Bendamustine is safe and effective for lymphodepletion before tisagenlecleucel in patients with refractory or relapsed large B-cell lymphomas.

BACKGROUND: Anti-CD19 chimeric antigen receptor T-cell immunotherapy (CAR-T) is now a standard treatment of relapsed or refractory B-cell non-Hodgkin lymphomas; however, a significant portion of patients do not respond to CAR-T and/or experience toxicities. Lymphodepleting chemotherapy is a critical component of CAR-T that enhances CAR-T-cell engraftment, expansion, cytotoxicity, and persistence. We hypothesized that the lymphodepletion regimen might affect the safety and efficacy of CAR-T. PATIENTS AND METHODS: We compared the safety and efficacy of lymphodepletion using either fludarabine/cyclophosphamide (n = 42) or bendamustine (n = 90) before tisagenlecleucel in two cohorts of patients with relapsed or refractory large B-cell lymphomas treated consecutively at three academic institutions in the United States (University of Pennsylvania, n = 90; Oregon Health & Science University, n = 35) and Europe (University of Vienna, n = 7). Response was assessed using the Lugano 2014 criteria and toxicities were assessed by the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 and, when possible, the American Society for Transplantation and Cellular Therapy (ASTCT) consensus grading. RESULTS: Fludarabine/cyclophosphamide led to more profound lymphocytopenia after tisagenlecleucel infusion compared with bendamustine, although the efficacy of tisagenlecleucel was similar between the two groups. We observed significant differences, however, in the frequency and severity of adverse events. In particular, patients treated with bendamustine had lower rates of cytokine release syndrome and neurotoxicity. In addition, higher rates of hematological toxicities were observed in patients receiving fludarabine/cyclophosphamide. Bendamustine-treated patients had higher nadir neutrophil counts, hemoglobin levels, and platelet counts, as well as a shorter time to blood count recovery, and received fewer platelet and red cell transfusions. Fewer episodes of infection, neutropenic fever, and post-infusion hospitalization were observed in the bendamustine cohort compared with patients receiving fludarabine/cyclophosphamide. CONCLUSIONS: Bendamustine for lymphodepletion before tisagenlecleucel has efficacy similar to fludarabine/cyclophosphamide with reduced toxicities, including cytokine release syndrome, neurotoxicity, infectious and hematological toxicities, as well as reduced hospital utilization.

Intracellular adenosine formation and release by freshly-isolated vascular endothelial cells from rat skeletal muscle: effects of hypoxia and/or acidosis.

Previous studies suggested indirectly that vascular endothelial cells (VECs) might be able to release intracellularly-formed adenosine. We isolated VECs from the rat soleus muscle using collagenase digestion and magnetic-activated cell sorting (MACS). The VEC preparation had >90% purity based on cell morphology, fluorescence immunostaining, and RT-PCR of endothelial markers. The kinetic properties of endothelial cytosolic 5'-nucleotidase suggested it was the AMP-preferring N-I isoform: its catalytic activity was 4 times higher than ecto-5'nucleotidase. Adenosine kinase had 50 times greater catalytic activity than adenosine deaminase, suggesting that adenosine removal in VECs is mainly through incorporation into adenine nucleotides. The maximal activities of cytosolic 5'-nucleotidase and adenosine kinase were similar. Adenosine and ATP accumulated in the medium surrounding VECs in primary culture. Hypoxia doubled the adenosine, but ATP was unchanged; AOPCP did not alter medium adenosine, suggesting that hypoxic VECs had released intracellularly-formed adenosine. Acidosis increased medium ATP, but extracellular conversion of ATP to AMP was inhibited, and adenosine remained unchanged. Acidosis in the buffer-perfused rat gracilis muscle elevated AMP and adenosine in the venous effluent, but AOPCP abolished the increase in adenosine, suggesting that adenosine is formed extracellularly by non-endothelial tissues during acidosis in vivo. Hypoxia plus acidosis increased medium ATP by a similar amount to acidosis alone and adenosine 6-fold; AOPCP returned the medium adenosine to the level seen with hypoxia alone. These data suggest that VECs release intracellularly formed adenosine in hypoxia, ATP during acidosis, and both under simulated ischaemic conditions, with further extracellular conversion of ATP to adenosine.

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.

Effect of eccentric contraction-induced injury on force and intracellular pH in rat skeletal muscles.

