Purinoceptor Subtypes Research Articles

Electron-immunocytochemical localization of P2X1 receptors in the rat cerebellum.

The distribution of the P2X1 subtype of purinoceptors associated with the extracellular activities of ATP was studied in the rat cerebellum at the electron-microscope level. Receptors were labelled with peroxidase-antiperoxidase and the avidin-biotin-peroxidase complex for immunocytochemistry. Immunoreactivity to P2X1 receptors was localized in subpopulations of synapses between varicosities of parallel fibres of granule cells and dendritic spines of Purkinje cells. Unlabelled varicosities of parallel fibres formed asymmetric synapses with labelled dendritic spines, whereas labelled varicosities of parallel fibres formed asymmetric synapses with unlabelled dendritic spines. P2X1 immunoreactivity was also localized in some astrocyte processes. The functional significance of these findings is discussed.

Differences in the sensitivity to purinergic stimulation of myelinating and non-myelinating Schwann cells in peripheral human and rat nerve.

Schwann cells of the peripheral nervous system are distinguished by morphological and functional criteria in myelinating and non-myelinating subtypes. We and others have previously reported that Schwann cells in isolated peripheral human and rat nerve respond to extracellular application of ATP with a rise in the intracellular free calcium concentration [Ca2+]i. In the present study, the receptors mediating these Ca2+ transients have been investigated in myelinating and non-myelinating Schwann cells of intact fascicles of isolated human sural nerves, rat ventral roots, and rat vagus nerves. Microfluorometry and confocal laser scanning was used on preparations stained with the Ca2+-sensitive dyes Calcium Green-1 and Fura Red. In myelinating Schwann cells of human and rat nerves, the ATP-induced rise of [Ca2+]i resulted from the activation of a P2Y2 purinoceptor subtype (rank order of potency: UTP > or = ATP >> 2-MeSATP = ADP). In contrast, in non-myelinating Schwann cells, Ca2+ transients were produced by activation of a P2Y1 purinoceptor subtype (rank order of potency: 2-MeSATP > ATP > ADP >> UTP). The P1 agonist adenosine and alpha,alpha-meATP did not evoke Ca2+ signals. Ca2+ transients in both types of Schwann cells were found to be due to Ca2+ release from cyclopiazonic acid-sensitive intracellular stores. However, inhibition by suramin was only found in non-myelinating Schwann cells. These findings indicate that mammalian Schwann cells express phenotype-specific P2Y receptor subtypes.

Functional Expression of P2UReceptors in Rat Spermatogenic Cells: Dual Modulation of a Ca2+-Activated K+Channel

Previous study has demonstrated functional expression of a sperm Ca2+-activated K+channel inXenopusoocytes injected with RNAs from the rat testes. Using the same expression system, the present study investigated the specific purinoceptor subtype involved in mediating the effect of extracellular ATP. The effect of ATP on an outwardly rectifying current, previously demonstrated to be mediated by a “Maxi” Ca2+-activated K+channel, was compared to the current responses to different nucleotides using the two-electrode voltage-clamp technique. An order of relative effectiveness appeared to be UTP > ATP > ADP > adenosine, consistent with the pharmacological classification of P2Ureceptors. ATP was also found to exert an inhibitory effect on the Ca2+-activated K+current, elicited by the Ca2+ionophore ionomycin. A similar inhibitory effect was observed with UTP, again suggesting the involvement of P2Ureceptor. RT-PCR study also confirmed the expression of P2Ureceptor mRNA in isolated spermatogenic cells. The present results demonstrate the expression of P2Ureceptor and its dual role, both stimulatory and inhibitory, in the modulation of the Ca2+-activated K+channel in spermatogenic cells. The present finding lends support to an important role of extracellular ATP in sperm functions.

Purinoceptor subtypes mediating contraction and relaxation of marmoset urinary bladder smooth muscle.

