Acute systemic hypoxia elevates venous but not interstitial potassium of dog skeletal muscle.

Potassium release through ATP-sensitive potassium (K(ATP)) channels contributes to hypoxic vasodilation in the skeletal muscle vascular bed: It is uncertain whether K(ATP) channels on muscle cells contribute to the process. Potassium from muscle cells must cross the interstitial space to reach the vascular tissues, whereas that from vascular endothelium would have a higher concentration in venous blood than in interstitial fluid. We determined the effect of systemic hypoxia on arterial, venous, and interstitial potassium in the constant-flow-perfused gracilis muscles of anesthetized dogs. Hypoxia reduced arterial Po(2) from 138 to 25 and Pco(2) from 28 to 26 mmHg. Arterial pH and potassium were well correlated (r(2) = 0.9): Both increased in early hypoxia and decreased during the postcontrol. In denervated muscles, perfusion pressure decreased from 95 to 76 mmHg by the end of the hypoxic period; neither venous nor interstitial potassium was elevated. In innervated muscles, perfusion pressure increased from 110 to 172 mmHg by the 11th min of hypoxia and then decreased to 146 mmHg by the end of the hypoxic period; venous potassium increased from 5.0 to 5.3 mM, but interstitial potassium remained unchanged. Glibenclamide abolished both the increase in venous potassium and the hypoxic vasodilation in the innervated muscle. Thus skeletal muscle cells were unlikely to have contributed to the release of potassium, which was suggested to originate from vascular endothelium. The sympathetic nerve supply may play a direct or indirect role in the opening of K(ATP) channels under hypoxic conditions.

The effect of systemic hypoxia on interstitial and blood adenosine, AMP, ADP and ATP in dog skeletal muscle.

1. We investigated the effect of moderate systemic hypoxia on the arterial, venous and interstitial concentration of adenosine and adenine nucleotides in the neurally and vascularly isolated, constant-flow perfused gracilis muscles of anaesthetized dogs. 2. Systemic hypoxia reduced arterial PO2 from 129 to 28 mmHg, venous PO2 from 63 to 23 mmHg, arterial pH from 7.43 to 7.36 and venous pH from 7.38 to 7.32. Neither arterial nor venous PCO2 were changed. Arterial perfusion pressure remained at 109 +/- 8 mmHg for the first 5 min of hypoxia, then increased to 131 +/- 11 mmHg by 9 min, and then decreased again throughout the rest of the hypoxic period. 3. Arterial adenosine (427 +/- 98 nM) did not change during hypoxia, but venous adenosine increased from 350 +/- 52 to 518 +/- 107 nM. Interstitial adenosine concentration did not increase (339 +/- 154 nM in normoxia and 262 +/- 97 nM in hypoxia). Neither arterial nor venous nor interstitial concentrations of adenine nucleotides changed significantly in hypoxia. 4. Interstitial adenosine, AMP, ADP and ATP increased from 194 +/- 40, 351 +/- 19, 52 +/- 7 and 113 +/- 36 to 764 +/- 140, 793 +/- 119, 403 +/- 67 and 574 +/- 122 nM, respectively, during 2 Hz muscle contractions. 5. Adenosine, AMP, ADP and ATP infused into the arterial blood did not elevate the interstitial concentration until the arterial concentration exceeded 10 microM. 6. We conclude that the increased adenosine in skeletal muscle during systemic hypoxia is formed by the vascular tissue or the blood cells, and that adenosine is formed intracellularly by these tissues. On the other hand, adenosine formation takes place extracellularly in the interstitial space during muscle contractions.

Interstitial adenosine concentration in rat red or white skeletal muscle during systemic hypoxia or contractions.

Interstitial adenosine concentrations in red soleus (SL) or white extensor digitorum longus (EDL) muscles of anaesthetised rats were determined using microdialysis and HPLC. Systemic hypoxia was induced by ventilating the animals with 10% oxygen in nitrogen for 15 min: arterial PO2 decreased from 111.8 +/- 10.9 to 42.2 +/- 4.3 mmHg (n = 4; P < 0.01) and mean systemic arterial blood pressure from 97.6 +/- 4.9 to 59.0 +/- 3.6 mmHg (n = 22; P < 0.001). The interstitial adenosine concentration was not significantly changed from its control values of 294 +/- 44 nM (n = 20) in EDL and 302 +/- 36 nM (n = 20) in SL during hypoxia or the recovery period. The interstitial lactate concentration did not change in the early part of the hypoxia but increased from 1.0 +/- 0.2 to 1.4 +/- 0.3 mM (n = 6; P < 0.05) in SL and from 2.0 +/- 0.4 to 2.4 +/- 0.4 mM (n = 6; P < 0.05) in EDL during the later part of the hypoxia, and remained elevated in the recovery period. Muscle contractions (2 Hz for 15 min) produced a transient increase in the interstitial adenosine concentration of SL from 150 +/- 35 to 244 +/- 75 nM (n = 10; P < 0.05) during the first 5 min of stimulation. In EDL the interstitial adenosine concentration increased from 145 +/- 50 to 435 +/- 144 nM (n = 10; P < 0.05) in the later part of the contraction and remained elevated in the early part of the recovery period. These data suggest that: (i) in systemic hypoxia adenosine does not appear in the interstitial space, which rules out its release from skeletal muscle, although it may be formed by the vascular tissues in this condition; (ii) adenosine is formed in the interstitial space of skeletal muscle during muscle contractions; (iii) there is slow clearance of adenosine from the interstitial space of white muscle, perhaps due to the low vascularity of the tissue.

