Degranulation of rat omental mast cells by A1 receptor agonists in vitro

The haemodynamic effects of adenosine are thought to result in part from a release of mast cell amines via A3 receptor stimulation. To investigate the nature of the receptors involved in adenosine-induced mast cell degranulation in the rat isolated omentum we have used adenosine analogues with varying specificities as activators of the A1, A2 and A3 receptors, and antagonists with differing specificities for A1 and A2 receptors. Analogues which act predominantly as A1 (e.g. N6-cyclopentyladenosine) or as mixed A1/A2 receptor agonists (e.g. adenosine, inosine, 5'-(Nethylcarboxamido) adenosine) caused mast cell degranulation, whereas a predominantly A3 receptor agonist (IB-MECA) was inactive. Pre-treatment of the omentum with the A1/A2 receptor antagonist 8-phenyltheophylline or with the more specific A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine significantly reduced agonist-induced degranulation. Pre-treatment with disodium cromoglycate or with BN52021 also reduced degranulation of mast cells in response to N6-cyclopentyladenosine. In the rat isolated omental mast cell we conclude that degranulation is an indirect result of A1 receptor stimulation. Platelet-activating factor release appears to mediate at least part of the degranulation.


Introduction
Adenosine has been shown to increase the 5 release of both histamine and 5-hydroxytryptamine (5HT) 6 from rat isolated mast cells. This occurs only after such cells have been prestimulated with, for example, an antigen 2,3,6 or the ionophore A23187.1,4,5 Recent evidence suggests that in antigenically stimulated mast cells in culture adenosine increases the release of amines stored in the granules via activation of adenosine type 3 (A3) receptors7 Some of the haemodynamic actions of injected adenosine in vivo also have been attributed to the release of histamine and/or 5HT from mast cells as a result of activation of A receptors8-m. The wide heterogeneity among mast cells located in 11 12 different tissues, however, prompted us to explore the extent to which an involvement of A receptors is a general phenomenon.
Various methods may be used to study mast cell degranulation. Many workers have used rat (C) 1996 Rapid Science Publishers isolated peritoneal mast cells, [1][2][3][4][5][6] but that artificially precludes interactions occurring between normally adjacent cell types. Such interactions 12 form an integral part of inflammation. The rat isolated omentum can be transilluminated and it contains large numbers of connective tissue type mast cells located in the so-called milky spots. 3'4 This makes it possible to observe mast cells in their normal tissue environment, surrounded by various other cell types. Mast cells normally stain metachromatically when they are exposed to toluidine blue, 15 but lose this property after degranulation. 14 In the present work we have investigated the mast cell degranulating influence of applied adenosine and some of its synthetic structural analogues in the rat omentum. The nature of the receptors that are involved in these responses has been explored with the aid of selective adenosine receptor antagonists. We have also investigated whether or not pre-treatment of the omentum with disodium cromoglycate (DSCG) can reduce the degranulating effect of a specific adenosine type 1 (A1) receptor agonist. In addition, we have investigated the possibility that adenosine receptor agonists indirectly degranulate the mast cells in this preparation by first releasing platelet-activating factor (PAF) or a cyclo-oxygenase product from within the omentum. We have shown previously that both of these types of agent occur endogenously and can degqanulate mast cells in the rat omentum in vitro.

Methods
Female rats weighing about 250 g were killed by inhalation of chloroform vapour. The lesser omentum was removed from the abdomen and divided into five pieces of approximately equal size. Each piece was spread out gently on a microscope slide and flooded with normal saline (NS) or with NS plus a putative antagonist, and then incubated in a moist atmosphere at 37C, as described previously. 14 After 5 min of incubation, the bathing fluid was drained away and replaced with fresh NS, or with NS plus agonist, or with NS plus antagonist plus agonist, or with NS plus antagonist. Incubation continued for a further 15 min. The specimens were then drained, washed with distilled water, and stained for 8min with a solution of toluidine blue (0.5%) in Mcllvaine's buffer (pH 4). After rinsing again with distilled water, the specimens were viewed under a microscope at 100 magnification. The number of metachromatically (pink/magenta)-stained mast cells in each omental milky spot was counted. The average count for all the milky spots in each piece of omentum was used to calculate the mean SEM for each treatment group. Omental pieces from a minimum of three rats were used for each treatment group. The number of milky spots examined varied from 15 to 40 per piece of omentum.

Statistics
Bonferroni's test was used for comparing sev- 24 eral treatment groups with one control group.