1. The effects of adenosine triphosphate (ATP), adenosine diphosphate (ADP), alpha,beta-methylene-ATP (alpha,beta-MeATP) and 2-methylthio-ATP (2-MeSATP) on longitudinally orientated smooth muscle strips from marmoset urinary bladder were investigated by use of standard organ bath techniques. 2. After being mounted in superfusion organ baths, 66.7% (n=249) of marmoset detrusor smooth muscle strips developed spontaneous tone, 48.2% of all strips examined developed tone equivalent to greater than 0.1 g mg(-1) of tissue and were subsequently utilized in the present investigation. 3. On exposure to ATP, muscle strips exhibited a biphasic response, a rapid and transient contraction followed by a more prolonged relaxation. Both responses were found to be concentration-dependent. ADP and 2-MeSATP elicited a similar response (contraction followed by relaxation), whereas application of alpha,beta-MeATP only produced a contraction. The potency order for each effect was alpha,beta-MeATP> >2-MeSATP> ATP>ADP (contractile response) and ATP=2-MeSATP> or = ADP> > alpha,beta-MeATP (relaxational response). 4. Desensitization with alpha,beta-MeATP (10 microM) abolished the contractile phase of the response to ATP, but had no effect on the level of relaxation evoked by this agonist. On the other hand, the G-protein inactivator, GDPbetaS (100 microM) abolished only the relaxation response to ATP. Suramin (general P2 antagonist, 100 microM) shifted both the contractile and relaxation ATP concentration-response curves to the right, whereas cibacron blue (P2Y antagonist, 10 microM) only antagonized the relaxation response to ATP. In contrast, the adenosine receptor antagonist, 8-phenyltheophylline (10 microM), had no effect on the relaxation response curve to ATP. 5. Incubation with tetrodotoxin (TTX, 3 microM) or depolarization of the muscle strip with 40 mM K+ Krebs failed to abolish the relaxation to ATP. In addition, neither Nomega-nitro-L-arginine (L-NOARG, 10 microM) nor methylene blue (10 microM) had any effect on the relaxation response curve. However, tos-phe-chloromethylketone (TPCK, 3 microM), an inhibitor of cyclicAMP-dependent protein kinase A (PKA), significantly (P<0.01) shifted the curve for the ATP-induced relaxation to the right. 6. It is proposed that marmoset detrusor smooth muscle contains two receptors for ATP, a classical P2X-type receptor mediating smooth muscle contraction, and a P2Y (G-protein linked) receptor mediating smooth muscle relaxation. The results also indicate that the ATP-evoked relaxation may occur through the activation of cyclicAMP-dependent PKA.

The inhibition of epithelial cell proliferation by ATP and ADP is mediated through a P2X purinoceptor subtype

The inhibition of epithelial cell proliferation by ATP and ADP is mediated through a P2X purinoceptor subtype

Purinergic transmitters inhibit bone formation by cultured osteoblasts

Adenosine triphosphate (ATP) and other purinoceptor agonists cause a transient rise in [Ca 2+] i in cultured osteoblast-like cells and have a mitogenic effect, as does parathyroid hormone (PTH), and there is evidence that ATP and PTH can act synergistically on osteoblasts. The likelihood that nucleotides, acting through purinoceptors, are important local factors in bone remodeling is therefore considerable. However, their effect on bone formation is unknown. We recently developed a culture system in which appositional bone formation occurs only in narrow grooves cut in a substratum. We have used this as an assay to measure the effects of ATP (50 and 500 μmol/L), ATPγS (20 μmol/L), 2-MeSATP (2 and 20 μmol/L), uridine triphosphate (UTP) (0.2, 2, and 20 μmol/L), adenosine (20 μmol/L), bovine PTH (0.25 and 0.5 IU/mL), rat PTH1–34 (10 −8 and 10 −7 mol/L), and rat PTHrP1–40 (10 −9 and 10 −8 mol/L) on bone formation by rat calvarial osteoblasts. The culture medium was renewed 3 times/week (every 2 or 3 days), and the number of bone loci and length and area of Alizarin red-stained mineralized bone formed in the grooves of each specimen in 16–29 days were measured. Compared with controls, ATPγS, 2-MeSATP, and ATP reduced the amount of bone formed in a 2–3 week culture period. Adenosine had no effect, and UTP either had no effect or at 2 μmol/L stimulated bone formation. PTH and PTHrP completely abolished bone formation in 4 week cultures. Our findings are consistent with evidence for more than one P2 purinoceptor subtype in bone, and show for the first time that the effect of ATP on appositional bone formation by osteoblasts in vitro is, like PTH and PTHrP, inhibitory.

