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Red Blood Cell–Derived ATP as a Regulator of Skeletal Muscle Perfusion

ELLSWORTH, MARY L.

Medicine & Science in Sports & Exercise: January 2004 - Volume 36 - Issue 1 - p 35-41
doi: 10.1249/01.MSS.0000106284.80300.B2
CLINICAL SCIENCES: Symposium—Protecting Muscle ATP during Fatigue: How Metabolites Contribute to Crisis Management
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ELLSWORTH, M. L. Red Blood Cell–Derived ATP as a Regulator of Skeletal Muscle Perfusion. Med. Sci. Sports Exerc., Vol. 36, No. 1, pp. 35–41, 2004. Blood flow to skeletal muscle is a complex process designed to provide adequate, yet not excessive, amounts of oxygen to meet the ever-changing metabolic needs of the tissue. To accomplish this goal, a mechanism must exist that couples the oxygen needs of the tissue with the oxygen delivery system. A number of mechanisms have been investigated that have focused primarily on the vessel or tissue supplied. However, because none of these was able to adequately explain the precision inherent in oxygen supply, we began to investigate the potential role of the mobile oxygen carrier itself, the red blood cell. This review will provide evidence in support of the idea that the red blood cell is able to both sense oxygen need and evoke changes in blood flow to meet that need. In this scheme, as a red blood cell enters a region of increased metabolic demand relative to supply, the fall in hemoglobin oxygen saturation evokes the release of ATP, found within the red blood cell in mM amounts. The released ATP binds to purinergic receptors located on the vascular endothelium and induces a vasodilation that is conducted upstream increasing oxygen supply to the region of tissue supplied by the vessel. Although this mechanism is likely only one component of a complex system, which precisely regulates blood flow, we suggest that it plays a vital role in the regulation of perfusion distribution within tissue.

Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, St. Louis, MO

Address for correspondence: Mary L. Ellsworth, Ph.D., Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, 1402 South Grand Blvd., St. Louis, MO 63104; E-mail: Ellsworthm@slu.edu.

Submitted for publication March 2003.

Accepted for publication August 2003.

The mammalian red blood cell is a small, anucleated cell that has a critical role as a supplier of oxygen to all regions of the body. Although the ability of the red blood cell to carry oxygen is undeniable, its role in determining where that oxygen is to be delivered is in the forefront of current investigation. We suggest that the red blood cell has the capacity to sense oxygen need and effect changes in oxygen supply to meet that need, a unique and intriguing role for this mobile oxygen carrier. The object of this presentation is to lay out our currently available evidence supporting the notion that the red blood cell is more than simply a carrier of oxygen but, by virtue of its ability to release ATP, serves an equally important role as a regulator of its own distribution through contributing to the control of tissue blood flow. We perceive that this mechanism would provide the fine tuning necessary to adequately perfuse all regions of complex organs enabling the precise matching of oxygen supply with demand at a localized level.

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BACKGROUND

Appropriate oxygen delivery to striated muscle is a complex process. The complexity is a consequence of both the variability in the metabolic needs of the tissue and the diffusive nature of the oxygen molecule itself. The precise regulation of oxygen supply to muscle results from a combination of neural (3,15,39), myogenic (27,29,30,36), and metabolic (19,28,47) controllers that serve to meet the continually changing metabolic demands. The accomplishment of this difficult task requires that a mechanism exist that can determine the oxygen needs of the tissue relative to its supply and subsequently induce either an increase in blood flow when supply is unable to meet demand or a decrease when supply exceeds demand. In addition, although total flow is an important component, a top priority must be the efficient distribution and redistribution of blood flow to meet the variable oxygen needs within the tissue while minimizing the work of the heart.

