuculline methiodide also blocked hSK1 and rSK2 in a voltage-dependent manner with IC(50) values of 15 and 25 microM when measured at +80 mV. In conclusion, the pharmacological separation between SK channel subtypes expressed in mammalian cells is too small to support the notion that the apamin-insensitive afterhyperpolarization of neurones is mediated by hSK1.     

Zolmitriptan stimulates a Ca(2+)-dependent K(+) current in C6 glioma cells stably expressing recombinant human 5-HT(1B) receptors

Stimulation of a Ca(2+)-dependent K(+) current by zolmitriptan, a 5-HT(1B/1D) receptor partial agonist, was investigated in C6 glioma cells stably expressing recombinant human 5-HT(1B) receptors. Outward K(+) currents (I(K)) were examined in non-transfected C6 glioma cells and in cells expressing cloned human 5-HT(1B) receptors using the patch-clamp technique in the whole-cell configuration. In C6 glioma cells expressing recombinant human 5-HT(1B) receptor, zolmitriptan increased I(K) in a concentration-dependent manner (maximum increase 16.3+/-7.8%, n=5, p<0.001) with a pD(2) value (geometric mean with 95% confidence intervals) of 7.03 (7.90-6.10). Zolmitriptan failed to elicit increases in I(K) in non-transfected C6 cells. In the presence of the mixed 5-HT(1B/1D) receptor antagonist, N-[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2(-methyl-4(5-methyl-1 ,2,4)-oxadiazol-3-yl)[1,1-biphenyl]-4-carboxamide 2HCl (GR 127935, 0. 1 mcM), zolmitriptan (1 mcM) failed to significantly increase I(K) in C6 cells expressing human 5-HT(1B) receptors confirming that zolmitriptan-evoked responses were indeed mediated by human 5-HT(1B) receptors. In C6 cells expressing cloned human 5-HT(1B) receptors, zolmitriptan-induced increases in I(K) were prevented by the calcium chelator, EGTA (5 mM) when included in the patch pipette (maximum increase -3.3+/-4.2%, n=4, P=NS). The Ca(2+)-dependent K(+) channel blockers, iberiotoxin (0.1 mcM) and tetraethylammonium (TEA, 1 mM), abolished zolmitriptan-induced increases in I(K) (4.5+/-7.3%, n=4 and -0.8+/-1.7%, n=4, respectively, P=NS in each case) in C6 cells expressing human 5-HT(1B) receptors, confirming the involvement of Ca(2+)-dependent K(+) channels. In conclusion, the 5-HT(1B/1D) receptor partial agonist, zolmitriptan, stimulates I(K/Ca) in C6 glioma cells stably transfected with human 5-HT(1B) receptors suggesting an increase of hyperpolarizing current.     

Differential effects of P2Y1 and P2Y12 nucleotide receptors on ERK1/ERK2 and phosphatidylinositol 3-kinase signalling and cell proliferation in serum-deprived and nonstarved glioma C6 cells

We have previously shown that, in glioma C6 cells, two nucleotide ADP-sensitive receptors coexist: P2Y1, coupled to PLC and responsible for Ca2+ release, and P2Y12, negatively coupled to adenylate cyclase. In the present study, we examined the effects of the stimulation of these two receptors on ERK1/2 and PI3-K activation, and cell proliferation in either serum-deprived or nonstarved C6 cells. In response to ADP and its analogues, in serum-starved cells, both p44 ERK1 and p42 ERK2 were activated in a time-dependent manner, as monitored by Western blot analysis using an antiphospho-p42/p44 MAPK antibody. The phosphorylation was reduced both by removal of the extracellular Ca2+ and partially or almost completely by MRS2179 or AR-C69931MX, specific antagonists of the P2Y1 and P2Y12 receptors, respectively. The inhibitory effect of antagonists was additive. These data indicate the involvement of both receptors, P2Y1 and P2Y12, in the ERK1/2 activation, but the P2Y12 receptor contribution predominates. ERK1/2 activity was positively correlated with cell proliferation of cultured glioma C6 cells. In nonstarved cells, ADP markedly decreased the PI3-K activity. In contrast, in serum-starved cells, ADP evoked an increase in the PI3-K activity. Blocking of the P2Y1 receptor by MRS2179 additionally increased this ADP response. These results suggest that the P2Y1 receptor has an inhibitory and the P2Y12 receptor a stimulatory effect on PI3-K signalling pathway. RT-PCR analysis revealed different mRNA expression of both receptors in starved and nonstarved cells. In nonstarved cells, the P2Y1 receptor mRNA predominates, whereas in serum-deprived cells the expression of P2Y12 mRNA becomes more pronounced.British Journal of Pharmacology (2004) 141, 497-507. doi:10.1038/sj.bjp.0705639