The effect of eccentric contraction on force generation and intracellular pH (pH(i)) regulation was investigated in rat soleus muscle. Eccentric muscle damage was induced by stretching muscle bundles by 30% of the optimal length for a series of 10 tetani. After eccentric contractions, there was reduction in force at all stimulation frequencies and a greater reduction in relative force at low-stimulus frequencies. There was also a shift of optimal length to longer lengths. pH(i) was measured with a pH-sensitive probe, 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein AM. pH(i) regulation was studied by inducing an acute acid load with the removal of 20-40 mM ammonium chloride, and the rate of pH(i) recovery was monitored. The acid extrusion rate was obtained by multiplying the rate of pH(i) recovery by the buffering power. The resting pH(i) after eccentric contractions was more acidic, and the rate of recovery from acid load post-eccentric contractions was slower than that from postisometric controls. This is further supported by the slower acid extrusion rate. Amiloride slowed the recovery from an acid load in control experiments. Because the Na(+)/H(+) exchanger is the dominant mechanism for the recovery of pH(i), this suggests that the impairment in the ability of the muscle to regulate pH(i) after eccentric contractions is caused by decreased activity of the Na(+)/H(+) exchanger.

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).

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

1. 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. 2. 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. 3. 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. 4. 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 K(m) of 5'N, which increased its capacity to form adenosine by 10-20% for every 0.5 unit decrease inpH 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. 5. 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. 6. 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.

L-NAME inhibits Mg(2+)-induced rat aortic relaxation in the absence of endothelium.

1 L-NG-nitro-arginine methyl ester (L-NAME; 100 microM), a nitric oxide synthase (NOS) inhibitor, reversed the relaxation induced by 3 microM acetylcholine (ACh) and 2-10 mM Mg2+ in endothelium-intact (+E) rat aortic rings precontracted with 1 microM phenylephrine (PE). In PE-precontracted endothelium-denuded (-E) rat aorta, 3 microM ACh did not, but Mg2+ caused relaxation which was reversed by L-NAME, but not by D-NAME. 2 The concentration response profiles of L-NAME in reversing the equipotent relaxation induced by 5 mM Mg2+ and 0.2 microM ACh were not significantly different. 3 L-NAME (100 microM) also reversed Mg(2+)-relaxation of -E aorta pre-contracted with 20 mM KCl or 10 microM prostaglandin F2alpha (PGF2alpha). L-NG-monomethyl-arginine (L-NMMA; 100 microM) was also effective in reversing the Mg(2+)-relaxation. 4 Addition of 0.2 mM Ni2+, like Mg2+, caused relaxation of PE-pre-contracted -E aorta, which was subsequently reversed by 100 microM L-NAME. 5 Reversal of the Mg(2+)-relaxation by 100 microM L-NAME in PE-precontracted -E aorta persisted following pre-incubation with 1 microM dexamethasone or 300 microM aminoguanidine (to inhibit the inducible form of NOS, iNOS). 6 Pretreatment of either +E or -E aortic rings with 100 microM L-NAME caused elevation of contractile responses to Ca2+ in the presence of 1 microM PE. 7 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.

Mechanism of action of prostaglandin E2 in the dog skeletal muscle circulation.

OBJECTIVE: To determine whether prostaglandin E2 (PGE2) influences the dog skeletal muscle circulation by a direct action on the vascular smooth muscle or via pre- or post-synaptic modulation of sympathetic neurotransmission. METHODS: In 18 anaesthetised dogs, a gracilis muscle was vascularly isolated and perfused at constant flow. Sympathetic vasoconstrictor tone on the muscles was reflexly controlled by alterations to the pressure at which the isolated carotid sinuses were perfused. The effects of PGE2 injection into the muscle were compared at low carotid sinus pressure, high carotid sinus pressure, and following denervation of the muscle, with or without noradrenaline infusion. RESULTS: At all levels of sympathetic tone, PGE2 produced significantly more vasodilation than the saline vehicle. However, at a carotid sinus pressure of 46.0 +/- 2.3 mmHg (1 mmHg = 0.133 kPa), PGE2 caused a decrease in femoral arterial perfusion pressure of 52.6 +/- 7.1 mmHg, which was significantly greater than the response at a carotid sinus pressure of 208.5 +/- 3.7 (33.6 +/- 4.2 mmHg decrease) or following denervation (25.6 +/- 3.7 mmHg decrease). In a separate group of denervated muscles, PGE2 caused a similar decrease in perfusion pressure in the presence or absence of a noradrenaline infusion. CONCLUSIONS: PGE2 appears to cause vasodilation through two separate mechanisms: one mechanism involves presynaptic inhibition of sympathetic vasoconstrictor tone, whilst the other is independent of the sympathetic nervous system, and is therefore presumably a direct action on the vascular smooth muscle or endothelium. Under our experimental conditions, both mechanisms contributed equally to the vasodilation.

