Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

Several studies have demonstrated an increase in the functional response to activation of adenosine A₃ receptors in rat basophilic leukemia (RBL-2H3) cells following exposure to dexamethasone (Collado-Escobar et al., 1990a,b; Qian and McCloskey, 1993; Ramkumar et al., 1995). An initial analysis of the response led to the suggestion that enhanced responsiveness was due in part to an increased A₃ receptor number (Collado-Escobar et al., 1990b). Direct evidence in support of this was provided by Ramkumar and colleagues (1995), who demonstrated that the augmented response to A₃ receptor activation following dexamethasone was associated with increases in both the level of mRNA and the number of A₃ receptors. Moreover, the levels of the G_i family of GTP-binding proteins were also increased following dexamethasone treatment, raising the possibility that such increases might contribute, at least in part, to the enhanced response to A₃ receptor activation (Ramkumar et al., 1995).

To date, up-regulation of A₃ receptor responsiveness by dexamethasone has been described only in studies with the RBL-2H3 mast cell line, and the data obtained may not reflect the regulation of this receptor in the whole animal. It was, therefore, of interest to explore the effects of dexamethasone on the responsiveness of mast cells to A₃ receptor activation under more physiological conditions in vivo. The adenosine A₃ receptor agonist N⁶-2-(4-aminophenyl)ethyladenosine (APNEA) induces hypotension in the anesthetized rat (Carruthers and Fozard, 1993a,b; Fozard and Hannon, 1994). The response is associated with a widespread degranulation of tissue mast cells and a substantial increase in plasma histamine (Hannon et al., 1995; Fozard et al., 1996). Pharmacological evidence points to mast cell activation as the primary mechanism contributing to A₃ receptor-mediated hypotension in the rat (Hannon et al., 1995; Van Schaick et al., 1996).

Here, we describe the effects of dexamethasone on the hypotension, histamine release, and degranulation of tissue mast cells induced by APNEA. Comparisons have been made with the effects on compound 48/80, a polycationic mast cell activator that interacts directly with the G_i/G_o families of trimeric GTP-binding proteins (Mousli et al., 1990; Tomita et al., 1991; Chahdi et al., 2000).

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Animals. Male Sprague-Dawley rats, supplied by Biological Research Laboratories (Füllinsdorf, Switzerland) and weighing 196 to 410 g, were used throughout. Groups of up to five animals were housed in sawdust-lined drawer cages (approximately 560 × 335 × 200-mm) and kept at an ambient temperature of 22 ± 2°C under 12-h normal phase light/dark cycles. All experiments were carried out with the approval of the Veterinary Authority of the City of Basel (Kantonales Veterinaeramt, Basel-Stadt).

Cardiovascular Studies. Animals were anesthetized with pentobarbitone sodium (60 mg kg¹, i.p.) and set up for recording blood pressure and heart rate and intrajugular venous administration of drugs, as previously described (Hannon et al., 1995). After a stabilization period of approximately 15 min, dose-response curves to APNEA, the selective adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA), the selective adenosine A_2A receptor agonist 2-[p-(2-carboxyethyl)phenylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680), or the mast cell degranulating agent compound 48/80 were established by cumulative bolus injection; the intervals between doses were sufficient to allow a plateau response to develop. In the case of APNEA, the A₁/A_2A/A_2B receptor antagonist 8-(p-sulfophenyl)theophylline (8-SPT) was injected intravenously at a dose of 40 mg kg¹ 5 min prior to establishing the agonist dose-response curve to "isolate" the A₃ receptor-mediated component of the response to APNEA (see Carruthers and Fozard 1993a; Fozard and Carruthers 1993; Fozard and Hannon, 1994). Only one agonist dose-response curve was generated per animal.

