Philippe Bodin

Purinergic Signalling: ATP Release

Adenosine triphosphate (ATP) has a fundamental intracellular role as the universal source of energy for all living cells. The demonstration of its release into the extracellular space and the identification and localisation of specific receptors on target cells have been essential in establishing, after considerable resistance, its extracellular physiological roles. It is now generally accepted that ATP is a genuine neurotransmitter both in the central and peripheral nervous systems. As such, there are numerous arguments which prove that the release of ATP by nerve terminals is by exocytosis. In some non-neuronal cells, however, recent evidence suggests that ATP release could also be carrier-mediated and would involve ATP-binding cassette proteins (ABC), an ubiquitous family of transport ATPases.

Evidence that release of adenosine triphosphate from endothelial cells during increased shear stress is vesicular.

In response to increased shear stress, vascular endothelial cells release adenosine triphosphate (ATP) by an unknown mechanism. We have investigated this mechanism using different approaches. First, we discovered that quinacrine, used to locate intracellular stores of ATP bound to peptides, displayed a granular fluorescence, typical of vesicular storage. Second, we found that two inhibitors of vesicular transport (monensin and N-ethylmaleimide) produced a highly significant reduction in the release of ATP from vascular endothelial cells in response to increased shear stress. Preliminary experiments using inhibitors of the cystic fibrosis transmembrane regulator, the sulfonylurea receptor, and the multidrug resistance protein showed no involvement of these ATP-binding cassette transporter proteins (previously characterized in endothelial cells) in the mechanism of release of ATP. We suggest, therefore, that the release of ATP from vascular endothelial cells, like that of nerve cells, is probably by vesicular exocytosis.

Increased flow‐induced ATP release from isolated vascular endothelial cells but not smooth muscle cells

1 Freshly harvested smooth muscle cells and endothelial cells isolated from the rabbit aorta were perfused (0.5 ml min−1) and stimulated twice by an increase of flow rate (3.0 ml min−1) in order to compare their ability to release adenosine 5′‐triphosphate (ATP). 2 In smooth muscle cells, the basal release of ATP (0.0265 ± 0.0033 pmol ml−1 per 106 cells) was not increased during periods of increased flow (P = 0.2). 3 In endothelial cells, the concentration of ATP in the perfusate during periods of low flow (0.0335 ± 0.0038 pmol ml−1 per 106 cells) was significantly increased by 14 times and 5 times during the first and second periods of increased flow, respectively. 4 The release of ATP by endothelial cells did not appear to be caused by the lysis of cells during the period of increased flow because it can be reproduced several times and because there was no difference between lactate dehydrogenase activity in perfused cells and that in non‐perfused cells. 5 These results show that, of the two major cell types of the vascular wall, only endothelial cells react to shear stress by releasing ATP.

Rapid release of endothelin and ATP from isolated aortic endothelial cells exposed to increased flow

Freshly harvested rabbit aortic endothelial cells on filters were exposed to two 3 min periods of a sixfold increase in flow rate of the perfusion buffer. This led to an increase in the levels of endothelin and ATP in the perfusate; arginine vasopressin remained at the basal level. Less ATP was released on the second exposure to high flow; however, endothelin release was not diminished. Using immunohistochemical techniques, endothelin and arginine vasopressin were localised in the same population of endothelial cells; endothelin and vasopressin were present in approximately 90% and 70% of endothelial cells, respectively, which suggests that there is some co-localisation. This is the first time that a stimulation has been shown to produce rapid release of endothelin.

INCREASED RELEASE OF ATP FROM ENDOTHELIAL CELLS DURING ACUTE INFLAMMATION

Abstract.Objective and Design: The effects of lipopolysaccharide (LPS), a potent inflammatory mediator, on the shear stress stimulated release of adenosine triphosphate (ATP) were investigated on endothelial cells from human umbilical vein in primary culture.¶Methods: Human umbilical vein endothelial cells (HUVEC) in primary cultures were subjected to shear stress using a cone and plate apparatus. ATP released by the cells was measured by luminometry, using a luciferin-luciferase assay.¶Results: Under conditions of shear stress alone (25 dyn/cm2), ATP accumulates into the culture medium and reaches a maximum after 3 to 5 min of stimulation (121.7 ± 13.2 pmol/ml). The shear stress-stimulated release of ATP was significantly increased after a 4  h pre-incubation of endothelial cells with 50 μg/ml (314.4 ± 26.7 pmol/ml) and 10 μg/ml lipopolysaccharide (207.7 ± 22.2 pmol/ml). Dexamethasone, an anti-inflammatory glucocorticoid, inhibited the effects of lipopolysaccharide.¶Conclusions: These results show that non-damaged endothelial cells release ATP under experimental inflammatory conditions and support an early role of extracellular ATP in the inflammatory process.

Effect of shear stress on the release of soluble ecto-enzymes ATPase and 5'-nucleotidase along with endogenous ATP from vascular endothelial cells.

Stimulation of endothelial cells from human umbilical vein by shear stress induced release of endogenous ATP which was accompanied by an extracellular increase in the activity of enzymes degrading both ATP (ATPases) and AMP (5′‐nucleotidases). The activity of soluble ATPase was progressively increased from 1.62±0.27 to 12.7±1.0 pmoles ml−1 h−1 after 60 min of stimulation by shear stress. The rate of [3H]‐ATP hydrolysis in the medium was inhibited by the purinergic agents suramin, Reactive blue 2 and pyridoxalphosphate‐6‐azophenyl‐2′4′‐disulphonic acid, and remained insensitive to the classic inhibitors of ion‐pumping and intracellular ATPases. Shear stress also increased the activity of 5′‐nucleotidase in the medium from 2.0±0.5 to 27.2±2.8 pmoles ml−1 h−1. When shear stress was applied after removal of ecto‐5′‐nucleotidase by phosphatidylinositol‐specific phospholipase C, the release of 5′‐nucleotidase was drastically reduced. These results show that soluble ATPase and 5′‐nucleotidase which are released during shear stress are not released from an intracellular compartment together with ATP but have an extracellular origin.