Local hypoxia does not elevate adenosine output from isolated gracilis muscle in anaesthetized dogs.

1. The influence of local hypoxia on adenosine and lactate output from isolated perfused gracilis muscle was studied in anaesthetized dogs. 2. Oxygen tension in the arterial blood supplying the muscle was reduced by a membrane lung from 145.9 +/- 28.9 to 52.9 +/- 2.6 (moderate hypoxia) or 30.0 +/- 1.2 mmHg (severe hypoxia). 3. Moderate hypoxia did not significantly alter vascular resistance, but severe hypoxia reduced arterial perfusion pressure from 199.0 +/- 13.6 to 122.6 +/- 8.7 mmHg. 4. Veno-arterial (V-A) lactate was 0.47 +/- 0.13 mmol/L in normoxia; neither level of hypoxia changed it significantly. Veno-arterial adenosine was 74 +/- 78 nmol/L in normoxia. Moderate hypoxia decreased this to -36 +/- 59 nmol/L (P < 0.05), but the level of V-A adenosine in severe hypoxia (52 +/- 96 nmol/L) was similar to that in normoxia. 5. These data confirm that hypoxia does not directly stimulate adenosine output from oxidative skeletal muscle.

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.

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. 2. Vessels were perfused initially (25 min) with physiological salt solution (PSS) at a pressure of 30 mmHg. Perfusion was then continued with calcium-free PSS containing either 3.0 micromol/L noradrenaline (NA) or 100 mmol/L K+, which had been made either acidotic or alkalotic. Contractile responses to graded concentrations of calcium were assessed. 3. A reduction in the intracellular or extracellular pH was induced by the addition of a weak acid (30 mmol/L sodium propionate) or reduction of the concentration of HCO3- in the PSS, respectively; an elevation of the intracellular or extracellular pH was produced by the addition of a weak base (10 mmol/L trimethylamine) or by increasing HCO3-, respectively. The PSS was bubbled with 5% CO2/95% O2. 4. Lowered intracellular pH did not alter NA- or K+-stimulated contractions. During lowered extracellular pH, contractile responsiveness and peak response were significantly reduced in K+-stimulated arteries, but were not affected in NA-stimulated arteries. 5. Elevated intracellular pH did not alter NA-induced contraction, but reduced the sensitivity to K+-stimulated contractions. Elevated extracellular pH had little effect on the magnitude of K+-induced contractions, but slightly enhanced (although not significantly) NA-induced contractions. 6. It is concluded that reduced contractile responses to K+ during extracellular acidosis are due to the modulation of potential-operated calcium channels (POC). Alkalotic vasodilatation is mediated by intracellular events and is POC-modulated, whereas alkalotic vasoconstriction appears to be due to extracellular events and is modulated by receptor-operated calcium channels (ROC).

The role of intracellular pH in the control of adenosine output from red skeletal muscle.

More than 30 years ago, it was proposed that adenosine was released from skeletal muscle in response to a decrease in the oxygen supply-to-demand ratio. It has subsequently been confirmed that adenosine is released from red muscles in proportion to the contraction frequency, but the mechanism that controls its release remains controversial. There is no direct evidence for the involvement of oxygen insufficiency in the process, and there is some indirect evidence that it is not involved. On the other hand, there is direct evidence that a decrease in pH, with no change in oxygen supply-to-demand ratio, can stimulate adenosine release, and the amounts of adenosine released are well correlated with the pH change in all situations tested. A direct analysis of the role of hypoxia in adenosine release is therefore urgently needed.

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.

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)

Adenosine and cholinergic-induced gastric contraction in rats.