To accomplish these precise regulatory tasks, the upstream vasculature must be able to sense that a portion of the muscle downstream of the supply vessels is in need of oxygen. Investigators have explored a number of interesting avenues, all of which have their supporters: 1) the arterioles themselves could be sensitive to blood or tissue oxygen levels (8,25,37,40); 2) the extravascular tissue could produce a metabolite which enters the circulation and affects a change in arteriolar tone via diffusion from a paired venule (1,4,20,21,22,43,45); and 3) a single specific site could exist within the tissue that senses oxygen utilization, the latter unlikely due to both the complex nature of the microvasculature (11,13,14) and the ease with which oxygen diffuses down its oxygen tension (PO2) gradient. Although one can make a case that each of these mechanisms could induce changes in vascular perfusion, none of them is able to completely explain the observed sensitivity of the vasculature to changes in oxygen requirements within the physiological range of oxygen tensions. Thus, we were left to speculate on an additional possibility, namely that the oxygen carrier itself plays a role in supplying the muscle with appropriate amounts of oxygen. Is it possible that the efficient, mobile red blood cell is able to affect its own distribution? Such an idea is intriguing and worthy of consideration.

Why did we think about the red blood cell? In 1993, Stein and Ellsworth (50) evaluated capillary oxygen exchange in two groups of hamsters, one with a normal P50 (∼26 mm Hg) and the second with a reduced P50 (∼16 mm Hg) induced by the chronic, short-term administration of sodium cyanate. In both groups of hamsters, the systemic hematocrit was reduced by 40% by the isovolemic exchange of 2 mL of blood with 2 mL of plasma from a donor hamster. In situ video microscopy of the hamster cheek pouch retractor muscle (52) was used to determine oxygen saturation and hemodynamics (9) at upstream (A) and downstream (V) ends of the capillary network during normoxic (room air or 30% O2) and low PO2 (10% O2) ventilation. We observed that the difference in oxygen saturation (A-V) was the same in the two groups during normoxic ventilation. However, during ventilation with 10% O2, the oxygen saturation difference in the animals in the untreated groups (normal P50) was markedly reduced compared with the treated animals (P50 ∼16 mm Hg) despite having higher values for PO2. This led us to speculate that oxygen content, rather than oxygen tension is the important determinant of oxygen supply in severe hypoxia. Because the red blood cell is the only component of the oxygen transport process that is influenced by oxygen content, it was reasonable to speculate that the red blood cell itself could be involved in the regulation of perfusion distribution.

What do we know about red blood cells? Mammalian red blood cells contain millimolar amounts of ATP (38) and additionally have the glycolytic machinery necessary to generate it. Bergfeld and Forrester reported in 1992 (2) that human red blood cells release ATP in response to low PO2 in the presence of hypercapnia. We also know that purinergic receptors are located on the endothelium which, when stimulated by ATP, results in vasodilation (41). These data led us to formulate the hypothesis that the red blood cell, in addition to serving as an efficient carrier of oxygen, is an important sensor and effector of alterations in vascular caliber enabling the appropriate matching of oxygen supply with demand. If this hypothesis is correct, then the following needs to be established: 1) ATP must be released from red blood cells in response to factors associated with oxygen demand in excess of oxygen supply; 2) intraluminal application of ATP at physiological levels must evoke an increase in oxygen supply sufficient to meet oxygen need; and 3) there must be a cause and effect relationship between these two events.

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DO RED BLOOD CELLS RELEASE ATP UNDER CONDITIONS WHICH MIGHT BE ENCOUNTERED IN A TISSUE REGION WHICH HAS A LOW PO2?