Functional classification of P1-purinoceptors in guinea-pig left and right atria: anomalous characteristics of antagonism by cyclopentyltheophylline.

The P1 purinoceptor subtype mediating the negative inotropic responses of guinea-pig left atria and the negative chronotropic responses of beating right atria were characterized. Guinea-pig isolated paced left atria (2Hz, 5ms, threshold voltage+50%) and spontaneously beating right atria were set up in Krebs-bicarbonate solution and isometric tension and rate of contraction, respectively, were recorded. Concentration-response curves for the reduction of tension and rate, respectively, by adenosine receptor agonists, N6-cyclopentyladenosine (CPA), the R- and S- stereoisomers of N6-(2-phenylisopropyl) adenosine (R-PIA and S-PIA), 5'-(N-carboxamido) adenosine (NECA) and 2-p-((carboxyethyl)-phenethylamino)-5'-(N-carboxamido) adenosine (CGS21680) were obtained. The orders of potency on the left atria (CPA = NECA > R-PIA > S-PIA > CGS21680) and right atria (CPA = R-PIA > S-PIA > CGS21680) were consistent with the responses being mediated via A1 receptors. Antagonism of the responses to CPA or R-PIA by 8-cyclo-1,3-dimethylxanthine (CPT) was examined by a full Schild analysis. Concentration-response curves for CPA or R-PIA were obtained in the absence or presence of five or six concentrations (10(-7)-10(-5) or 3 x 10(-5)M) of CPT. The shift in the concentration-response by CPT was expressed as the concentration-ratio (CR) and plotted as -log(CR-1) against log molar concentration of CPT (Schild plot). pA2 values were calculated from the intercept on the concentration axis and by application of the equation; pA2 = log(antagonist concentration) -log (CR-1). The Schild plots had unity slopes indicating competitive antagonism and the pA2 values derived therefrom indicated that the responses were mediated via A1-receptor. Closer inspection of the Schild plots, however, showed that at the higher concentrations of CPT there was a limit to the displacement of the concentration-response curves of the left and right atria to CPA and of the left atria to R-PIA. There were also significant differences in the apparent pA2 values calculated from the equation, when different concentrations of antagonist were examined. These results indicated that at higher concentrations of agonist there may be a component of the response that is resistant to antagonism by CPT. Whether this is related to the proposal that cardiac responses are mediated via A3 receptors is discussed.

Effects of extracellular ATP on cytosolic Ca 2+ concentration and secretory responses in rat parotid acinar cells

At concentrations > 50 μM, extracellular ATP dose-dependently increased cytosolic Ca 2+ concentration ([Ca 2+] i) via activation of P 2Z purinoceptors in rat parotid acinar cells. This increase in [Ca 2+] i is primarily due to entry of Ca 2+ from the extracellular medium. Inositol trisphosphate formation and Ca 2+ release from intracellular Ca 2+ stores were slightly stimulated by ATP. These responses seem to result from the activation of a P 2 purinoceptor subtype different from P 2Z because the maximum responses were induced by much lower concentrations (< 0.25 mM) of ATP than those (> 1 mM) required to produce the maximum activation of Ca 2+ entry. ATP did not stimulate amylase release, supporting the view that an increase in [Ca 2+] i is not a sufficient stimulus. In addition, ATP evoked a marked K + release in the presence and absence of extracellular Ca 2+, while in carbachol-stimulated cells, only a transient release of K + was observed in the absence of extracellular Ca 2+. The ATP-induced K + release may be mediated by a mechanism independent of the opening of the Ca 2+-sensitive K + channels.

