Purines are important modulators of bone cell biology. ATP is metabolized into adenosine by human primary osteoblast cells (HPOC); due to very low activity of adenosine deaminase, the nucleoside is the end product of the ecto-nucleotidase cascade. We, therefore, investigated the expression and function of adenosine receptor subtypes (A(1) , A(2A) , A(2B) , and A(3) ) during proliferation and osteogenic differentiation of HPOC. Adenosine A(1) (CPA), A(2A) (CGS21680C), A(2B) (NECA), and A(3) (2-Cl-IB-MECA) receptor agonists concentration-dependently increased HPOC proliferation. Agonist-induced HPOC proliferation was prevented by their selective antagonists, DPCPX, SCH442416, PSB603, and MRS1191. CPA and NECA facilitated osteogenic differentiation measured by increases in alkaline phosphatase (ALP) activity. This contrasts with the effect of CGS21680C which delayed HPOC differentiation; 2-Cl-IB-MECA was devoid of effect. Blockade of the A(2B) receptor with PSB603 prevented osteogenic differentiation by NECA. In the presence of the A(1) antagonist, DPCPX, CPA reduced ALP activity at 21 and 28 days in culture. At the same time points, blockade of A(2A) receptors with SCH442416 transformed the inhibitory effect of CGS21680C into facilitation. Inhibition of adenosine uptake with dipyridamole caused a net increase in osteogenic differentiation. The presence of all subtypes of adenosine receptors on HPOC was confirmed by immunocytochemistry. Data show that adenosine is an important regulator of osteogenic cell differentiation through the activation of subtype-specific receptors. The most abundant A(2B) receptor seems to have a consistent role in cell differentiation, which may be balanced through the relative strengths of A(1) or A(2A) receptors determining whether osteoblasts are driven into proliferation or differentiation.

1 In the present work, we investigated the action of adenosine originating from extracellular catabolism of adenine nucleotides, in two preparations where synaptic transmission is modulated by both inhibitory Al and excitatory A2,-adenosine receptors, the rat hippocampal Schaffer fibres/CAl pyramid synapses and the rat innervated hemidiaphragm. 2 Endogenous adenosine tonically inhibited synaptic transmission, since 0.5-2 uml-' of adenosine deaminase increased both the population spike amplitude (30+4%) and field excitatory post-synaptic potential (f.e.p.s.p.) slope (27±4%) recorded from hippocampal slices and the evoked [3H]-acetylcholine ([3H]-ACh) release from the motor nerve terminals (25+2%).3 a,,B-Methylene adenosine diphosphate (AOPCP) in concentrations (100-200 gM) that almost completely inhibited the formation of adenosine from the extracellular catabolism of AMP, decreased population spike amplitude by 39+5% and f.e.p.s.p. slope by 32+3% in hippocampal slices and [3H]-ACh release from motor nerve terminals by 27+3%. 4 Addition of exogenous 5'-nucleotidase (5 uml-') prevented the inhibitory effect of AOPCP on population spike amplitude and f.e.p.s.p. slope by 43-57%, whereas the P2 antagonist, suramin (100 gM), did not modify the effect of AOPCP.5 In both preparations, the effect of AOPCP resulted from prevention of adenosine formation since it was no longer evident when accumulation of extracellular adenosine was hindered by adenosine deaminase (0.5-2 uml-'). The inhibitory effect of AOPCP was still evident when Al receptors were blocked by 1,3-dipropyl-8-cyclopentylxanthine (2.5-5 nM), but was abolished by the A2 antagonist, 3,7-dimethyl-1-propargylxanthine (10 gM).6 These results suggest that adenosine originating from catabolism of released adenine nucleotides preferentially activates excitatory A2 receptors in hippocampal CAl pyramid synapses and in phrenic motor nerve endings.

1 The effects of the adenosine analogues, 5'-N-ethyl-carboxamide adenosine (NECA), R-N6-phenylisopropyladenosine (R-PIA), 2-chloroadenosine (CADO), and CGS 21680C on electrically evoked tritium outflow from preparations loaded with [3H]-choline and on evoked endplate potentials (e.p.ps), as well as the ability of the xanthines, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) and PD 115,199 to antagonize the effects of the adenosine analogues, were investigated in phrenic nerve-diaphragm preparations. 2 NECA, R-PIA and CADO decreased, in a concentration-dependent manner, the evoked tritium outflow from preparations loaded with [3H]-choline. NECA and R-PIA were about equipotent and more potent than CADO. 3 DPCPX shifted to the right in a near parallel fashion the concentration-response curve for the inhibitory effect of R-PIA on evoked tritium outflow. 4 In the presence of DPCPX, NECA increased, rather than decreased, evoked tritium outflow. PD 115,199 antagonized, in a concentration-dependent manner, this excitatory effect of NECA. 5 CGS 21680C, in low nanomolar concentrations, increased evoked tritium outflow, an effect also antagonized by PD 115,199. 6 CGS 21680C increased, and R-PIA decreased, the amplitude of e.p.ps recorded from preparations paralysed with tubocurarine. Both effects could be observed in the same endplate.7 It is concluded that both inhibitory (probably Al) and excitatory (probably A2) adenosine receptors coexist at the rat neuromuscular junction, modulating the evoked release of acetylcholine.