Forrester and colleagues (16,17) had previously demonstrated a dramatic increase in ATP levels in venous effluent from active human forearm muscle suggesting a link to reduced PO2. To investigate this further, Bergfeld and Forrester (2) measured ATP release from human red blood cells exposed to a low PO2. Although their studies were complicated by a marked hypercapnia, their results clearly demonstrated that low PO2 causes human red blood cells to release ATP. In an attempt to isolate the impact of low PO2, we subsequently employed an in vitro assay system shown schematically in Figure 1. In these experiments (10,12), hamster red blood cells were exposed to TRIS-buffered Ringer solutions that had been equilibrated with gases of known composition. In one series, an aliquot of washed red blood cells was placed into one of three tissue baths that differed in their PO2 (∼85, ∼35, and ∼11 mm Hg) with PCO2 and pH remaining constant at ∼35 mm Hg and ∼7.36, respectively. After a 1-min exposure, an aliquot of cells was removed and immediately assayed for ATP using the luciferin/luciferase technique (51). We determined that exposing the cells to a PO2 of ∼35 mm Hg resulted in a 40% increase in ATP release compared with control (PO2 ∼85 mm Hg). An even greater increase in ATP release was observed when the cells were exposed to a PO2 of ∼11 mm Hg (Fig. 2). The release of ATP in response to reduced PO2 has been subsequently shown for rat, rabbit, and healthy human red blood cells. More recently, Jagger et al. (26) used carbon monoxide to dissociate the effects of oxygen saturation from PO2. They clearly established that ATP release from rat red blood cells is a consequence of a fall in oxygen saturation and not PO2, which confirms our original observation (50). In addition, they evaluated the role of glycolysis in ATP release and provided data suggestive of a possible link that, if confirmed, would provide supporting evidence that two pools of ATP exist within the red blood cell (24) with the releasable pool closely linked with the cell membrane and associated signal transduction pathways. In a very recent study, González-Alonso and colleagues (18) have established that in humans, blood flow and oxygen delivery to skeletal muscle are clearly influenced by the oxygen-desaturation dependent release of ATP from red blood cells. What is left to be established is the mechanism by which dissociation of oxygen from the hemoglobin molecule and the associated changes in confirmation of the hemoglobin molecule is linked to the signal transduction pathway within the red blood cell, which is ultimately responsible for the release of ATP (2,48,49).

FIGURE 1

FIGURE 1

FIGURE 2

FIGURE 2

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DOES INTRALUMINAL APPLICATION OF ATP AT PHYSIOLOGICAL LEVELS EVOKE AN INCREASE IN OXYGEN SUPPLY SUFFICIENT TO MEET OXYGEN NEEDS?

For ATP to have an important impact on tissue oxygen supply, it must be able to induce a conducted vasodilator response, defined as one which extends well beyond the site of initiation and serves to significantly augment the impact of the local response (33,46). Using the Saran-covered hamster cheek pouch retractor muscle (52) and in vivo video microscopy, McCullough et al. (34) evaluated the effectiveness of applying ATP into arterioles at concentrations ranging from 10−8 M to 10−4 M. In these studies, ATP was introduced into the lumen of first- and second-order arterioles via a glass pipette having a tip diameter of 1–2 μm (Fig. 3). The solution was ejected from the pipette using a Picospritzer II (General Valve, Fairfield, NJ) at a pressure of 10 psi for durations of either 50 or 500 ms, which corresponds to volumes of 40 and 400 pL, respectively. Videotaped recordings of the same arteriole 45–1750 μm upstream from the site of application were used to determine the magnitude of the conducted response. We observed that at all concentrations tested, upstream arteriolar diameter increased with the maximum change in diameter observed at 10−6 M (Fig. 4). Figure 5 shows an example of an arteriole before and after application of 10−6 M ATP ∼150 μm downstream from and out of view of the site being observed. In this example, ATP induced localized increases in vessel diameter that ranged from 16 to 65% at the sites indicted by the arrows. It is of interest that a similar concentration of adenosine had no significant effect on upstream vessel diameter nor is there any effect of a control solution. Although on the surface it appears that ATP has only a minor effect on vessel diameter (average ∼8% increase), it is important to remember that according to Poiseuille’s law, flow is proportional to the fourth power of the radius (Q = πr4ΔP/8ηL); thus, the impact of intraluminal ATP is markedly greater than it might first appear. In addition, because the vasodilation is conducted upstream, the impact on perfusion is again enhanced as predicted by Segal and Duling (46) and confirmed in subsequent studies by Kurjiaka and Segal (33) and Collins et al. (5).

