Heather J. Ballard

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 interstital 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 μm. 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.

Development of T-tubular vacuoles in eccentrically damaged mouse muscle fibres

Single fibres were dissected from mouse flexor digitorum brevis muscles and subjected to a protocol of eccentric stretches consisting of ten tetani each with a 40 % stretch. Ten minutes later the fibres showed a reduced force, a shift in the peak of the force‐length relation and a steepening of the force‐frequency relation. Addition of the fluorescent dye sulforhodamine B to the extracellular space enabled the T‐tubular system to be visualized. In unstimulated fibres and fibres subjected to 10 isometric tetani, the T‐tubules were clearly delineated. Sulforhodamine B diffused out of the T‐tubules with a half‐time of 18 ± 1 s. Following the eccentric protocol, vacuoles connected to the T‐tubules were detected in six out of seven fibres. Sulforhodamine B diffused out of the vacuoles of eccentrically damaged fibres extremely slowly with a half‐time of 6.3 ± 2.4 min and diffused out of the T‐tubules with a half‐time of 39 ± 4 s. Vacuole production was eliminated by application of 1 mm ouabain to the muscle during the eccentric protocol. On removal of the ouabain, vacuoles appeared over a period of 1 h and were more numerous and more widely distributed than in the absence of ouabain. We propose that T‐tubules are liable to rupture during eccentric contraction probably because of the relative movement associated with the inhomogeneity of sarcomere lengths. Such rupture raises intracellular sodium and when the sodium is pumped from the cell by the sodium pump, the volume load of Na+ and water exceeds the capacity of the T‐tubules and causes vacuole production. The damage to the T‐tubules may underlie a number of the functional changes that occur in eccentrically damaged muscle fibres.

Evidence for control of adenosine metabolism in rat oxidative skeletal muscle by changes in pH.

We investigated the effects of pH elevation or depression on adenosine output from buffer‐perfused rat gracilis muscle, and kinetic properties of adenosine‐forming enzymes, 5′‐nucleotidase (5′N) and non‐specific phosphatase (PT), and adenosine‐removing enzymes, adenosine kinase (AK) and adenosine deaminase (AD), in homogenates of muscle. Depression of the perfusion buffer pH from 7.4 to 6.8, by addition of sodium acetate, reduced arterial perfusion pressure from 8.44 ± 1.44 to 7.33 ± 0.58 kPa, and increased adenosine output from 35 ± 5 to 56 ± 6 pmol min−1 (g wet wt muscle)−1 and AMP output from 1.8 ± 0.3 to 9.1 ± 3.9 pmol min−1 (g wet wt muscle)−1. Elevation of the buffer pH to 7.8, by addition of ammonium chloride, reduced arterial perfusion pressure from 8.74 ± 0.57 to 6.96 ± 1.37 kPa, and increased adenosine output from 25 ± 5 to 47 ± 8 pmol min−1 (g wet wt muscle)−1 and AMP output from 3.7 ± 1.1 to 24.6 ± 6.8 pmol min−1 (g wet wt muscle)−1. Activity of membrane‐bound 5′N was an order of magnitude higher than that of either cytosolic 5′N or PT: pH depression reduced the Km of 5′N, which increased its capacity to form adenosine by 10–20% for every 0.5 unit decrease in pH within the physiological range. PT was only found in the membrane fraction: its contribution to extracellular adenosine formation increased from about 5% at pH 7.0 to about 15% at pH 8.0. Cytosolic 5′N had a low activity, which was unaffected by pH; the rate of intracellular adenosine formation was an order of magnitude lower than the rate of adenosine removal by adenosine kinase or adenosine deaminase, which were both exclusively intracellular enzymes. We conclude that (i) adenosine is formed in the extracellular compartment of rat skeletal muscle, principally by membrane‐bound 5′N, where it is protected from enzymatic breakdown; (ii) adenosine is formed intracellularly at a very low rate, and is unlikely to leave the cell; (iii) enhanced adenosine formation at low pH is driven by an increased extracellular AMP concentration and an increased affinity of membrane‐bound 5′N for AMP; (iv) enhanced adenosine formation at high pH is driven solely by the elevated extracellular AMP concentration, since the catalytic capacity of membrane 5′N is reduced at high pH.

Influence of stimulation parameters on the release of adenosine, lactate and CO2 from contracting dog gracilis muscle.

