Kinetic studies

Two unusual findings of interest were observed in the kinetic studies which require interpretation. Firstly, the association kinetics of [^HjCHA and fHjDPCPX have a very slow component (Figures 3.4,3.5) which, in the case of [^HjDPCPX, is not present when the assay is carried out in the presence of GTP (Figure 3.4). Secondly, the off-rate kinetics of fHjCHA and [^H]PIA show two components, a slow component, (t% ~20 min) and a pseudoirreversible component. However,

both components represent agonist-receptor-G-protein complexes as GTP enhances their off-rates. The molecular explanation for the existence of two components is not known but could for example result from the Ai receptor coupling to more than one G-protein and thus generating two kinetically different complexes. This has been suggested as the explanation for the biphasic dissociation kinetics of [^^®I]HPIA seen in solubilised porcine atrial adenosine Ai receptors (Leid et al. 1989). However, their experiments were carried out using receptors solubilised with detergent so it is possible that the presence of detergent may have altered the dissocation kinetics. Other G protein coupled receptors, such as cloned m l, m2 muscarinic receptors and human dopamine D1 receptors have been shown to interact with more than one G protein (Ashkenazi at ai. 1987, Lazareno at ai. 1993, Kimura at ai. 1995). Alternatively the biphasic dissociation knietics could be the result of a slow isomérisation process of an agonist-receptor- G-protein complex. A very slow dissociation component has also been observed in autoradiographic studies of Ai receptors (Parkinson & Fredholm; 1992).

Conflicting data have been published on agonist dissociation from Ai adenosine receptors in the presence and absence of guanine nucleotides. Lohse at ai.

(1984) found that in the absence of GTP, the dissociation of [^H]PIA was monophasic but in the presence of GTP dissociation was biphasic. A similar result has been obtained using the agonist N®-3([^^^l]iodo-4-hydroxyphenyl isopropyl)adenosine ( [^^^l]HPIA) on the solubilised porcine atrial Ai adenosine receptor (Leid at ai. 1988, Leid at ai. 1989). However, the enhancement of agonist dissociation rate by addition of guanine nucleotides has been shown by Stiles (1985) using the agonist f^^l]HPIA, binding to soluble Ai adenosine receptors. The conflicting data may be the result of artifacts of detergent solubilisation which would alter the membrane environment in which the receptor exists.

3.S.3.2 Effects of GTP on antagonist binding

Conflicting results have been obtained by various groups for the effects of GTP on antagonist binding to adenosine receptors. In bovine and rat brain, GTP did not alter the antagonist binding (Lohse et al. 1984, Klotz et al. 1986, Olah and Stiles, 1990). In other studies an increase in antagonist affinity has been seen in rat adipocytes (Ramkumar and Stiles; 1988) and increased Bmax values have been observed in rat and bovine brain (Yeung and Green; 1983, Green; 1984, Klotz et al. 1990, Stiles; 1988).

Ramkumar and Stiles (1988) proposed that the tight receptor-G protein association constrains the receptor conformation causing a decreased affinity of the receptor for antagonists. When the receptor is uncoupled from the G protein, by the addition of GTP, the receptor conformation changes, resulting in increased receptor affinity for antagonist with no change in receptor number. If endogenous adenosine had been completely removed in this study, the results would indicate that GTP affects the binding affinity of antagonists at Ai adenosine receptors. This would lead to the expectation that some adenosine receptor antagonists could alter tissue function in the absence of receptors occupied by agonists and act as ‘inverse agonists’.

A model has been suggested that postulates two forms of the receptor - one with high affinity for antagonist and low affinity for agonist and the other with low affinity for antagonist and high affinity for agonist. The model predicts that addition of guanine nucleotides initiates a conversion between the two states of the receptor and results in an increase in antagonist affinity with no change in Bmax (reviewed in Schütz and Freissmuth; 1992). This explanation would not hold for the studies that observed increases in Bm a xwith no effect on antagonist affinity (Green; 1984, Klotz

etal. 1990, Stiles; 1988).

If adenosine is still present in the assay, then the results can be attributed to a decrease in the affinity of endogenous adenosine for the Ai adenosine receptors in the presence of GTP, and thus a reduction in its ability to inhibit antagonist binding

to receptors. This does however still support the model predicting a change in antagonist affinity with no change in Bmax- It is difficult to remove endogenous adenosine entirely, which in fact may be generated constantly (Linden; 1989) so the conflicting results are probably due to variations in the endogenous adenosine levels in the assays performed.

In the studies described in this thesis, GTP enhances the binding of the antagonist [^H]DPCPX even in the presence of ADA. The effect is to increase the Bmax without affecting the antagonist affinity constant. These phenomena have been observed in this work and in several previous Ai receptor binding studies as mentioned above (e.g. Stiles; 1988, Leung et al. 1990, Klotz et al. 1990, Parkinson & Fredholm; 1992, Prater ef a/. 1992). The latter effect has been explained previously in terms of Ai receptors, R, being precoupled to G-proteins, G, to form R.G and, in that state, having a lower affinity than the uncoupled receptor for the antagonist, L (Ki > K2, Model 1 ). However, this model does not explain the data.