Differential effects of diadenosine phosphates on purinoceptors in the rat isolated perfused kidney

1. The activation of various purinoceptors in rat renal vasculature by P1,P2-diadenosine pyrophosphate (Ap2A), P1,P3-diadenosine triphosphate (Ap3A), P1,P4-diadenosine tetraphosphate (Ap4A), P1,P5-diadenosine pentaphosphate (Ap5A), P1,P6-diadenosine hexaphosphate (Ap6A) was studied by measuring their effects of perfusion pressure of a rat isolated perfused kidney. 2. The vasoconstrictive response to Ap5A was completely due to P2x purinoceptor activation, that to Ap4A and Ap6 was P2x purinoceptor mediated to a large extent, as evidenced by the inhibitory effects of suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid tetrasodium (PPADS). 3. The vasoconstrictive effects of Ap2A and Ap3A were mostly due to stimulation of A1-receptors, as shown by the inhibitory effect of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). 4. The vasoconstrictive response to Ap6A was partially insensitive to A1 and P2x purinoceptor blockers. 5. In raised tone preparations Ap2A and Ap3A evoked vasodilatation, which was blocked by the A2 receptor blocker, 3,7-dimethyl-1-propargylxanthine (DMPX). 6. In raised tone preparations Ap4A evoked vasodilatation when the P2-purinoceptors were blocked by suramin. 7. The activation of different purinoceptor subtypes by diadenosine phosphates critically depends on the number of phosphate groups.

Receptors responsive to extracellular pyrimidine nucleotides.

Extracellular adenine nucleotides have long been known to mediate pharmacological responses via several subtypes of P2 purinoceptors. More recently, however, similar responses to uracil nucleotides have been demonstrated. Here, Didier Communi and Jean-Marie Boeynaems discuss the evidence to suggest that distinct pyrimidinoceptors do indeed exist, although they do not appear to constitute a separate receptor family of their own.

Functional evidence for multiple purinoceptor subtypes in the rat medial vestibular nucleus

Extracellular recording techniques were used in brain slices to characterize excitatory responses produced by purine nucleotides in the rat medial vestibular nucleus, an area where functional purinoceptors have not previously been described. In the continued presence of the adenosine antagonist 8-cyclopentyl-1,3-dipropylxanthine, which alone caused a small increase in the spontaneous firing rate, the P2 purinoceptor agonists α,β-methyleneadenosine 5′-triphosphate and adenosine 5′- O-(2-thiodiphosphate) caused concentration-dependent increases in spontaneous firing rate, with ec 50 values of 1.7 and 41.8 μM, respectively. Only approximately 35% of all neurons studied displayed excitatory responses to these agents. Responses waned in the continued presence of high concentrations of the latter, but not the former agonist. Furthermore, in the continued presence of a maximal concentration of α,β-methyleneadenosine 5′-triphosphate, adenosine 5′- O-(2-thiodiphosphate) produced further increases in the firing rate of these neurons. The P2 antagonist, suramin, ablated responses to α,β-methyleneadenosine 5′-triphosphate, but did not affect responses to adenosine 5′- O-(2-thiodiphosphate), whereas pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid antagonized responses to both agonists. The nucleotide analogue α,β-methyleneadenosine 5′-diphosphate, which displays affinity for putative P2X receptors in brain, also produced concentration-dependent increases in firing frequency, which were also markedly antagonized in the presence of suramin, this agonist being only slightly less potent than α,β-methyleneadenosine 5′-triphosphate. In conclusion, a subpopulation of rat medial vestibular neuronal responses mediated by both P2X and P2Y purinoceptors can be distinguished, in addition to the presence of P1 receptors for adenosine. Comparison of these P2 purinoceptors with the properties of recombinantly expressed P2X and P2Y receptors suggests that these endogenous P2 purinoceptors differ in several important aspects from heterologously expressed recombinant receptors identified from cloning studies.

Presence of P2X1 Purinoceptors in Human Platelets and Megakaryoblastic Cell Lines

Human platelets are thought to possess at least two subtypes of purinoceptor, one of which, coupled to G-proteins, could be the P2Y1 receptor (Leon et al. 1997). However, it has been suggested that the unique rapid calcium influx induced by ADP in platelets could involve P2X1 ionotropic receptors (MacKenzie et al. 1996) and the aim of this study was thus to investigate the presence of P2X purinoceptors in platelets and megakaryoblastic cells. Using PCR experiments, we found P2X1 mRNA to be present in human platelets and megakaryoblastic cell lines. In platelets, the selective P2X1 agonist alphabetaMeATP induced a rise in intracellular calcium only in the presence of external calcium and this effect was antagonized by suramin and PPADS. Repeated addition of alphabetaMeATP desensitized the P2X1 purinoceptor but only slightly affected the ADP response, while no calcium response to alphabetaMeATP was observed in megakaryoblastic cells. These results support the existence of functional P2X1 purinoceptors on human platelets and the presence of P2X1 transcripts in megakaryoblastic cell lines.