Changes in the regulation of connective tissue ATP-mediated mechano-transduction and remodeling may be an important link to the pathogenesis of chronic pain. It has been demonstrated that mast cell-derived histamine plays an important role in painful fibrotic diseases. Here we analyzed the involvement of ATP in the response of human subcutaneous fibroblasts to histamine. Acute histamine application caused a rise in intracellular Ca2+ ([Ca2+]i) and ATP release from human subcutaneous fibroblasts via H1 receptor activation. Histamine-induced [Ca2+]i rise was partially attenuated by apyrase, an enzyme that inactivates extracellular ATP, and by blocking P2 purinoceptors with pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt and reactive blue 2. [Ca2+]i accumulation caused by histamine was also reduced upon blocking pannexin-1 hemichannels with 10Panx, probenecid, or carbenoxolone but not when connexin hemichannels were inhibited with mefloquine or 2-octanol. Brefeldin A, an inhibitor of vesicular exocytosis, also did not block histamine-induced [Ca2+]i mobilization. Prolonged exposure of human subcutaneous fibroblast cultures to histamine favored cell growth and type I collagen synthesis via the activation of H1 receptor. This effect was mimicked by ATP and its metabolite, ADP, whereas the selective P2Y1 receptor antagonist, MRS2179, partially attenuated histamine-induced cell growth and type I collagen production. Expression of pannexin-1 and ADP-sensitive P2Y1 receptor on human subcutaneous fibroblasts was confirmed by immunofluorescence confocal microscopy and Western blot analysis. In conclusion, histamine induces ATP release from human subcutaneous fibroblasts, via pannexin-1 hemichannels, leading to [Ca2+]i mobilization and cell growth through the cooperation of H1 and P2 (probably P2Y1) receptors.

This study aimed at investigating the expression and function of uracil nucleotide-sensitive receptors (P2Y(2), P2Y(4), and P2Y(6)) on osteogenic differentiation of human bone marrow stromal cells (BMSCs) in culture. Bone marrow specimens were obtained from postmenopausal female patients (68 ± 5 years old, n = 18) undergoing total hip arthroplasty. UTP and UDP (100 µM) facilitated osteogenic differentiation of the cells measured as increases in alkaline phosphatase (ALP) activity, without affecting cell proliferation. Uracil nucleotides concentration-dependently increased [Ca(2+)](i) in BMSCs; their effects became less evident with time (7 > 21 days) of the cells in culture. Selective activation of P2Y(6) receptors with the stable UDP analog, PSB 0474, mimicked the effects of both UTP and UDP, whereas UTPγS was devoid of effect. Selective blockade of P2Y(6) receptors with MRS 2578 prevented [Ca(2+)](i) rises and osteogenic differentiation caused by UDP at all culture time points. BMSCs are immunoreactive against P2Y(2), P2Y(4), and P2Y(6) receptors. While the expression of P2Y(6) receptors remained fairly constant (7∼21 days), P2Y(2) and P2Y(4) became evident only in less proliferative and more differentiated cultures (7 < 21 days). The rate of extracellular UTP and UDP inactivation was higher in less proliferative and more differentiated cell populations. Immunoreactivity against NTPDase1, -2, and -3 rises as cells differentiate (7 < 21 days). Data show that uracil nucleotides are important regulators of osteogenic cells differentiation predominantly through the activation of UDP-sensitive P2Y(6) receptors coupled to increases in [Ca(2+)](i) . Endogenous actions of uracil nucleotides may be balanced through specific NTPDases determining whether osteoblast progenitors are driven into proliferation or differentiation.

