Involvement of A1 adenosine receptors in altered vascular responses and inflammation in an allergic mouse model of asthma

Abstract

Poor lung function and respiratory disorders like asthma have a positive correlation with the development of adverse cardiovascular events. Increased adenosine levels are associated with lung inflammation that could lead to altered vascular responses and systemic inflammation. We hypothesized that asthmatic lung inflammation has systemic effects through A(1) adenosine receptors (A(1)AR) and investigated the effects of aerosolized adenosine on vascular reactivity and inflammation, using A(1)AR knockout (A(1)KO) and corresponding wild-type (A(1)WT) mice that were divided into three experimental groups each: control (CON), allergen sensitized and challenged (SEN), and SEN + aerosolized adenosine (SEN + AD). Animals were sensitized with ragweed (200 microg ip; days 1 and 6), followed by 1% ragweed aerosol challenges (days 11 to 13). On day 14, the SEN + AD groups received one adenosine aerosol challenge (6 mg/ml) for 2 min, and aortae were collected on day 15. 5'-N-ethylcarboxamidoadenosine (NECA; nonselective adenosine analog) induced concentration-dependent aortic relaxation in the A(1)WT CON group, which was impaired in the A(1)WT SEN and SEN + AD groups. All groups of A(1)KO mice showed similar (no significant difference) concentration-dependent relaxation to NECA. The A(1)WT SEN and SEN + AD groups had a significantly higher contraction to selective A(1) agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA) compared with the CON group. Western blot data showed that aortic A(1)AR expression was significantly increased in WT SEN and SEN + AD mice compared with CON mice. Gene expression of ICAM-1 and IL-5 was significantly increased in allergic A(1)WT aorta and were undetected in the A(1)KO groups. A(1)WT allergic mice had significantly higher airway hyperresponsiveness (enhanced pause) to NECA, with adenosine aerosol further enhancing it. In conclusion, allergic A(1)WT mice showed altered vascular reactivity, increased airway hyperresponsiveness, and systemic inflammation. These data suggest that A(1)AR is proinflammatory systemically in this model of allergic asthma.

Fig. 1.

Concentration-response curves for 5′-N-ethylcarboxamidoadenosine (NECA)-mediated aortic relaxation/contraction in A₁ adenosine receptor (A₁AR) wild-type (A₁WT; A) and A₁AR knockout (A₁KO; B) with control (CON), allergic (SEN), and adenosine aerosolized allergic (SEN + AD) groups. Values are expressed as means ± SE; n = 10 mice. On y-axis, positive and negative values indicate relaxation and contraction, respectively. *P < 0.05 compared with A₁WT CON; #P < 0.05 compared with A₁WT SEN.

Fig. 2.

Concentration-response curves for 2-chloro-N⁶-cyclopentyladenosine (CCPA)-mediated aortic contraction in A₁WT CON, SEN, and SEN + AD groups. Values are expressed as means ± SE; n = 6 mice. On y-axis, positive and negative values indicate relaxation and contraction, respectively. *P < 0.05 compared with A₁WT CON.

Fig. 3.

Protein expression of A₁AR in mice aorta from A₁WT CON, A₁WT SEN, and A₁WT SEN + AD groups. Values are expressed as means ± SE; n = 3 mice after normalizing A₁AR protein expression with β-actin in each lane. *P < 0.05 compared with A₁WT CON.

Fig. 4.

Expression of ICAM-1 (A) and IL-5 (B) by real-time PCR in A₁WT mouse aorta from CON, SEN, and SEN + AD groups. For gene expression by comparative C_T method using real-time PCR, first column was made as the calibrator against which all other groups were compared. Values are expressed as means ± SE; n = 4 mice. *P < 0.05 compared with A₁WT CON; #P < 0.05 compared with A₁WT SEN.

Fig. 5.

Airway responsiveness [enhanced pause (P_enh)] to methacholine (MCh) in A₁KO and A₁WT mice. Values are expressed as means ± SE; n = 4–8 mice. On y-axis, P_enh is expressed as percentage of the vehicle. *P < 0.05, A₁WT SEN and SEN (S) + AD compared with A₁WT CON; #P < 0.05, A₁KO SEN and SEN + AD compared with A₁KO CON.

Fig. 6.

Airway responsiveness (P_enh) to adenosine analog NECA in A₁KO and A₁WT mice. Values are expressed as means ± SE; n = 4–8 mice. On y-axis, P_enh is expressed as percentage of the vehicle. *P < 0.05 compared with all other groups; $P < 0.05 compared with A₁WT CON and all A₁KO groups.