Electrophysiological effects of ATP on brain neurones.

1. The electrophysiological effects of ATP on brain neurones are either due to the direct activation of P2 purinoceptors by the unmetabolized nucleotide or to the indirect activation of P1. purinoceptors by the degradation product adenosine. 2. Two subtypes of P2 purinoceptors are involved, a ligand-activated ion channel (P2X) and a G protein-coupled receptor (P2Y). Hence, the stimulation of P2X purinoceptors leads to a cationic conductance increase, while the stimulation of P2Y purinoceptors leads to a G protein-mediated opening or closure of potassium channels. 3. ATP may induce a calcium-dependent potassium current by increasing the intracellular Ca2+ concentration. This is due either to the entry of Ca2+ via P2X purinoceptors or to the activation of metabotropic P2Y purinoceptors followed by signaling via the G protein/phospholipase C/inositol 1,4,5-trisphosphate (IP3) cascade. Eventually, IP3 releases Ca2+ from its intracellular pools. 4. There is no convincing evidence for the presence of P2U purinoceptors sensitive to both ATP and UTP, or pyrimidinoceptors sensitive to UTP only, in the central nervous system (CNS). 5. ATP-sensitive P2X and P2Y purinoceptors show a wide distribution in the CNS and appear to regulate important neuronal functions.

Multiple roles of ATP and adenosine in somatosensory processing: Therapeutic implications

A number of lines of evidence support roles for adenosine and adenosine 5′-triphosphate (ATP) in the transmission and modulation of somatosensory information in the dorsal horn of the spinal cord. Adenosine and ATP have also been demonstrated to have nociceptive (pain-related) and antinociceptive actions in the periphery. Thus, adenosine and ATP have multiple functions in somatosensory processing, which are dependent upon the site and on the relative activation of P1 and P2 purinoceptor subtypes on neurons and non-neuronal cells. The multiple roles provide a rich number of potential therapeutic targets for producing analgesia in a number of clinical pain states. However, because of the multiple and potentially contrasting pro- and anti-nociceptive roles of adenosine and ATP, pharmacological targeting of specific sites and purinoceptor subtypes will be necessary to achieve maximum effectiveness while minimizing side effects. Drug Dev. Res. 39:279–288, 1996. © 1997 Wiley-Liss, Inc.

The possible role of ATP and PACAP as mediators of apaminsensitive NANC inhibitory junction potentials in circular muscle of guinea-pig colon.