1 The coexistence of both inhibitory A 1 and facilitatory A 2 adenosine receptors in the rat myenteric plexus prompted the question of how adenosine activates each receptor subtype to regulate cholinergic neurotransmission. 2 Exogenously applied adenosine (0.3-300 mM) decreased electrically evoked [ 3 Increasing endogenous adenosine levels, by the addition of (1) the adenosine precursor AMP (30-100 mM), (2) the adenosine kinase inhibitor 5 0 -iodotubercidin (10 mM) or (3) inhibitors of adenosine uptake (dipyridamole, 0.5 mM) and of deamination (erythro-9(2-hydroxy-3-nonyl)adenine, 50 mM), enhanced electrically evoked [ ) attenuated endogenous adenosine formation from AMP, analysed by HPLC, the corresponding reduction in [ 3 H]ACh release only became evident when stimulation of the myenteric plexus was prolonged to over 250 s. 5 In summary, we found that endogenously generated adenosine plays a predominantly tonic facilitatory effect mediated by prejunctional A 2A receptors. Extracellular deamination and cellular uptake may restrict endogenous adenosine actions to the neuro-effector region near the release/ production sites.

The direct detrusor relaxant effect of β-adrenoceptor agonists as a primary mechanism to improve overactive bladder symptoms has been questioned. Among other targets, activation of β-adrenoceptors downmodulate nerve-evoked acetylcholine (ACh) release, but there is insufficient evidence for the presence of these receptors on bladder cholinergic nerve terminals. Our hypothesis is that adenosine formed from the catabolism of cyclic AMP in the detrusor may act as a retrograde messenger via prejunctional A receptors to explain inhibition of cholinergic activity by β-adrenoceptors. Isoprenaline (1 µM) decreased [H]ACh release from stimulated (10 Hz, 200 pulses) human (-47 ± 5%) and rat (-38 ± 1%) detrusor strips. Mirabegron (0.1 µM, -53 ± 8%) and CL316,243 (1 µM, -37 ± 7%) mimicked isoprenaline (1 µM) inhibition, and their effects were prevented by blocking β-adrenoceptors with L748,337 (30 nM) and SR59230A (100 nM), respectively, in human and rat detrusor. Mirabegron and isoprenaline increased extracellular adenosine in the detrusor. Blockage of A receptors with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 100 nM) or the equilibrative nucleoside transporters (ENT) with dipyridamole (0.5 µM) prevented mirabegron and isoprenaline inhibitory effects. Dipyridamole prevented isoprenaline-induced adenosine outflow from the rat detrusor, and this effect was mimicked by the ENT1 inhibitor, -(4-nitrobenzyl)-6-thioinosine (NBTI, 30 µM). Cystometry recordings in anesthetized rats demonstrated that SR59230A, DPCPX, dipyridamole, and NBTI reversed the decrease in the voiding frequency caused by isoprenaline (0.1-1,000 nM). Data suggest that inhibition of cholinergic neurotransmission by β-adrenoceptors results from adenosine release via equilibrative nucleoside transporters and prejunctional A-receptor stimulation in human and rat urinary bladder.

Nicotinic receptor (nAChR) subtypes involved in pre- and postjunctional actions underlying tetanic fade were studied in rat phrenic-nerve hemidiaphragms. We investigated the ability of subtype-specific nAChR antagonists to depress nerve-evoked contractions and [(3)H]-acetylcholine ([(3)H]-ACh) release. Muscle tension was transiently increased during brief high frequency trains (50 Hz for 5 sec). The rank potency order of nAChR antagonists to reduce tetanic peak tension was alpha-bungarotoxin > d-tubocurarine >> mecamylamine > hexamethonium. Reduction of maximal tetanic tension produced by dihydro-beta-erythroidine (0.03-10 microM), methyllycaconitine (0.003-3 microM), and alpha-conotoxin MII (0.001-0.3 microM) did not exceed 30%. Besides reduction of peak tension d-tubocurarine (0.1-0.7 microM), mecamylamine (0.1-300 microM), and hexamethonium (30-3,000 microM) also caused tetanic fading. With alpha-conotoxin MII (0.001-0.3 microM) and dihydro-beta-erythroidine (0.03-10 microM), tetanic fade was evident only after decreasing the safety factor of neuromuscular transmission (with high magnesium ions, 6-7 mM). The antagonist rank potency order to reduce evoked (50 Hz for 5 sec) [(3)H]-ACh release from motor nerve terminals was alpha-conotoxin MII (0.1 microM) > dihydro-beta-erythroidine (1 microM) approximately d-tubocurarine (1 microM) > mecamylamine (100 microM) > hexamethonium (1,000 microM). When applied in a concentration (0.3 microM) above that producing tetanic paralysis, alpha-bungarotoxin failed to affect [(3)H]-ACh release. Data obtained suggest that postjunctional neuromuscular relaxants interact with alpha-bungarotoxin-sensitive nicotinic receptors containing alpha1-subunits, whereas blockade of neuronal alpha3beta2-containing receptors produce tetanic fade by breaking nicotinic autofacilitation of acetylcholine release.