Measurement of Plasma Histamine Concentrations and Histological Analysis. Rats were pretreated with vehicle (saline, 1 ml kg¹, i.p.) or dexamethasone (1 mg kg¹, i.p.) 24 h prior to being anesthetized and prepared for intrajugular venous administration of drugs, as described above. A cannula was placed in the right carotid artery for blood collection. A stabilization period of approximately 15 min was started after completion of all surgery. A total of 29 animals was allocated into 6 groups: group 1 was pretreated with the vehicle for dexamethasone (saline, 1 ml kg¹, i.p.) and 24 h later received APNEA (10 µg kg¹ i.v., followed 5 min later by 20 µg kg¹ i.v.); group 2 was pretreated with dexamethasone (1 mg kg¹ i.p.) and 24 h later received APNEA (10 µg kg¹ i.v., followed 5 min later by 20 µg kg¹ i.v.); group 3 was pretreated with vehicle for dexamethasone (saline, 1 ml kg¹, i.p.) and 24 h later received compound 48/80 (100 µg kg¹ i.v., followed 5 min later by 200 µg kg¹, i.v.); group 4 was pretreated with dexamethasone (1 mg kg¹, i.p.) and 24 h later received compound 48/80 (100 µg kg¹, i.v., followed 5 min later by 200 µg kg¹, i.v.); group 5 was pretreated with vehicle (saline, 1 ml kg¹, i.p.) and 24 h later received i.v. injections of the vehicle for APNEA/compound 48/80; and the animals in group 6, which were pretreated with dexamethasone (1 mg kg¹, i.p.), received injections of the vehicle for APNEA/compound 48/80. Blood was collected between 3 to 5 min after each injection of APNEA, compound 48/80, or vehicle by allowing it to drip into potassium EDTA-coated tubes. Plasma samples were prepared and stored at 30°C until assayed for histamine using a commercially available enzyme-linked immunosorbent assay test system (see Hannon et al., 1995, 2001).

Materials. Pentobarbitone sodium was obtained from Sanofi Sante Animale (Libourne, France). Compound 48/80 (condensation product of N-methyl-p-methoxyphenylethylamine with formaldehyde) and dexamethasone 21-phosphate (disodium salt) were obtained from Sigma-Aldrich (Buchs, Switzerland). APNEA, CPA, CGS 21680, and 8-SPT were synthesized at Novartis Pharma AG (Basel, Switzerland). The adenosine receptor agonists were dissolved in 50% dimethyl sulfoxide in distilled water and diluted immediately before use in 0.9% w/v NaCl. 8-SPT (40 mg) was dissolved in 0.2 ml of 0.4 N NaOH and diluted with distilled water to 20 mg ml¹. Compound 48/80 and dexamethasone were made up in 0.9% w/v NaCl.

Data Presentation. Mean values (± S.E.M.) from n individual experiments are presented. Details of the statistical analyses are given in the text or in the legends to the figures. A P value <0.05 was considered significant.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Effect of Dexamethasone on the Cardiovascular Responses to APNEA, Compound 48/80, CPA, and CGS 21680. Intravenous injection of APNEA (1-30 µg kg¹) to rats in which the A₃ receptor-mediated response had been isolated by pretreatment with 8-SPT (40 mg kg¹, i.v., 5 min) induced falls in blood pressure at the two highest doses accompanied by small falls in heart rate (Fig. 1). The effect of dexamethasone given intraperitoneally at different doses and times of pretreatment on the cardiovascular response to APNEA are shown in Fig. 1; the corresponding baseline mean arterial blood pressure (BP) and heart rate (HR) values prior to starting the APNEA injection sequences are shown in Table 1. Pretreatment with dexamethasone (1 mg kg¹) for 24 h induced significant but limited blockade of the hypotensive response to APNEA. The doses of APNEA that reduced blood pressure by 30 mm Hg (ED₃₀) were 6.5 ± 2.2 (n = 5) and 17.7 ± 2.0 (n = 5) µg kg¹ for the animals given vehicle or dexamethasone, respectively (p < 0.05; Student's t test with Hommel-Hochberg correction). A dose of 0.3 mg kg¹ of dexamethasone was without effect (ED₃₀; 6.3 ± 0.3 µg kg¹, n = 4), and no further blockade could be achieved by increasing the dose of dexamethasone from 1 to 3 mg kg¹ (ED₃₀; 21.3 ± 6.0 µg kg¹, n = 4). A 3-h pretreatment with dexamethasone (1 mg kg¹) was without significant effect (ED₃₀; 9.8 ± 2.2 µg kg¹, n = 3). Moreover, although pretreatment with 1 mg kg¹ on 3 successive days resulted in significant (p < 0.05) suppression of responses to APNEA (ED₃₀; 23.2 ± 2.3 µg kg¹, n = 4), the degree of blockade was not significantly greater than that seen at 24 h after a single dose.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Effects of dexamethasone on the cardiovascular responses to APNEA in anesthetized rats. A, dose-response relationship for dexamethasone given intraperitoneally 24 h prior to establishing the dose-response curve to APNEA. , vehicle-pretreated controls; , , and , animals pretreated with 0.3, 1, or 3 mg kg1 dexamethasone. B, effects of dexamethasone, 1 mg kg1, given intraperitoneally at different times prior to establishing the dose-response curve to APNEA. , vehicle-pretreated controls (pretreatment time, 24 h); , pretreatment time 3 h; , pretreatment time 24 h; and , dexamethasone given on 3 successive days, the last dose being given 24 h prior to APNEA. Points represent mean values (±S.E.M., where these exceed the size of the point) of the number of animals shown in Table 1.