Results
Adenosine (1 M), inosine (100 M), NECA (0.1 xM) and CPA (0.1 M) were each found to cause significant degranulation of the mast cells in milky spots of the rat isolated omentum (Fig.  l a,b). Ten-fold lower concentrations of each of these compounds, however, failed to exert a significant degranulating effect (Fig. l a,b). Tenfold higher concentrations of adenosine (10 txM) and of NECA (1 xM) were also without a significant degranulating effect (Fig. l a,b), whereas a ten-fold increase in the concentration of CPA (to 1 btM) caused almost the same amount of degranulation as the lower concentration of O. 1 xM (Fig. l b). IB-MECA was ineffective in the present experiments in a wide concentration range of O. 1 1 O0 bI,M (Fig. 1 a).
Pretreatment of the omentum with DSCG (100 bM) or with BN52021 (10 bM) significantly antagonized the effects of added CPA (0.1 bM), whereas indomethacin (1 bM) was inactive under similar conditions (Fig. l b). DSCG (100 bM) alone had no significant effect on degranulation (mast cell count 16.9-+-0.7), BN52021 and indomethacin alone having been shown previously to have no degranulating effect at these concentrations.14

Discussion
The present results show that adenosine, inosine, NECA and CPA each degranulate rat omental mast cells in vitro. Adenosine, 18 inosine 6 and NECA 6' 7 have each been reported to stimulate both the A and A2 types of receptor, with NECA being more potent than adenosine in this respect, 17 but with adenosine in turn being more potent than inosine. 6'25 The degranulating effect of each of these agonists in the present experiments was counteracted by pretreating the omentum with 8PT, a mixed A/ A2 receptor antagonist 18 (Fig. l a,b). In contrast, IB-MECA, which is a much more powerful activator of A 3 receptors than of A or A2 receptors, 6 unexpectedly showed no effect, even when tested at a concentration of 100 (Fig. l a), suggesting that A 3 receptor stimulation was not involved. This is in marked contrast to the observation that A3 activation can cause murine mast cells to degranulate in vivo. 9 Moreover, the application of CPA, a relatively specific A1 receptor agonist, 17 caused a significant degree of mast cell degranulation in the present experiments (Fig. l b). It is important to note also that pretreatment with DPCPX, a relatively specific A1 receptor antagonist, 9 significantly reduced the degranulating effects of both CPA and NECA (Fig. l b). These results, therefore, strongly suggest that involvement of A receptors predominated in the present situation. A2 receptor activation, on the other hand, is unlikely to have been important. A raised level of cyclic AMP, which one might expect to result from any A2 receptor stimulation, does not cause a release of amines from mast cells. [2][3][4] This also accords with the fact that any A2stimulating action of NECA which might have been unmasked after blocking its A receptormediated effect with DPCPX, actually produced no observable effect on degranulation in the present experiments (Fig. lb). It is worth noting here that other workers have found that 10 adenosine was less active than 1 bM adenosine in reducing vascular luminal diameter, which is another phenomenon thought to be due to mast cell degranulation. 25 The affinity constants at adenosine-sensitive binding sites shown by both adenosine and NECA are higher at A2 than at A1 receptors. 18 The lesser degranulating effect of the higher concentrations (10 bM and 1 M respectively) of these two compounds may have been due to partial mast cell stabilization exerted via a stimulation of A2 (adenylate cyclase stimulating) receptors that appeared only at these higher concentrations. Those AI receptors which seem to be responsible for the present observations may reside in the mast cell membranes or they may be located in other cells within the omentum.
Activation of the A1 receptors in various types of cell can lead to either an increase or a decrease in the coupling of surface receptors to their G proteins. 26 Hence, mast cell degranulation that was observed here may have been produced indirectly, rather than as a direct result of adenosine receptor stimulation of the mast cell itself. Other workers have put forward a similar proposition.
In support of this hypothesis, it was found that pretreatment of the omentum with BN52021, a PAF-receptor antagonist, 22 significantly reduced the degranulating effects of added CPA, although indomethacin, a cyclo-oxygenase inhibitor, 23 did not (Fig.  l b). Under the present experimental conditions, therefore, a release of PAF may mediate part of the degranulation that was seen. This would correlate well with our earlier observation using this preparation, that PAE and possibly thromboxane A2, appeared to be involved as mediators of the mast cell degranulation that was induced by the NO-synthase inhibitor N-nitro-L-arginine methyl ester. 14 However, in addition to histamine, 5-hydroxytryptamine and PAE various pro-inflammatory cytokines are known to be released from stimulated mast cells. 11 '12 We have no evidence to implicate or rule out an involvement of cytokines or other such substances here. It is possible that protection against the degranulating effect of CPA that was shown by DSCG (Fig. l b) may have resulted from reduced release of one of these other substances, 27 but equally it may have been due 344 Mediators of Inflammation Vol 5 1996 to an adenosine receptor blockin action of the type described by earlier workers. * Finally, it must be noted that mast cells vary considerably in their responsiveness to stimulation by different agents. I1'12 Onecannot extrapolate with any degree of certainty, therefore, between different organs or species. Mast cells in the rat omentum may turn out to be exceptional. They certainly differ from those in rat skin 29 in being refractory to degranulation with a potent A 3 receptor-specific agonist such as IB-MECA, but in being sensitive to a potent Al-receptor selective agonist such as CPA. Further work is needed to explore the pharmacological responsiveness of mast cells located in different rat tissues.