1. The addition of adenosine, CO2 and lactate to the venous blood draining an isolated constant‐flow perfused gracilis muscle was studied in anaesthetized and artificially ventilated dogs during twitch and tetanic contractions. 2. Venous adenosine concentration increased from 154 +/‐ 33 nM (mean +/‐ S.E.M.) to 279 +/‐ 121 or 280 +/‐ 125 nM after 10 min of 1.5 or 3 Hz twitch contractions and to 240 +/‐ 120 or 276 +/‐ 139 nM after 10 min of 1 or 5 s tetani occurring at 0.1 Hz. Twitch contractions at 0.1 or 0.5 Hz for 10 min did not significantly elevate venous adenosine. 3. Venous lactate concentration was significantly increased after 10 min of 1.5 or 3 Hz twitches or 5 s tetani at 0.1 Hz. There was a good correlation (r = 0.70; P < 0.001) between venous adenosine and lactate concentrations. 4. Venous partial pressure of CO2 (PCO2) was significantly elevated after 10 min of 1.5 or 3 Hz twitch contractions or 1 or 5 s tetani at 0.1 Hz. There was also a good correlation (r = 0.58; P < 0.001) between venous adenosine concentration and PCO2. 5. Venous partial pressure of O2 (PO2) decreased during all contractions except those at 0.1 Hz, but the oxygen cost per unit of tension x time was similar during every pattern of stimulation, and the percentage of the total energy production achieved by anaerobic means during muscle contractions did not exceed that at rest, indicating that there had been no limitation to the oxygen supply. Venous PO2 was poorly correlated with venous adenosine concentration (r = 0.28), but quite well correlated with venous lactate concentration (r = 0.53; P < 0.001). If the indirect influence of PO2 on venous adenosine concentration via an increase in lactate concentration was eliminated by partial correlation, then the coefficient for the relationship between venous adenosine concentration and venous PO2 became 0.15. 6. There was a significant correlation between the venous adenosine concentration and the venous pH (r = 0.53; P < 0.001). If the influence of oxygenation on venous adenosine and pH was eliminated by partial correlation, the coefficient for the relationship between venous adenosine and pH increased to 0.95.(ABSTRACT TRUNCATED AT 400 WORDS)

The influence of lactic acid on adenosine release from skeletal muscle in anaesthetized dogs.

1. In anaesthetized and artificially ventilated dogs, a gracilis muscle was vascularly isolated and perfused at a constant flow rate of 11.9 +/‐ 2.2 ml min‐1 100 g‐1 (mean +/‐ S.E.M., n = 16; equivalent to 170.2 +/‐ 21.3% of its resting free flow). 2. Stimulation (3 Hz) of the obturator nerve produced twitch contractions of the gracilis muscle, reduced venous pH from 7.366 +/‐ 0.027 to 7.250 +/‐ 0.031 (n = 5), increased oxygen consumption from 0.62 +/‐ 0.24 to 2.76 +/‐ 0.46 ml min‐1 100 g‐1 (n = 5) and increased adenosine release from ‐0.40 +/‐ 0.14 (net uptake) to 1.36 +/‐ 0.50 nmol min‐1 100 g‐1 (n = 8). 3. Infusion of lactic acid (4.2 mM) into the artery reduced venous pH to 7.281 +/‐ 0.026 (n = 5) and increased adenosine release to 0.96 +/‐ 0.40 nmol min‐1 100 g‐1 (n = 8), but did not significantly alter oxygen consumption (0.80 +/‐ 0.19 ml min‐1 100 g‐1; n = 5). Stimulation (3 Hz) in the presence of lactic acid infusion produced no further significant changes in venous pH or adenosine release, but increased oxygen consumption to 2.53 +/‐ 0.37 ml min‐1 100 g‐1 (n = 5). 4. Infusion of a range of lactic acid concentrations (> or = 1.83 mM) produced dose‐dependent increases in adenosine release. The maximum lactic acid concentration tested (5.95 mM) reduced venous pH to 7.249 +/‐ 0.023 (n = 5) and increased adenosine release to 2.64 +/‐ 1.26 nmol min‐1 100 g‐1 (n = 6). 5. A strong correlation existed between the adenosine release and the venous pH (r = ‐0.92); points obtained during muscle stimulation and/or lactic acid infusion fell on a single correlation line. 6. The vasoactivity of adenosine administered by close‐arterial injection was unaltered by infusion of either lactic acid (7.2 mM) or saline. 7. These results suggest that the release of adenosine from skeletal muscle can be induced by a decrease in pH (probably at an intracellular site), and that this mechanism may contribute to the release of adenosine during muscle contractions.

Evidence For The Existence Of A Constitutive Nitric Oxide Synthase In Vascular Smooth Muscle

1. We have identified a neuronal nitric oxide synthase (NOS)‐like constitutive form of NOS in vascular smooth muscle (VSM) using a functional contractility approach as well as immunohistochemical methods.