1. In the presence of atropine (1 microM), guanethidine (3 microM), indomethacin (3 microM), nifedipine (1 microM), L-nitroarginine (L-NOARG, 100 microM), and the selective tachykinin NK1 and NK2 receptor antagonists, SR 140,333 and GR 94,800, respectively (0.1 microM each), a single pulse of electrical field stimulation (EFS) produced a monophasic non-adrenergic non-cholinergic (NANC) inhibitory junction potential (i.j.p., about 10 mV in amplitude) in the circular muscle of guinea-pig proximal colon, recorded by the modified single sucrose gap technique. 2. The P2 purinoceptor agonist, alpha, beta methylene ATP (alpha, beta mATP, 100 microM) and the pituitary adenylyl cyclase activating peptide (PACAP, 1 microM) both produced hyperpolarization (11 +/- 0.8 mV, n = 14 and 10.2 +/- 0.8 mV, n = 19, respectively) and relaxation (1.1 +/- 0.2 mV, n = 14 and 1.5 +/- 0.2 mN, n = 19, respectively) of the circular muscle. 3. Apamin (0.1 microM) nearly abolished (about 90% inhibition) the NANC i.j.p. and the alpha, beta mATP-induced hyperpolarization, markedly reduced the alpha, beta mATP-induced relaxation (73% inhibition) and the PACAP-induced hyperpolarization (65% inhibition), while the PACAP-induced relaxation was unaffected. 4. Tetraethylammonium (TEA, 10 mM) increased the EFS-evoked i.j.p. and revealed an excitatory junction potential (e.j.p.). In the presence of TEA, alpha, beta mATP induced a biphasic response: transient depolarization and contraction followed by hyperpolarization and relaxation. The hyperpolarization to PACAP was reduced by TEA (45% inhibition) but the relaxation was unaffected. 5. The combined application of apamin (0.1 microM) and TEA (10 mM) abolished the i.j.p. and single pulse EFS evoked a pure e.j.p. with latency three times longer than that of the i.j.p. In the majority of strips tested, alpha, beta mATP and PACAP elicited a biphasic response : depolarization and small contraction followed by hyperpolarization and relaxation. 6. The P2 purinoceptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) inhibited the NANC i.j.p. in concentration-dependent manner and inhibited the alpha, beta mATP-induced hyperpolarization and relaxation, without affecting the hyperpolarization and relaxation induced by PACAP. On the other hand, the P2 purinoceptor antagonist, suramin (100 microM) inhibited to a similar extent (60-80%) the NANC i.j.p. and the hyperpolarization and relaxation induced by alpha, beta mATP or PACAP. 7. PPADS and suramin reduced the NANC e.j.p. evoked by a single pulse EFS in the presence of apamin and TEA (100 microM of PPADS and 300 microM of suramin inhibited the e.j.p. by about 40%). 8. We conclude that ATP, but not PACAP, mediates the apamin-sensitive NANC i.j.p. in the circular muscle of the guinea-pig colon. After blockade of the NANC i.j.p., ATP may act as an excitatory transmitter by activating excitatory P2 purinoceptors. The subtypes of P2 purinoceptor involved in the inhibitory and excitatory responses remain to be established. The data suggest that excitatory P2 purinoceptors may be located extrajunctionally.

Functional characterisation of P2 purinoceptors in PC12 cells by measurement of radiolabelled calcium influx.

The aim of this study was to determine whether 45Ca2+ influx could be used as a quantitative measure of channel activation for functional characterisation of P2X purinoceptors in cell lines. In undifferentiated PC12 cells, grown in suspension, ATP (EC50 = 45 microM), ATP gamma S (EC50 = 50 microM) and 2-meSATP (EC50 = 81 microM) but not alpha beta meATP (1 mM) stimulated 45Ca2+ influx 2-5 fold. This effect did not appear to be due to activation of P2U or P2Y purinoceptors since 1 mM UTP, ADP or ADP beta S did not produce any significant effect. Similarly, the effects of ATP were not apparently mediated through activation of P2Z purinoceptors since dibenzylATP behaved as a weak (EC50 = 191 microM) partial agonist (Maximal effect 29.5% of ATP maximum) and there was no detectable ATP-stimulated ethidium bromide uptake in the PC12 cells. ATP-stimulated 45Ca2+ influx was not affected by nifedipine suggesting that it was not secondary to activation of L-type calcium channels and rather reflected influx through a P2X purinoceptor present in these cells. The ATP-stimulated 45Ca2+ influx could be reduced by monovalent cations, presumably as a result of direct competition for influx through the cation channel, with the following rank order of potency:- guanidinium (EC50 = 16 mM) > sodium > Tris > choline > N-methyl-D-glucamine = sucrose). A number of P2 purinoceptor antagonists inhibited ATP-stimulated 45Ca2+ influx. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (3-300 microM), pyridoxal 5-phosphate (3-300 microM) and d-tubocurarine (30-300 microM) produced an insurmountable antagonism of responses to ATP, with no marked change in agonist EC50. Suramin (100-300 microM) and cibacron blue (30-300 microM) produced a surmountable antagonism while DIDS (4,4'-diisothiocyanatostilbene-2,2'disulfonic acid) only antagonised responses to ATP at concentrations in excess of 300 microM. The general properties of the P2X purinoceptor population identified in these cells were consistent with them being P2X2 purinoceptors. These findings suggest that ATP-stimulated 45Ca2+ influx may be used as a reliable and quantitative functional assay for characterisation of P2X purinoceptor subtypes in cell lines.

Extracellular nucleotides act through P2U purinoceptors to elevate [Ca2+]i and enhance basic fibroblast growth factor-induced proliferation in sheep chondrocytes.