View this table: [in this window] [in a new window]   TABLE 1 Effect of dexamethasone (DEXA) or vehicle on the baseline mean arterial BP and HR responses prior to APNEA administration in anesthetized rats DEXA was administered intraperitoneally either 24 h (0.3, 1, or 3 mg kg1) or alternatively at a dose of 1 mg kg1 on 3 successive days prior to establishing the dose-response curve to APNEA. Values represent mean ± S.E.M. of the number (n) of animals shown.

1

1

1

1

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Effects of dexamethasone on the cardiovascular responses to compound 48/80 (A), CPA (B), and CGS 21680 (C) in anesthetized rats. , vehicle-pretreated controls; , animals pretreated with dexamethasone, 1 mg kg1, given intraperitoneally 24 h prior to establishing the agonist dose-response curves; and , animals pretreated with dexamethasone, 1 mg kg1 , given intraperitoneally on 3 successive days with the last dose being given 24 h prior to compound 48/80. Points represent mean values (±S.E.M., where these exceed the size of the point) of the number of animals shown in Table 2.

View this table: [in this window] [in a new window]   TABLE 2 Effect of dexamethasone (DEXA) or vehicle on the baseline mean arterial BP and HR responses prior to compound 48/80, CPA, or CGS 21680 administration in anesthetized rats DEXA was administered intraperitoneally 24 h (1 mg kg1) prior to establishing the agonist dose-response curves and, in the case of compound 48/80, at a dose of 1 mg kg1 on 3 successive days. Values represent mean ± S.E.M. of the number (n) of animals shown.

Plasma Histamine Concentrations and Mast Cell Degranulation Following APNEA and Compound 48/80: Effects of Dexamethasone. The plasma histamine concentrations increased dose dependently following intravenous injection of APNEA (10 and 30 µg kg¹) or compound 48/80 (100 and 300 µg kg¹) (Table 3). Pretreatment with dexamethasone, 1 mg kg¹, i.p. 24 h prior to the experiment did not alter baseline plasma histamine concentrations per se. The increases in the plasma histamine concentrations induced by the 10 µg kg¹ dose of APNEA and the 100 µg kg¹ dose of compound 48/80 were significantly reduced in animals pretreated with dexamethasone (1 mg kg¹, 24 h) compared with vehicle-pretreated controls. In contrast, the response to the higher doses of APNEA and compound 48/80 were not significantly altered by dexamethasone pretreatment (Table 3).

