Cellular mechanisms by which adenosine induces vasodilatation in rat skeletal muscle: significance for systemic hypoxia

Abstract

1. In anaesthetized rats, we recorded arterial blood pressure (ABP), heart rate (HR), femoral blood flow (FBF) and femoral vascular conductance (FVC). We tested the effects of the nitric oxide (NO) synthesis inhibitor L-NAME (nitro-L-arginine methyl ester), or the ATP-sensitive K+ (KATP) channel inhibitor glibenclamide, on responses evoked by systemic hypoxia (breathing 8% O2 for 5 min) or i.a. infusion for 5 min of adenosine, the NO donor sodium nitroprusside (SNP), the adenosine A1 receptor agonist CCPA (2-chloro-N6-cyclopentyladenosine) or the adenosine A2A receptor agonist CGS 21680 (2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoadeno sin e hydrochloride). 2. L-NAME (10 mg kg-1 i.v.) greatly reduced the increase in FVC induced by hypoxia or adenosine, as we have shown before, but had no effect on the increase in FVC evoked by SNP. In addition, L-NAME abolished the increase in FVC evoked by CCPA and greatly reduced that evoked by CGS 21680. These results substantiate the view that muscle vasodilatation induced by systemic hypoxia and infused adenosine are largely NO dependent. They also indicate that muscle dilatation induced by A1 receptor stimulation is entirely NO dependent while that induced by A2A receptors is largely NO dependent; dilatation may also be induced by direct stimulation of A2A receptors on the vascular smooth muscle. 3. Glibenclamide (10 or 20 mg kg-1 i.v.) reduced the increase in FVC induced by hypoxia, preferentially affecting the early part (< 1 min). In addition, glibenclamide greatly reduced the increase in FVC induced by adenosine, but it had no effect on that evoked by SNP. Further, glibenclamide abolished the increase in FVC evoked by CCPA and greatly reduced that evoked by CGS 21680. These results substantiate the view that hypoxia-induced muscle vasodilatation is initiated by KATP channel opening. They also indicate that NO does not induce muscle vasodilatation by opening KATP channels on the vascular smooth muscle, but indicate that the dilatation induced by adenosine and by A2A receptor stimulation is largely dependent on KATP channel opening, while that induced by A1 receptor stimulation is wholly dependent on KATP channel opening. 4. These results, together with previous evidence that hypoxia-induced vasodilatation in skeletal muscle is largely mediated by adenosine acting on A1 receptors, lead us to propose that adenosine is released from endothelium during systemic hypoxia and acts on endothelial A1 receptors to open KATP channels on the endothelial cells and cause synthesis of NO, which then acts on the vascular smooth muscle to cause dilatation. During severe systemic hypoxia we propose that adenosine may also act on A2A receptors on the endothelium to cause dilatation by a similar process and may act on A2A receptors on the vascular smooth muscle to cause dilatation by opening KATP channels.

Figure 1. Effects of L-NAME on cardiovascular responses evoked by systemic hypoxia (A; breathing 8 % O₂ for 5 min) and adenosine (B; 1.2 mg kg⁻¹ min⁻¹; i.a. infusion for 5 min)

A, means ±s.e.m. recorded at the times indicated; hypoxia began at time 0. ▾, control values; •, values after L-NAME (10 mg kg⁻¹i.v.). B, means ±s.e.m. recorded before (0), and at the 5th minute of infusion as indicated. □, control; , after L-NAME. In B, ***P < 0.001, *P < 0.05, significant difference between values recorded at 0 and 5 min. ²²²P < 0.001, ²²P < 0.01, significant difference between baseline values (at 0 min) before and after L-NAME. For hypoxia, the corresponding values are indicated in the text. In A and B, †††P < 0.001, significant difference between change recorded during hypoxia or adenosine infusion before and after L-NAME. Abbreviations: ABP, arterial blood pressure; HR, heart rate; FBF, femoral blood flow; FVC, femoral vascular conductance.

Figure 2. Effects of L-NAME on cardiovascular responses evoked by SNP (0.016 mg kg⁻¹ min⁻¹; i.a. infusion for 5 min)

Means ±s.e.m. at 0 and 5 min as indicated. □, control; , after L-NAME (10 mg kg⁻¹i.v.). ***P < 0.001, **P < 0.01, significant difference between values recorded at 0 and 5 min. ‡‡‡P < 0.001, ‡‡P < 0.01, significant difference between baseline values (at 0 min) before and after L-NAME. ††P < 0.01, significant difference between change recorded during SNP infusion before and after L-NAME. Abbreviations as in Fig. 1.