The effect of lactic acid on intracellular pH and adenosine output from superfused rat soleus muscle fibres.

We investigated the effects of graded doses of lactic acid on the intracellular pH and adenosine output from superfused bundles of about 15 skeletal muscle fibres. Intracellular pH was determined using the fluorescent intracellular dye, 2',7'-bis-(2-carboxyethyl)-5-(and,6-) carboxyfluorescein (BCECF), and adenosine efflux was measured by HPLC. Intracellular pH was 7.07 +/- 0.05 under control conditions, which was around 0.35 units lower than extracellular pH, and adenosine output was 63 +/- 10 pmol/min/g. Lactic acid produced dose-dependent decreases in intracellular pH and dose-dependent increases in adenosine output: 10 mM lactic acid decreased intracellular pH to 6.57 +/- 0.04 and increased adenosine output to 159 +/- 34 pmol/min/g. The adenosine output and the intracellular pH were well correlated (r2 = 0.988; P < 0.01).

Alpha-adrenoceptor subtypes of dog saphenous vein: another unusual property.

The functional relationship between vascular smooth muscle alpha1- and alpha2-adrenoceptor (AR) subtypes was investigated by simultaneous measurement of contractile and fluorescence ratio in fura-2 loaded rings of dog saphenous vein (DSV). Prazosin, as well as rauwolscine, at 0.1 microM, substantially antagonized contractions and associated cytosolic [Ca2+] rises induced by UK 14304, while rauwolscine, as well as prazosin, antagonized similar effects of phenylephrine (PE). These antagonisms were characterized by a parallel rightward shift of the concentration-response curves. In the absence of extracellular Ca2+, PE as well as UK 14304 caused simultaneous transient elevation of contractile force and cytosolic [Ca2+], although the UK 14304 responses were smaller than PE responses. We propose that DSV smooth muscle cells possess interacting alpha1- and alpha2-ARs which have overlapping functional domain sensitive to the agonists and antagonists of either alpha-AR subtype. Both alpha-AR subtypes appear to utilize similar signaling mechanisms via Ca2+ release from the same intracellular stores and Ca2+ entry across the plasma membrane.

Intracellular lactate controls adenosine output from dog gracilis muscle during moderate systemic hypoxia.

The influence of systemic hypoxia on lactate and adenosine output from isolated constant-flow-perfused gracilis muscle was determined in anesthetized dogs. The lactate transport inhibitor alpha-cyano-4-hydroxycinnamic acid (CHCA) was employed to distinguish the direct effects of hypoxia on adenosine output from the effects produced indirectly by a change in lactate concentration. Reduction of arterial PO2 from 135 +/- 4 to 39 +/- 2 mmHg raised arterial lactate from 1.26 +/- 0.32 to 2.22 +/- 0.45 mM but decreased venoarterial lactate difference from 0.53 +/- 0.09 to -0.13 +/- 0.19 mM, indicating that lactate output from the muscle was abolished. Arterial adenosine did not change, but venoarterial adenosine difference increased from 20.6 +/- 10.1 to 76.5 +/- 14.4 nM. CHCA infusion during hypoxia abolished adenosine output from gracilis muscle (venoarterial adenosine difference = -20.5 +/- 40.6 nM). In isolated rat soleus muscle fibers, intracellular pH increased from 6.96 +/- 0.04 to 7.71 +/- 0.14 in response to a reduction of PO2 from 459 +/- 28 to 53 +/- 3 mmHg. Correspondingly, adenosine output decreased from 3.71 +/- 0.15 to 3.04 +/- 0.27 nM. These data suggest that hypoxia did not directly stimulate adenosine output from red oxidative skeletal muscle, but rather systemic hypoxia increased lactate delivery and the resulting increase in intracellular lactate decreased intracellular pH, which stimulated adenosine output.

Adenosine output from dog gracilis muscle during systemic hypercapnia and/or amiloride-SITS infusion.

The influence of acidosis on adenosine output from the isolated constant-flow-perfused gracilis muscle was studied in anesthetized dogs. Depression of intracellular pH (pHi) by supplementation of the inspired air with 10% CO2-90% O2 increased arterial PCO2 from 34.2 +/- 1.0 to 53.5 +/- 1.9 mmHg, arterial PO2 from 138.3 +/- 3.9 to 256.6 +/- 17.6 mmHg, and venoarterial adenosine concentration from 14 +/- 15 to 47 +/- 19 nM. Twitch contractions of the muscle at 2 Hz increased venoarterial adenosine concentration to 165 +/- 63 and 204 +/- 62 nM in normocapnia and hypercapnia, respectively. Venoarterial lactate concentration increased from 0.42 +/- 0.07 to 0.90 +/- 0.15 mM during normocapnic contractions but remained unchanged during hypercapnic contractions (0.42 +/- 0.11 mM). Depression of pHi by infusion of amiloride and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid increased venoarterial adenosine concentration from -2 +/- 27 to 124 +/- 48 nM in normocapnia and from 16 +/- 24 to 236 +/- 119 nM in hypercapnia. These results indicate that adenosine output from red oxidative skeletal muscle was stimulated by procedures that depress pHi.