1

View larger version (34K): [in this window] [in a new window]   Fig. 3.   The degranulation status of mast cells in different tissues following pretreatment with vehicle (saline, 1 ml kg1, i.p., 24 h; left panels) or dexamethasone (1 mg kg1, i.p., 24 h; right panels) and subsequent administration of vehicle, APNEA (30 µg kg1 i.v.), or compound 48/80 (300 µg kg1 i.v.) to anesthetized rats. % cells, percentage of mast cells per tissue (means ± S.E.M. of sections from 4-5 rats) with a status defined according to the following scale: 0 = essentially intact mast cells with no, or only marginal, degranulation; 1 = mast cells showing unequivocal signs of degranulation; and 2 = degranulated mast cell with no cell body visible. , p < 0.05, , p < 0.01 that the value differs from that of vehicle/vehicle-treated animals; , p < 0.05, , p < 0.01 that the value differs from that of dexamethasone-pretreated animals (Student's unpaired t test; Mann-Whitney U test).

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The hypotensive response to APNEA in the Sprague-Dawley rat pretreated with 8-SPT is a consequence of activation of adenosine A₃ receptors (Fozard and Carruthers 1993; Fozard and Hannon 1994). Pharmacological, biochemical (Hannon et al., 1995), and histological (Fozard et al., 1996) evidence indicates that this fall in blood pressure is a consequence of mediator release from mast cells. The major finding of the present study is that pretreatment with dexamethasone suppresses the hypotensive response to APNEA and the associated increase in plasma histamine concentrations and tissue mast cell degranulation.

Reduction of the hypotensive response to APNEA by dexamethasone was selective in that the cardiovascular responses to adenosine A₁ receptor activation (with CPA) or A_2A receptor activation (with CGS 21680) were unaltered by pretreatment with dexamethasone. The doses of CPA and CGS 21680 (0.3-10 µg kg¹ i.v. in each case) were selected based on the observations of Fozard and Carruthers (1993) who demonstrated, in pithed rats with blood pressures supported by angiotensin II, pronounced bradycardic or hypotensive responses via the activation of adenosine A₁ or A_2A adenosine receptors, respectively. The observation rules out nonspecific suppression of cardiovascular reactivity as an explanation for the reduced hypotensive response to APNEA following dexamethasone.

Blockade of the hypotensive response to APNEA by dexamethasone was limited in scope. Thus, inhibition was maximal with 1 mg kg¹ given 24 h prior to administration of APNEA, and neither an increase in the dose of dexamethasone nor an increase in the length of pretreatment produced further inhibition. Thus, there appear to be both dexamethasone-sensitive and -insensitive components to the hypotensive response to APNEA. Since the response to APNEA, at the doses used, seems to be entirely mast cell-mediated (Hannon et al., 1995; Fozard et al., 1996), the observation suggests the existence of populations of mast cells with differential sensitivities to dexamethasone. The heterogeneity of mast cells is well established and includes biochemical and pharmacological differences (Metcalfe et al., 1997). It would not be too unusual for this heterogeneity to extend to susceptibility to inhibition by dexamethasone. However, further studies would be needed to verify the fact. Finally, it bears emphasis that a 3-h pretreatment with 1 mg kg¹ was insufficient to establish blockade of APNEA. Three hours is generally adequate for the effects of glucocorticosteroids, through altered gene expression, to manifest (Adcock and Ito, 2000). Clearly, suppression of the hypotensive response to APNEA does not reflect short-term changes in gene expression.

There can be little doubt that suppression of the hypotensive response to APNEA by dexamethasone reflects down-regulation of the A₃ receptor-mediated mast cell degranulation, which underlies the hypotensive response to APNEA in the presence of 8-SPT in the Sprague-Dawley rat (Fozard and Carruthers, 1993; Fozard and Hannon, 1994; Fozard et al., 1996). Thus, blockade was associated with a decrease in the plasma histamine concentrations and reduced mast cell degranulation in the thymus. Moreover, the fact that histamine levels associated with the lower but not the higher dose of APNEA were significantly affected is consistent with the observed limited blockade by dexamethasone of the hypotensive response to APNEA. The choice of tissues for histological analysis was based on the results from earlier experiments (Fozard et al., 1996) and represents those tissues where the most marked changes were evident following APNEA. The lack of effect of APNEA on mast cells in the skin was therefore unexpected considering our earlier finding. It probably reflects the fact that a lower dose of APNEA (30 µg kg¹) was used in the present study compared with that used in the earlier study (100 µg kg¹) and that the tissues were removed 10 min after the APNEA injection in the earlier study as opposed to 3 to 5 min in the present study. Finally, the tissues utilized in the histological analyses were taken from the animals used in the experiment to measure plasma histamine concentrations, where the administered dose reflects the complete dose range in the cardiovascular studies. Since the suppressant effects of dexamethasone were manifested against the lower effects of APNEA, the relationship between the histological findings, the cardiovascular effects, and the changes in plasma histamine levels should be considered qualitative rather than quantitative.