R R.G

K,

I

11

L.R ^ = 7 LR.G

Model 1

If R is in equilibrium with R.G (and/or L.R is in equilibrium with LR.G) then the effect of GTP would be to increase the affinity of L without changing the Bmax and to change the dissociation rate constant of L. This is not observed.

If R and L.R are not in equilibrium with R.G and L R.G respectively during the time frame of the binding experiment but do equilibrate in the presence of GTP, then it might be possible to observe a GTP induced increase in Bmax affecting the Kd of L or its dissociation rate. This would only be true if K i» K2 and the concentration of

L was such that only insignificant amounts of LR.G were generated (K2[L]«1). Because of these multiple restrictions this model is somewhat implausible.

A more reasonable explanation is that, by analogy with the CHA and PIA kinetic studies, adenosine can form slowly reversible and pseudoirreversible adenosine- receptor-G-protein complexes. These complexes could be generated during the preparation of the membranes or even (because of the pseudoirreversible nature of the binding process) in the presence of ADA. Such complexes would not bind a radioligand, agonist or antagonist, until adenosine dissociated.

Dissociation of adenosine could be very slow in the absence of GTP, explaining the observed slow association component in the f HJagonist or fH]DPCPX kinetic assays. In the presence of GTP, dissociation of adenosine would be fast and effectively generate extra receptor sites which would be detected as an increase in Bmax but not affinity of [^H]DPCPX. There is no necessity to postulate Ai receptor precoupling to G-proteins in the absence of agonist. Finally, because adenosine, as long-lived adenosine-receptor-G-protein complexes, is not metabolised by ADA it is not surprising that GTP induces increases in [^H]DPCPX binding even in the presence of ADA (Table 3.1). This phenomenon, sometimes described as that of a “locked agonist” has been reported for other receptors (e.g. Severne et al. 1987).

Thus, saponin treatment of membranes does not remove endogenous adenosine. It does appear to allow a more ready access of guanine nucleotides to G-proteins capable of coupling to Ai receptors. This procedure may facilitate the quantitative analysis and interpretation of receptor-G-protein coupling by eliminating an "inaccessibility factor". The presence of long-lived adenosine-receptor-G-protein complexes may explain a number of unusual findings that have been reported in Ai adenosine receptor binding studies.

3.S.3.3 Comparison of affinity constant values obtained in this thesis with those obtained previously.

Despite > 90% receptor identity at the amino acid level, the pharmacology of adenosine Ai receptors differs markedly between species (Tucker and Linden; 1993). There are differences in the affinities for different ligands as well as in rank order potencies (Klotz et al. 1991. Ukena et al. 1986). Bovine and canine receptors differ the most, with bovine Ai adenosine receptors having higher affinity for compounds such as PIA and DPCPX (Tucker and Linden; 1993, Tucker et al. 1992). Rat and human receptors are intermediate in their binding characterisitics (Klotz et al. 1991, Ferkany et al. 1986, Murphy and Snyder; 1982). Binding data from recombinant receptors expressed in cell lines appears to correlate well with binding data of native receptors suggesting that structural differences in the Ai adenosine receptors among species cause the differences in structure-activity profiles (Tucker et al. 1994).

The affinities of ligands for Ai receptors has also been reported to vary between different tissues from the same species (Linden; 1991). For example, NECA has been reported to be 100-fold less potent than PIA in rat adipose tissue (Londos et al. 1980) whereas it is nearly equipotent with PIA in binding to Al receptors of rat heart and brain (Bruns et al. 1987). Thus it is very important to make comparisons of binding data from the same species, and in the same tissue if possible.

The discrepancies between many of the binding results on adenosine Ai receptors also arise by performing the different binding assays under non­ identical buffer and assay conditions. For example, DPCPX bound to OHO whole cells transfected with the human Ai adenosine receptor with a dissociation constant of 0.56± 0.1 InM when a Tris ions buffer was used, and with a dissociation constant of 0.96± 0.20nM when a modified Krebs Buffer was used (Townsend-Nicholson and Shine; 1992). Libert and co-workers (1992), in their characterisation of a human Ai adenosine receptor expressed in OHO cells used an identical buffer to the one that I have used (HEPES buffer pH7.4,

10mM MgCl2,100mM NaCI). It would seem that my results should correspond well to theirs. The log affinity values for DPCPX binding are similar in both cases; 8.97 for my results and 8.59 for theirs, assuming that their experiments were performed in the absence of adenosine deaminase ( it is not mentioned in the entire paper). However, they took no measurements of agonist binding to the high affinity state of the receptor and did not look at the agonist CHA at all so no further comparisons can be made.