FIGURE 3

FIGURE 3

FIGURE 4

FIGURE 4

FIGURE 5

FIGURE 5

It became important to determine if the concentration of ATP at which we observed the maximal conducted response (10−6 M) was physiologically possible. To this end, we computed the amount of ATP that would be released by red blood cells within a vessel of similar size. For these calculations, we used literature values for flow in the hamster retractor muscle (31,32,53) and the amount of ATP released from red blood cells exposed to low PO2 from our in vitro studies (12). Considering that the red blood cells remain in the vessel for a finite period of time, we computed the exposure of the red cells to the vessel wall based on red blood cell velocity (31,32,53). We determined that the expected local concentration of ATP would be on the order of 10−6 M, which correlated with the data of McCullough et al. (34), giving us assurance that we were studying a physiological event.

Because it would be anticipated that significant oxygen desaturation would occur more frequently on the venous side of the circulation, it became paramount to ascertain if a conducted response of the same magnitude could likewise be evoked in the supply arterioles by application of similar amounts of ATP into the collecting venules. Using the same animal model and basic protocol (Fig. 3), Collins et al. (5) observed that application of 10−6 M ATP into collecting venules induced an even larger increase in vessel diameter (∼12%) in the upstream supplying arterioles. Measurements of red blood cell flux, made using fluorescently labeled red blood cells (44), showed increases of 63% in second-order arterioles, 50% in terminal arterioles, and 92% in capillaries (Fig. 4). In addition, these data suggested that the response to the ATP was necessarily conducted via the endothelium because the signal traversed the intervening capillaries. However, the precise mechanism by which the conduction was achieved remains to be determined.

We were interested to determine the mediator involved in the conducted response that lead both McCullough et al. (34) and Collins et al. (5) to evaluate the role of nitric oxide. In each study, the systemic administration of Nω-nitro-L-arginine methyl ester (L-NAME, 0.4 mg·100 g−1 body weight; iv, Sigma, St. Louis, MO), an inhibitor of nitric oxide synthase, abolished the conducted vasodilator response to intraluminal ATP at concentrations ranging from 10−8 to 10−5 M. However, L-NAME did not completely abolish the response to intra-arteriolar ATP (34) at the highest concentration (10−4), suggesting that a second mediator may be involved as ATP concentrations increase, possibly a product of cyclooxygenase as suggested by Hammer et al. (20).

Although an increase in red blood cell flux is indicative of an augmentation of tissue oxygen supply, it was crucial that we demonstrate that 10−6 M ATP enhanced tissue oxygenation. In a subsequent study, capillary red blood cell flux and tissue PO2 in the region surrounding the capillary were measured simultaneously before and after the administration of 400 pL of 10−6 M ATP into the collecting venule (Fig. 3). We observed that in response to the ATP, red blood cell flux increased significantly followed within 5–7 s by a measurable increase in tissue PO2 (Fig. 6). Both parameters subsequently returned to baseline levels. These results further confirm the idea that ATP, by inducing a conducted vasodilator response, augments tissue PO2 in a manner consistent with our hypothesis further supporting ATP as an important player in the determination of blood flow distribution and tissue oxygenation. Based on the above studies, we have established that ATP is both released from red blood cells in response to their exposure to a low PO2 environment and that it evokes an increase in tissue oxygenation. What becomes of importance is to ascertain whether there is a direct link between these two events.

FIGURE 6

FIGURE 6

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IS THERE A CAUSE AND EFFECT RELATIONSHIP BETWEEN THE EXPOSURE OF THE RED BLOOD CELLS TO LOW PO2 AND THE SUBSEQUENT VASODILATION OF AN ARTERIOLE?