L‐NAME inhibits Mg2+‐induced rat aortic relaxation in the absence of endothelium

L‐NG‐nitro‐arginine methyl ester (L‐NAME; 100 μM), a nitric oxide synthase (NOS) inhibitor, reversed the relaxation induced by 3 μM acetylcholine (ACh) and 2–10 mM Mg2+ in endothelium‐intact (+E) rat aortic rings precontracted with 1 μM phenylephrine (PE). In PE‐precontracted endothelium‐denuded (−E) rat aorta, 3 μM ACh did not, but Mg2+ caused relaxation which was reversed by L‐NAME, but not by D‐NAME. The concentration response profiles of L‐NAME in reversing the equipotent relaxation induced by 5 mM Mg2+ and 0.2 μM ACh were not significantly different. L‐NAME (100 μM) also reversed Mg2+‐relaxation of −E aorta pre‐contracted with 20 mM KCl or 10 μM prostaglandin F2α (PGF2α). L‐NG‐monomethyl‐arginine (L‐NMMA; 100 μM) was also effective in reversing the Mg2+‐relaxation. Addition of 0.2 mM Ni2+, like Mg2+, caused relaxation of PE‐pre‐contracted −E aorta, which was subsequently reversed by 100 μM L‐NAME. Reversal of the Mg2+‐relaxation by 100 μM L‐NAME in PE‐precontracted −E aorta persisted following pre‐incubation with 1 μM dexamethasone or 300 μM aminoguanidine (to inhibit the inducible form of NOS, iNOS). Pretreatment of either +E or −E aortic rings with 100 μM L‐NAME caused elevation of contractile responses to Ca2+ in the presence of 1 μM PE. Our results suggest that L‐NAME exerts a direct action on, as yet, unidentified vascular smooth muscle plasma membrane protein(s), thus affecting its reactivity to divalent cations leading to the reversal of relaxation. Such an effect of L‐NAME is unrelated to the inhibition of endothelial NOS or the inducible NOS.

cAMP/Protein Kinase A Activates Cystic Fibrosis Transmembrane Conductance Regulator for ATP Release from Rat Skeletal Muscle during Low pH or Contractions

We have shown that cystic fibrosis transmembrane conductance regulator (CFTR) is involved in ATP release from skeletal muscle at low pH. These experiments investigate the signal transduction mechanism linking pH depression to CFTR activation and ATP release, and evaluate whether CFTR is involved in ATP release from contracting muscle. Lactic acid treatment elevated interstitial ATP of buffer-perfused muscle and extracellular ATP of L6 myocytes: this ATP release was abolished by the non-specific CFTR inhibitor, glibenclamide, or the specific CFTR inhibitor, CFTRinh-172, suggesting that CFTR was involved, and by inhibition of lactic acid entry to cells, indicating that intracellular pH depression was required. Muscle contractions significantly elevated interstitial ATP, but CFTRinh-172 abolished the increase. The cAMP/PKA pathway was involved in the signal transduction pathway for CFTR-regulated ATP release from muscle: forskolin increased CFTR phosphorylation and stimulated ATP release from muscle or myocytes; lactic acid increased intracellular cAMP, pCREB and PKA activity, whereas IBMX enhanced ATP release from myocytes. Inhibition of PKA with KT5720 abolished lactic-acid- or contraction-induced ATP release from muscle. Inhibition of either the Na+/H+-exchanger (NHE) with amiloride or the Na+/Ca2+-exchanger (NCX) with SN6 or KB-R7943 abolished lactic-acid- or contraction-induced release of ATP from muscle, suggesting that these exchange proteins may be involved in the activation of CFTR. Our data suggest that CFTR-regulated release contributes to ATP release from contracting muscle in vivo, and that cAMP and PKA are involved in the activation of CFTR during muscle contractions or acidosis; NHE and NCX may be involved in the signal transduction pathway.

Exercise makes your brain bigger: skeletal muscle VEGF and hippocampal neurogenesis

The hippocampus plays an important role in learning and memory. Unlike most brain cells (whose numbers are fixed before birth), thousands of new hippocampal granule cells continue to be generated every day throughout adult life. This article is protected by copyright. All rights reserved

INFLUENCE OF EXTRACELLULAR pH, SODIUM PROPIONATE AND TRIMETHYLAMINE ON EXCITATION‐CONTRACTION COUPLING IN THE RAT TAIL ARTERY

1. The effects of extracellular or intracellular pH changes on agonist‐ or depolarization‐induced contractions of the rat tail artery were investigated.