ATP-stimulated release of ATP by human endothelial cells

We investigated the effects of several concentrations of extracellular ATP on the release of intracellular ATP by human umbilical vein endothelial cells (HUVEC) in primary cultures. When ATP is added to the medium of cultured EC at a concentration of 1 microM, it is readily degraded by extracellular enzymes; 10 microM ATP added to the culture medium provokes a transient but significant increase, followed by a decrease in the concentration of extracellular ATP. At a concentration of 100 microM, there was a significant release of ATP and its level was maintained in the culture medium throughout the experiment. Our results show that extracellular ATP leads to a sustained release of intracellular ATP by HUVEC. Such sustained self-perpetuating release of ATP is likely to play an important part in physiological and pathological local vascular control mechanisms.

Synergistic effect of acute hypoxia on flow-induced release of ATP from cultured endothelial cells.

Human umbilical vein endothelial cells (HUVECs) in primary cultures were perfused under normoxic or hypoxic conditions. These cells were stimulated twice for 3 min by increased flow (from 0.5 to 3.0 ml/min). Under hypoxic conditions the basal release of ATP was the same as under normoxic conditions, but during increased flow the release was greater (0.58±0.07>0.32±0.04 pmoles/ml/106 cells (+78%), for the first period of stimulation; 0.39±0.05>0.22±0.03 pmoles/ml/106 cells (+79%) for the second period). Further experiments with sequential increments in flow rate showed that under both normoxic and hypoxic conditions, a positive correlation existed between ATP release and the rate of flow but there was always more ATP released under hypoxic conditions regardless of the flow rate. HUVECs in secondary culture (second passage) were similarly stimulated. No differences were observed between normoxic and hypoxic conditions. In both cases, the quantity of ATP released during high flow (0.050±0.004 pmoles/ml/106 cells) was significantly smaller than the quantity of ATP released during low flow (0.09±0.01 pmoles/ml/106 cells). To conclude, since hypoxia alone did not affect ATP release, there appears to be a synergistic relationship between increased shear stress and hypoxia in the stimulation of ATP release from HUVECs. Moreover, the release of ATP under these conditions seems to be a property of highly differentiated endothelial cells.

Blockade by glibenclamide of the flow-evoked endothelial release of ATP that contributes to vasodilatation in the pulmonary vascular bed of the rat

1 The effect of step augmentation of flow rate on the level of adenosine −5′‐triphosphate (ATP) measured in the Krebs perfusate was investigated, and the effect of glibenclamide on the release of ATP was tested in the rat pulmonary vascular bed. 2 For flow rates between 10.38 ± 1.18 and 28.88 ± 2.08 ml min−1 (n = 8) 1 μm suramin, a P2‐purinoceptor antagonist, significantly (P < 0.05) increased vascular resistance under conditions of step augmentation of flow rate. This suggests that endogenous ATP released during increases in flow rate dilates pulmonary vessels. 3 In response to a step augmentation in flow rate from 9.13 ± 0.97 to 18.3 ± 1.69 ml min−1 (n = 4) ATP levels were up to 23 fold higher (P < 0.05) for 15 s, and gradually dropped to a level of about half the initial rise. Once the ATP levels had stabilized, another step augmentation of flow rate to 27.00 ± 3.49 ml min−1 was able to evoke a corresponding increase of ATP release. The ability of the vascular bed to respond with increased ATP release after the initial ATP responses had tapered, demonstrates that the drop in ATP levels after the initial rise is not due to depletion of ATP. Furthermore, the maximal ATP response directly precedes the vasodilatation observed following each jump in perfusion pressure produced with each step increase in flow rate. 4 In response to two 3 fold step augmentations of flow rate (8.41–27.29 ml min−1) spaced 30 min apart there were two increases in the level of ATP which were not significantly different from each other. However, perfusion with 1 μm glibenclamide between the first and the second step augmentation of flow rate (8.08–24.67 ml min−1) significantly (P < 0.05; n = 6) blocked the increase in ATP release. This suggests that the release of intracellular ATP is mediated by glibenclamide‐sensitive K+ channels. 5 A concentration of 1 μm glibenclamide perfused for 30 min was without effect on vascular pressure at constant flow. However, under conditions where flow was augmented in a stepwise manner (between 11.50 and 36.45 ml min−1) perfusing with 1 μm glibenclamide increased vascular resistance (P < 0.10). 6 It is concluded that flow‐induced ATP release is mediated by a glibenclamide‐sensitive K+ channel, and that the release of ATP from endothelial cells probably functions to vasodilate the pulmonary vascular bed of the rat.

Chronic Hypoxia Changes the Ratio of Endothelin to ATP Release from Rat Aortic Endothelial Cells Exposed to High Flow

Endothelial cells isolated from the thoracic aorta of normoxic and chronically hypoxic rats were perfused (0.5 ml min-1) and stimulated by increased flow rate (3.0 ml min-1). The release of ATP, endothelin and vasopressin was investigated. During periods of high flow rate, endothelial cells isolated from normoxic rats increased their release of ATP and endothelin. In comparison, in hypoxic rats, ATP release during the period of high flow rate was less, whereas endothelin release was reater. Vasopressin release was not increased during periods of stimulation in either group of animals. These results suggest that, under conditions of reduced arterial oxygen tension, a dynamic balance between ATP and endothelin release could regulate the response of vessels to shear stress.