Comparison of the potency of adenosine as an agonist at human adenosine receptors expressed in Chinese hamster ovary cells.

The potency of adenosine and inosine as agonists at human adenosine receptors was examined in a functional assay using changes in cyclic AMP (cAMP) formation in intact Chinese hamster ovary (CHO) cells stably transfected with the human A1, A2A, A2B, and A3 receptors. Adenosine increased cAMP formation in cells expressing the A2A (EC(50): 0.7 microM) and A2B (EC(50): 24 microM) receptors and inhibited forskolin (0.3-3 microM)-stimulated cAMP formation in cells expressing the A1 (EC(50): 0.31 microM) and A3 receptors (EC(50): 0.29 microM). The potency of adenosine at the A2A and A2B receptors was not altered by the presence of the uptake inhibitor nitrobenzylthioinosine (NBMPR), whereas it was increased about 6-fold by NBMPR at the A1 and A3 receptors. In the presence of NBMPR, inosine was a potent agonist (EC(50): 7 and 0.08 microM at the A1 and A3 receptors, respectively), but with low efficacy especially at the A3 receptors. No effect of inosine was seen at the A(2) receptors. Caffeine, theophylline, and paraxanthine shifted the dose-response curve for adenosine at the A1, A2A, and A2B receptors. These results indicate that adenosine is the endogenous agonist at all human adenosine receptors and that physiological levels of this nucleoside can activate A1, A2A, and A3 receptors on cells where they are abundantly expressed, whereas pathophysiological conditions are required to stimulate A2B receptors to produce cyclic AMP.

P2Y receptors contribute to ATP-induced increases in intracellular calcium in differentiated but not undifferentiated PC12 cells.

ATP-induced Ca2+ transients were examined in individual PC12 cells of a well defined clone, before and after treatment with nerve growth factor (NGF) to induce a neurone-like phenotype. Using reverse transcriptase PCR these cells were found to express mRNA for several P2 receptors. In undifferentiated cells the ATP-induced Ca2+ response was entirely dependent on Ca2+ influx, could not be mimicked by UTP, alpha,beta-methylene ATP or dibenzoyl ATP or be blocked by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). ATP had no significant effect on levels of cyclic AMP or inositol 1,4,5-trisphosphate (InsP3). These results suggest that in undifferentiated PC12 cells ATP mainly acts on a P2X receptor, possibly the P2X4 subtype. After treatment with NGF for 7 days the ATP response was increased and partially sensitive to PPADS. A component of the ATP-induced Ca2+ increase was due to mobilisation of intracellular Ca2+ stores and another to capacitative Ca2+ entry. UTP caused an increase in intracellular Ca2+, and InsP3 formation could be stimulated by ATP and UTP. ATP also caused a small increase in cyclic AMP, but this was abolished in the presence of indomethacin. Thus, after NGF treatment ATP acts partially via a P2Y receptor, possibly the P2Y2 subtype.

Comparison of CGS 15943, ZM 241385 and SCH 58261 as antagonists at human adenosine receptors.

Three structurally related non-xanthine compounds, CGS 15943, ZM 241385 and SCH 58261, are potent A2A adenosine receptor antagonists and have been used as tools in many pharmacological studies. We have now characterized their affinity and selectivity profile on human adenosine receptors stably transfected into either CHO cells (A1 and A2B receptors) or HEK-293 cells (A2A and A3 receptors). In binding studies using [3H]SCH 58261 as a radioligand, the three compounds were equally potent at A2A receptors, their K(i) value being less than 1 nM. Affinity for A1 and A3 receptors was measured using [3H]DPCPX and [125I]AB-MECA as radioligands. Given the lack of selective ligands, interaction with A2B receptors was assessed using the cAMP accumulation assay following stimulation by the adenosine receptor agonist N-ethylcarboxamidoadenosine (NECA). CGS 15943 was almost as potent at A1 receptors (K(i)3.5 nM) as at A2A receptors, showed moderate affinity for A3 receptors (K(i) 95 nM) and also interacted with A2B receptors (K(i) 44 nM; pA2 7.5). ZM 241385 showed little affinity for A1 receptors (K(i) 255 nM), and did not interact with A3 receptors (K(i)>10 microM); however, it displayed moderate affinity for A2B receptors (K(i) 50 nM; pA2 7.3). SCH 58261 had weak affinity for A1 receptors (K(i) 287 nM), no interaction with A3 receptors (K(i)>10 microM), and showed negligible interaction with A2B receptors (K(i) 5 microM; pA2 6.0). These data indicate that SCH 58261 is the most selective A2A antagonist currently available. Moreover, the different receptor selectivity of these three chemically related compounds provides useful information to progress with structure-activity relationship studies.