Extracellular nucleotides interact with specific cell surface receptors to mediate a variety of biological responses, including elevation of the cytosolic free Ca2+ concentration ([Ca2+]i) in a number of cell types. Although extracellular ATP has been shown to affect chondrocyte function, the underlying mechanisms are poorly understood. In the present study, we investigated whether Ca2+-mobilizing purinoceptors are present on sheep chondrocytes. Chondrocytes were isolated from the proximal tibial growth plate of day 120-130 sheep fetuses. Early passage cells were loaded with indo-1 or fluo-3, and [Ca2+]i was monitored by fluorescence spectrophotometry. ATP (0.3-100 microM) induced transient elevation of [Ca2+]i, lasting approximately 1 min. Half-maximal elevation of [Ca2+]i was observed at an ATP concentration of 5.0 +/- 0.2 microM. Responses were still observed in the absence of extracellular Ca2+, and were abolished by pretreatment with thapsigargin, consistent with the release of Ca2+ from intracellular stores. Several nucleotides were tested for their ability to elevate [Ca2+]i. In order of potency, these were UTP approximately ATP >> ADP approximately 2-methylthio-ATP. No responses were elicited by benzoylbenzoic-ATP, a P2Z-selective agonist; alpha,beta-methylene-ATP, an agonist selective for certain P2X purinoceptors; AMP; adenosine; or pyrophosphate (all at 100 microM), demonstrating specificity. Taken together, these data indicate that nucleotides elevate [Ca2+]i in chondrocytes through interaction with the P2U purinoceptor subtype. Although pretreatment with pertussis toxin virtually abolished the Ca2+ response to lysophosphatidic acid, the response to UTP was relatively insensitive, suggesting that P2U purinoceptors are not linked to a pertussis toxin-sensitive G protein in chondrocytes. In contrast, the Ca2+ response to UTP was markedly inhibited by the biologically active phorbol ester 12-O-tetradecanoyl-beta-phorbol 13-acetate, but not by the inactive control compound 4 alpha-phorbol 12,13-didecanoate, suggesting that a 12-O-tetradecanoyl-beta-phorbol 13-acetate-sensitive isoform of protein kinase C regulates P2U purinoceptor signaling in these cells. UTP (10 microM) enhanced the proliferative response to basic fibroblast growth factor. The response to basic fibroblast growth factor was also enhanced by ATP, but not by 2-methylthio-ATP, consistent with involvement of P2U purinoceptors. Nucleotides released during trauma, inflammation, or cell death may act through P2U purinoceptors to regulate chondrocyte function in an autocrine or paracrine manner.

Purinergic receptors and metabolic function

Blood glucose concentration is a signal for the endocrine pancreas to release insulin or glucagon. Insulin lowers the level of glucose by inhibiting the hepatic glucose production and stimulating glucose utilization by skeletal muscles and adipocytes. In these latter cells, insulin also inhibits lipolysis and the subsequent release of glycerol (a gluconeogenic precursor) and non esterified fatty acids (energy substrate for hepatic gluconeogenesis). Glucagon opposes the effects of insulin, particularly in the liver, where it stimulates glycogenolysis and neoglucogenesis. A variety of purinoceptor subtypes are expressed in these different tissues, all involved in the regulation of glucose homeostasis: 1) P2Y and A 1 receptors in the pancreatic B cell, mediating stimulation or inhibition of insulin secretion respectively, A 2 receptors in the pancreatic A cell mediating glucagon secretion; 2) different P2 receptors in liver cells mediating glycogenolysis through multiple transducing systems; 3) A 1 receptors in adipocytes mediating antilipolytic action. The thorough characterization of these receptors, together with a better understanding of their metabolic role and position in this integrated physiological regulation may be useful in designing ligands aimed at stimulating insulin secretion or reducing insulin resistance, which are both critical in the pathophysiology of non-insulin-dependent diabetes mellitus (NIDDM). Thus, purinoceptors may constitute new targets for pharmacological compounds with potential therapeutic applications in NIDDM, such as P2Y agonists as insulin secretagogues and A 1 agonists as antilipolytic agents for improvement of insulin sensitivity.