The lack of effect of dexamethasone on hypotensive responses to compound 48/80 is of particular interest. The finding is unlikely to reflect a mast cell-independent mechanism contributing to the fall in blood pressure, since, like APNEA, the effects of compound 48/80 have been shown to be largely a consequence of mast cell activation in this model (Hannon et al., 1995; Fozard et al., 1996). There is an apparent anomaly in that despite their being no effect on the hypotensive response, the histamine release induced by the lower dose of compound 48/80 was significantly inhibited. Moreover, a significant, albeit slight, reduction in the degranulation response in thymus, skin, and skeletal muscle is seen. The explanation may be related to the fact that the hypotensive response to compound 48/80 manifests a steep dose-response relationship; indeed, it is all-or-nothing between 30 and 100 µg kg¹. Consequently, the effect of dexamethasone pretreatment may be sufficient to reduce the histamine release but not to the point where an effect on blood pressure can be distinguished. From the studies with APNEA, the concentration of plasma histamine associated with a fall in blood pressure lies between 154 and 227 ng ml¹, which is well below the 828 ng ml¹ present after administration of compound 48/80 following treatment with dexamethasone. This provides further evidence that dexamethasone inhibition of mast cell degranulation is limited in scope.

Our findings indicate suppression, by dexamethasone, of a mechanism(s) common to both the A₃ receptor- and compound 48/80-induced mast cell mediator release. Both stimuli manifest their effects on mast cells via the trimeric GTP-binding protein, G_i, although there are fundamental differences in that APNEA involves coupling to G_i2/3 following A₃ receptor activation (Fredholm et al., 2000), whereas compound 48/80 directly activates G_i3 (Aridor et al., 1993). Thus, the logical explanation for the effects would be that dexamethasone down-regulates the level and/or activity of the G_i protein(s). However, where such activity on G_i proteins has been sought, dexamethasone pretreatment generally had minimal effects (Gerwins and Fredholm, 1991; McLellan et al., 1992; Kalavantavanich and Schramm, 2000) or, in the RBL-2H3 cell line, even increased the expression of certain G_i protein subunits (Ramkumar et al., 1995).

The present findings stand in marked contrast to those from the RBL-2H3 cell line, where pretreatment with dexamethasone caused up-regulation of the response to A₃ receptor activation associated with an increase in expression of both the adenosine A₃ receptor and certain G_i protein subunits (Collado-Escobar et al., 1990a,b; Qian and McCloskey, 1993; Ramkumar et al., 1995). An explanation for this difference must remain speculative until further information is available. Clearly, however, it underlines the very real differences that may arise between results obtained with experiments in isolated cells and those generated in integrated organ systems.

Finally, our data may have relevance to the clinical situation in asthma, where the bronchoconstrictor response to adenosine (in the form of AMP) is mast cell-mediated (Fozard and Hannon, 2000; Meade et al., 2001). Treatment with glucocorticosteroids partially suppresses the responsiveness to AMP in asthmatics (Holgate et al., 2000; Van Schoor et al., 2000). Interestingly, in a study with mometasone, inhibition was incomplete and could not be further increased by increasing the dose (Holgate et al., 2000). The results of the present study would predict that suppression of a mast cell-mediated functional response would occur following glucocorticosteroid administration, but that the degree of inhibition would be limited. The nonspecific decrease in mast cell responsiveness identified in the present study may contribute to the suppression of adenosine-induced bronchoconstriction by glucocorticosteroids in asthma.