Adenosine A1 receptor-mediated modulation of dopamine D1 receptors in stably cotransfected fibroblast cells.

The antagonistic interactions between adenosine A1 and dopamine D1 receptors were studied in a mouse Ltk- cell line stably cotransfected with human adenosine A1 receptor and dopamine D1 receptor cDNAs. In membrane preparations, both the adenosine A1 receptor agonist N6-cyclopentyladenosine and the GTP analogue guanyl-5'-yl imidodiphospate induced a decrease in the proportion of dopamine D1 receptors in a high affinity state. In the cotransfected cells, the adenosine A1 agonist induced a concentration-dependent inhibition of dopamine-induced cAMP accumulation. Blockade of adenosine A1 receptor signal transduction with the adenosine A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine or with pertussis toxin pretreatment increased both basal and dopamine-stimulated cAMP levels, indicating the existence of tonic adenosine A1 receptor activation. Pretreatment with pertussis toxin also counteracted the effects of low concentrations of the A1 agonist on D1 receptor-agonist binding. The results suggest that adenosine A1 receptors antagonistically modulate dopamine D1 receptors at the level of receptor binding and the generation of second messengers.

5-Hydroxytryptamine, angiotensin and bradykinin transiently increase intracellular calcium concentrations and PKC-alpha activity, but do not induce mitogenesis in human vascular smooth muscle cells.

The potent vasoconstrictor substances, 5-hydroxytryptamine (5-HT), angiotensin II (A II), and bradykinin bind to G-protein coupled receptors and activate phospholipase C-beta. Using the Fura-2 technique and microfluorometry we found that all three agonists induce a transient increase in intracellular calcium ([Ca2+]i) by releasing stored calcium in human renal artery smooth muscle cells. Using binding of [3H]-phorbol dibutyrate (PDBu) to quantify membrane-associated protein kinase C (PKC) we also showed that 5-HT, A II and bradykinin induced a rapid but transient translocation of protein kinase C (PKC) to the plasma membrane. The time-course of the rise in [Ca2+]i was similar to that of the increase in [3H]-PDBu binding, suggesting transient activation of the calcium dependent alpha-isoform of PKC. Following prolonged pre-treatment with tetradecanoyl phorbol acetate (100 nmol L-1), which down-regulates PKC-alpha and delta, the angiotensin-induced PKC translocation was lost. 5-HT, A II or bradykinin were unable to increase cell proliferation or act as a co-mitogens with platelet-derived growth factor in human vascular smooth muscle cells. Thus, transient increases in [Ca2+]i or PKC activity by a vasoconstrictor agent are insufficient to cause vascular smooth muscle proliferation.

Endothelin-1 causes a prolonged protein kinase C activation and acts as a co-mitogen in vascular smooth muscle cells.

Endothelin-1 (ET-1) is known to act via G-protein coupled receptors. It has therefore been suggested that any mitogenic activity it may possess, is due to activation of phospholipase C and protein kinase C (PKC). We have therefore examined both the ability of ET-1 to act as a mitogen and its ability to activate PKC. We found that ET-1 significantly increased thymidine incorporation and enhanced platelet-derived growth factor-induced DNA synthesis, as well as causing a prolonged translocation of PKC to the cell membrane. ET-1 significantly increased PKC dependent phosphorylation of two specific substrates. The phosphorylation of MBP4-14 (from myelin basic protein) was partially dependent on extracellular Ca2+, implicating activation of PKC-alpha, whereas phosphorylation of the so called epsilon-peptide was Ca(2+)-independent and prolonged. This could be due either to the delta or zeta isoform of PKC, known to be present in these cells. However, ET-1 induced little proliferation of PKC activity in a transformed smooth muscle cell line, DDT1 MF-2, which lacks expression of the PKC-alpha isoform, but expresses the zeta-isoform. Thus, it would appear the ET-1-induced mitogenicity in smooth muscle cells may be related to the sustained, Ca(2+)-independent activation of PKC-delta.