Comparison of effects of MgCl2 and Gpp(NH)p on antagonist and agonist radioligand binding to adenosine A1 receptors.

AIM: To investigate modulation of antagonist and agonist binding to adenosine A1 receptors by MgCl2 and 5 -guanylimidodiphosphate (Gpp(NH)p) using rat brain membranes and the A1 antagonist [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) and the A1 agonist [3H]-2-chloro-N6-cyclopentyladenosine ([3H]CCPA). METHODS: Parallel saturation and inhibition studies were performed using well-characterised radioligand binding assays and a Brandel Cell Harvester. RESULTS: MgCl2 produced a concentration-dependent decrease (44%), whereas Gpp(NH)p increased [3H]DPCPX binding (19%). In [3H]DPCPX competition studies, agonist affinity was 1.5-14.6-fold higher and 4.6-10-fold lower in the presence of 10 mmol/L MgCl2 and 10 micromol/L Gpp(NH)p respectively; antagonist affinity was unaffected. The decrease in agonist affinity with increasing Gpp(NH)p concentrations was due to a reduction in the proportion of binding to the high affinity receptor state. In contrast to [3H]DPCPX, MgCl2 produced a concentration-dependent increase (72%) and Gpp(NH)p a decrease (85%) in [3H]CCPA binding. Using [3H]CCPA, agonist affinities were 5-17-fold higher than those for [3H]DPCPX, consistent with binding only to the high affinity receptor state. Agonist affinity was 1.3-10.5-fold higher and 2.4-4.7-fold lower on adding MgCl2 or Gpp(NH)p respectively; antagonist affinities were as for [3H]DPCPX. CONCLUSION: The inconsistencies surrounding the effects of MgCl2 and guanine nucleotides on radioligand binding to adenosine A1 receptors were systematically examined. The effects of MgCl2 and Gpp(NH)p on agonist binding to A1 receptors are consistent with their roles in stimulating GTP-hydrolysis at the G-protein alpha-subunit and in blocking formation of the high affinity agonist-receptor-G protein complex.

Target discovery and validation in the post-genomic era.

The recent publication of the human genome sequence provides an opportunity both to combat diseases that are presently considered as pharmaceutically intractable and also to improve current therapies for many common human diseases. The identification of every human gene by ongoing bioinformatic efforts has the potential, when combined with functional genomic approaches, to pinpoint the molecular basis of every human disease, and to discover appropriate intervention points. This exciting prospect is directly relevant to the successful development of effective therapeutics because the past record of drug discovery suggests that 30%-40% of experimental drugs fail because an inappropriate biological target was pursued. The major impact of genomic information may therefore be to reduce this biological failure rate by earlier definition of drug targets related to disease susceptibility or progression. This paper briefly reviews some of the approaches that can be used to identify biologically relevant drug targets.

Nitric-oxide-synthase-mediated cyclic guanosine monophosphate production in neonatal rat cerebellar prisms is resistant to calcineurin inhibition.

Although the macrolide immunosuppressant tacrolimus (FK506) is neuroprotective in animal models of focal and global cerebral ischaemia, the mechanism of this action is not known. FK506 inhibits the protein phosphatase calcineurin, whose substrates can include nitric oxide synthase (NOS), and the neuroprotective effect of FK506 has been attributed to inhibition of NOS activity. We have examined nitric oxide-mediated cyclic guanosine monophosphate (cGMP) accumulation in neonatal rat cerebellar prisms. As expected, N-methyl-D-aspartate (NMDA) induced a rapid, concentration dependent accumulation of cGMP that was inhibited by the NMDA receptor antagonist dizocilpine (MK801) and the NOS inhibitor L-nitro-arginine methyl ester. Phosphoserine immunopositivity following NMDA exposure was increased in the presence of FK506, confirming inhibition of calcineurin. However, FK506 had no effect on NMDA-stimulated cGMP accumulation. These findings suggest that the neuroprotective effect of FK506 may be mediated by mechanisms other than increased NOS phosphorylation.