Adenosine is known for its modulatory effects on gastric secretory function and mucosal blood flow in rats. However, its action on gastric motility has not been defined. The influence of adenosine on gastric contractions provoked by cholinergic drugs and direct vagal stimulation have, therefore, been examined. Bethanechol (25, 50 or 100 micrograms/kg i.v.) and electrical vagal stimulation dose and voltage dependently increased the number and the amplitude of gastric contractions. An adenosine-A1-receptor agonist, L-phenylisopropyladenosine (10 or 50 micrograms/kg s.c.), given 30 min beforehand, did not affect the changes in gastric parameters but decreased the basal mean blood pressure and lessened the reduction in blood pressure evoked by bethanechol. The adenosine-A2-receptor agonist N-ethylcarboxaminoadenosine (1 or 5 micrograms/kg s.c.), 30 min beforehand, however, significantly increased the number but not the force of gastric contractions; a lower dose of this drug increased the basal blood pressure and potentiated the depressive action of bethanechol on systemic blood pressure. Adenosine administration (7.5 mg/kg s.c.) significantly increased its plasma levels at 30 and 60 min after injection; pretreatment with it (2.5, 7.5 or 12.5 mg/kg s.c.), 30 min beforehand, did not affect the gastric and vascular actions of bethanechol. The highest dose of adenosine potentiated the contractile response of vagal stimulation. In the isolated fundus preparation, adenosine added to the organ bath (10(-6), 10(-4), 10(-2) M) also did not affect the contractions induced by acetylcholine.(ABSTRACT TRUNCATED AT 250 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.

Lack of interaction between adenosine-induced vasodilatation and carotid baroreflex-induced changes in sympathetic activity in dog hindlimb artery.

In anaesthetized dogs, a hindlimb was vascularly isolated and perfused at a constant flow rate of 7.7 +/- 1.9 ml min-1 100 g-1 (mean +/- S.E.M.; n = 5) through the femoral artery. The carotid sinuses were isolated and perfused at high (greater than 145 mmHg) or low (less than 75 mmHg) pressure to enable reflex sympathetic tone on the hindlimb vessels to be controlled. Both vagi were sectioned in the neck and mean aortic blood pressure was held constant by connection of the aorta to a reservoir. The responses to infusion of three doses of adenosine at high and low carotid sinus pressures were not significantly different: infusion of 0.60 +/- 0.16 microM-adenosine reduced femoral arterial perfusion pressure (FAPP) by 11.6 +/- 3.2% (n = 6) at high carotid sinus pressure and by 12.6 +/- 5.1% (n = 4) at low carotid sinus pressure, while 4.71 +/- 0.49 microM-adenosine reduced FAPP by 20.8 +/- 4.8% (n = 6) at high carotid sinus pressure and by 20.7 +/- 4.8% (n = 6) at low carotid sinus pressure; 50.1 +/- 7.3 microM-adenosine reduced FAPP by 36.7 +/- 5.5% (n = 6) at high carotid sinus pressure and by 27.7 +/- 7.8% (n = 5) at low carotid sinus pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

Proton permeability and lipid dynamics of gastric and duodenal apical membrane vesicles.

The passive proton permeability (Pnet) of apical membrane vesicles from rabbit parietal cells (gastric) and duodenal and renal cortical brush-border membranes (BBM) was determined by acridine orange fluorescence quenching. Values of Pnet were found to be gastric (4 x 10(-4) cm/s) less than duodenal (10(-3) cm/s) much less than renal (10(-2) cm/s). Arrhenius plots of the temperature profile of proton permeation of gastric vesicles was linear, whereas that of duodenal BBM displayed a discontinuity at 30-33 degrees C. Alcohols (octyl, benzyl, ethyl) increased Pnet in a concentration-dependent manner, with efficacy related to their oil-water partition coefficients. In a parallel series of experiments, structural parameters of the vesicle membrane lipids (fluidity) were monitored from both the steady-state and time-resolved fluorescence anisotropy of diphenylhexatriene. Fluidity of the membranes was unrelated to Pnet (renal congruent to duodenal less than gastric). Gastric vesicles demonstrated a linear Arrhenius plot of temperature dependence for fluidity, whereas duodenal BBM demonstrated a discontinuity. Membrane fluidity of gastric and duodenal vesicles was increased by alcohols, with the same potency as for Pnet, and these two variables were significantly correlated after perturbation with alcohols. Thus the fluidity of the lipid bilayer is not the major determinant of Pnet, but alteration of its structural parameters, as reflected by fluidity, produces parallel changes in Pnet.