Figure 3. Effect of L-NAME on cardiovascular responses evoked by the A₁ receptor agonist CCPA (A; 0.35 μg kg⁻¹ min⁻¹) and the A_2A receptor agonist CGS 21680 (B; 1.2 μg kg⁻¹ min⁻¹) (i.a. infusion for 5 min in each case)

Means ±s.e.m. recorded before (0) and at the 5th min of infusion as indicated. In A and B:▪, control data recorded in experiments of Bryan & Marshall (1999); , values recorded after L-NAME (10 mg kg⁻¹i.v.). ***P < 0.001, **P < 0.01, significant difference between values recorded at 0 and 5 min; †††P < 0.001, ††P < 0.01, †P < 0.05, significant difference between change evoked by agonist before and after L-NAME. Abbreviations as in Fig. 1.

Figure 4. Effect of the K_ATP channel antagonist glibenclamide at 10 mg kg⁻¹ (A) or 20 mg kg⁻¹ (B) i.v. on responses evoked by systemic hypoxia (8 % O₂ for 5 min)

Means ±s.e.m. recorded at the times indicated; hypoxia began at time 0. ▾, control values; •, values after glibenclamide. †††P < 0.001, ††P < 0.01, †P < 0.05, significant difference between change recorded during hypoxia before and after glibenclamide. Abbreviations as in Fig. 1.

Figure 5. Effect of the K_ATP channel antagonist glibenclamide (10 mg kg⁻¹i.v.) on responses evoked by adenosine (A; 1.2 mg kg⁻¹ min⁻¹) and SNP (B; 0.016 mg kg⁻¹ min⁻¹) (i.a. infusion for 5 min in each case)

Means ±s.e.m. recorded before (0) and at the 5th minute of infusion as indicated. □, control; , after glibenclamide. ***P < 0.001, **P < 0.01, *P < 0.05, significant difference between values recorded at 0 and 5 min. †††P < 0.001, ††P < 0.01, significant difference between change recorded during adenosine infusion before and after glibenclamide. Abbreviations as in Fig. 1.

Figure 6. Effect of the K_ATP channel antagonist glibenclamide (10 mg kg⁻¹i.v.) on responses evoked by the A₁ receptor agonist CCPA (A; 0.35 μg kg⁻¹ min⁻¹) and the A_2A receptor agonist CGS 21680 (B; 1.2 μg kg⁻¹ min⁻¹) (i.a. infusion for 5 min in each case)

Means ±s.e.m. recorded before (0) and at the 5th minute of infusion as indicated. ▪, control data recorded in experiments of Bryan & Marshall (1999); , values recorded after glibenclamide. ***P < 0.001, **P < 0.01, *P < 0.05, significant difference between values recorded at 0 and 5 min. ††P < 0.01, †P < 0.05, significant difference between change recorded during CCPA or GCS 21680 infusion before and after glibenclamide. Abbreviations as in Fig. 1.

Figure 7. Effects of the K_ATP channel antagonist glibenclamide on cardiovascular changes evoked by the K_ATP channel opener levcromakalim

Means ±s.e.m.□, control values; ▪, values recorded at plateau of response to levcromakalim infusion (1 mg kg⁻¹ min⁻¹i.v.); , values recorded after vehicle for glibenclamide; , values recorded after glibenclamide at 10 mg kg⁻¹i.v.;, values recorded after a further dose of glibenclamide at 10 mg kg⁻¹i.v., to give a final dose of 20 mg kg⁻¹. ***P < 0.001, **P < 0.01, significant effect of levcromakalim. †††P < 0.001, ††P < 0.01, significant change from value recorded during levcromakalim infusion. Abbreviations as in Fig. 1.

Figure 8. Schematic diagram showing proposed sites, and mechanisms of action of adenosine in rat skeletal muscle vasculature

Systemic hypoxia causes vascular endothelium to release adenosine which then acts on A₁ receptors on the endothelial cells that are coupled to K_ATP channels, thereby increasing the synthesis of NO which relaxes the vascular smooth muscle. In addition, systemic hypoxia causes adenosine to be released from skeletal muscle fibres and this may then act on A₁ and A_2A receptors on those fibres to open K_ATP channels and release K⁺ which is a vasodilator. Evidence suggests that A_2A receptors are also present on vascular smooth muscle and endothelium and can induce vasodilatation, but these are not stimulated during moderate systemic hypoxia.