Purines and cell death

Increasing evidence suggests that adenosine and ATP not only modulate cell growth and differentiation, but may also act as inducers of cell death. In the session Purines and cell death, held during the Purines '96 Symposium and chaired by Claudio Franceschi (Modena, Italy) and Geoffrey Burnstock (London, UK) presentation and discussion of new studies on the modulation of cell death by adenosine and ATP raised novel implications for a possible role of purines in both development and in the pathophysiology of various diseases. The cloning of a new ligand-gated P2X receptor (the P2X 7 ) was reported, and pharmacological studies have demonstrated that this is the unique P 2Z receptor known to cause cell death by cytolysis. During the session, induction of apoptosis by ATP was reported in a murine macrophage cell line (confirming a role for ATP in inflammation and immunomodulation), in cultures of renal mesangial cells and of endothelial cells from bovine main pulmonary artery; furthermore, ATP was shown to modulate glutamate neurotoxicity in cerebellar granule cells, suggesting that apoptosis by ATP is not only restricted to cells of the immune lineage, but may represent a more general means to regulate cell survival. Adenosine and its derivatives have been known for years to induce apoptosis of human lymphoid tissues, but it has been demonstrated only recently that these effects can also occur in other cell types, and that they can be due either to the activation of extracellular adenosine receptors or to an intracellular mechanism of action. During the session Purines and cell death, various authors reported apoptosis by adenosine analogues in human peripheral blood mononuclear cells, but also in chick sympathetic neurons, rat chromaffin cells, rat cerebellar granule neurons, intact chick embryos and rat microglial cells, suggesting that adenosine may play an important role not only in modulation of survival of lymphoid cells but also in the development and remodelling of the nervous system. A dual role for the adenosine A 3 receptor in cell survival was also demonstrated in cells of the astroglial lineage, as shown by induction of apoptosis at high concentrations of A 3 receptor agonists and protection from spontaneous cell death at low concentrations, suggesting that this receptor may promote either cell death or survival likely depending on the metabolic and functional state of the tissue. It was also reported that, following an ischemic episode, an alterated metabolism of adenine nucleotides and nucleosides in the heart may be the basis of the lack of recovery of phosphorylated forms of adenosine due to oxidative stress, therefore contributing to heart damage. Taken together, all these data confirm the involvement of purines in modulation of cell survival in various organs and systems; moreover, based on these data, it is expected that the characterization of the specific purinoceptor subtypes involved in these actions may lead to the development of entirely new therapeutic approaches to several diseases.

P2 purinoceptors in rat cortical astrocytes: expression, calcium-imaging and signalling studies.

Extracellular ATP is known to activate intracellular enzymes in astrocytes via P2 purinoceptors that appear to play important physiological and pathological roles in these supporting brain cells. In this study, major P2 purinoceptor subtypes on astrocytes of neonatal rat cerebral cortices were identified in receptor expression experiments, when astrocytic messenger RNA was injected into Xenopus oocytes and recombinant P2 purinoceptors were characterized pharmacologically. In messenger RNA-injected oocytes, ATP evoked inward chloride currents (ICl,Ca) typical of stimulating metabotropic receptors that release intracellular Ca2+. Half-maximal activation with ATP occurred at 40 nM: the Hill coefficient was 0.5, which indicated that ATP stimulated two subtypes of P2 purinoceptor. UTP and 2-methylthioATP were the most active (and equipotent) of a series of nucleotides activating recombinant P2 purinoceptors. These results indicated that the two P2 purinoceptors expressed by astrocytic messenger RNA were of P2U and P2Y subtypes. Responses to ATP were antagonized by the P2 purinoceptor antagonist (suramin) but not by the P1 purinoceptor blocker (sulphophenyltheophylline). Findings in expression studies were confirmed in assays of intracellular signalling systems using primary cultures of rat astrocytes. UTP and 2-methylthioATP stimulated mitogen-activated protein kinase to the same extent as ATP, although UTP was less potent than either ATP or 2-methylthioATP. Both UTP and ATP increased intracellular Ca2+ (as measured by fura-2/AM luminescence) which, in cross-desensitization experiments, indicated the involvement of two subtypes of P2 purinoceptors. In conclusion, rat cortical astrocytes express two major subtypes (P2U and P2Y) of metabotropic ATP receptor which, when activated, raise intracellular Ca2+ and also stimulate mitogen-activated protein kinase.