Confirming the presence of a relationship between the release of ATP from red blood cells exposed to a low PO2 environment and the induction of a conducted vasodilator response required that a system be utilized in which as many variables can be controlled as possible. Thus, we employed an isolated rat penetrating cerebral arteriole, which had been previously well characterized, found to exhibit responses to ATP similar to those of the hamster retractor muscles (6), and was available as a model system. In these studies (7), arterioles (length = 1868 ± 162 μm) were cannulated at each end, pressurized, and allowed to develop spontaneous tone, resulting in vessels having a diameter of 58 ± 3 μm. These vessels were perfused at a constant flow (3 μL·min−1) with albumin containing MOPS buffer followed by either washed autologous rat red blood cells (hematocrit of 15–20%) or 6% dextran (MW = 65,500; Sigma). Arteriolar diameter was measured continuously and the venous effluent collected for determination of ATP using the luciferin/luciferase assay (51). Initially the solution bathing the vessel was equilibrated with gas providing a PO2 ∼120 mm Hg. Reducing the bath PO2 to ∼35 mm Hg induced both an increase in vessel diameter and effluent ATP only when the vessels were perfused with red blood cells (Fig. 7). These results confirm that exposure of red blood cells within a vessel to a low PO2 environment does induce ATP release from the red blood cells and that this ATP has the effect of increasing vessel diameter, interestingly to the same extent as in our in vivo studies (∼7–8%). Taking data from a single 50-μm arteriole (∼2 mm in length) perfused with red blood cells at 50 nL·s−1 resulting in a mean velocity of 2.55 mm·s−1, we determined that the entire response from initial exposure of the red blood cells within the vessel to the low PO2 environment to the increase in vessel diameter took less than 1 s. This observation that the time course for release of the ATP and the subsequent vasodilatory effect was sufficiently short (<1 s) is vital if we are to conclude that this system of regulation is physiologically relevant.

FIGURE 7

FIGURE 7

From these studies, we have established that exposure of red blood cells to low PO2 induces the release of ATP. We have also demonstrated in the studies utilizing the hamster retractor muscle that intraluminal application of physiological levels of ATP into arterioles and collecting venules induces a conducted vasodilator response upstream with a concomitant increase in blood flow and oxygen supply. Thus, it is reasonable to assume that ATP released from the red blood cells entering a region of tissue in which oxygen demand was exceeding supply would also induce such a conducted response.

It is unlikely, however, that this is the only means by which substances present in the venules can influence muscle oxygen supply. Studies by Hester and his colleagues (20,23,35,42) have provided evidence to support the idea that a mediator, such as red blood cells derived ATP (20), exerts its effect by diffusion from the venule to the paired arteriole. Although on the surface it may appear that this mechanism diminishes the role of the conducted response, the reality is that it clearly complements it providing a secondary level of control. One could speculate that when a small region of the tissue is in need of oxygen, ATP is released from red blood cells evoking a conducted response redistributing the existing flow to meet the excess demand in a confined region. However, when tissue oxygen demand is more widespread or long lasting, the amount of ATP released by the red blood cells into the venule would be greater inducing a supplemental, diffusion-mediated increase in total flow. These two mechanisms, working in concert, would be able to supply oxygen to the tissue appropriately at all levels of need. Support for this two-tier control system can be derived from the disparity in the concentrations of ATP (10−6 (34) vs 10−4 M (35)) employed in these two studies. One complication with ATP playing such a role is the observation that application of ATP to the smooth muscle side of the arteriole (as would occur were it to diffuse from the venule) induces a powerful vasoconstriction (34). Therefore, a secondary mediator, most likely a product of cyclo-oxygenase, would be required (20). Interestingly, it would appear that different secondary mediators are involved in these responses with nitric oxide important for the conducted response (5,34) and derivatives of cyclo-oxygenase involved in diffusive flow control (23). Although this dual-control system has yet to be supported by direct studies, it is a reasonable explanation that would reconcile the two sets of data.

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CONCLUSION

We have demonstrated that red blood cell–derived ATP is involved in the control of blood flow distribution. In this construct, exposure of red blood cells to a tissue region that has a low PO2 induces the release of ATP, which subsequently binds to purinergic receptors on the vascular endothelium inducing a conducted vasodilator response. We have established that this response can be initiated in either the arterioles or venules, with the result an augmentation of blood flow and tissue oxygen tension. Importantly, we have established that there is a cause and effect relationship between the exposure of the red blood cells to low PO2 and the subsequent vasodilator response with a time course that establishes its physiological relevance for flow regulation. Clearly this mechanism is only one of a broad group of redundant systems responsible for the control of skeletal muscle perfusion. However, it is unique in that it provides a direct link between the sensing of localized oxygen need and the induction of alterations in supply to precisely meet that need, a link previously lacking. Thus, the mobile oxygen carrier takes on an even greater role, not only delivering the oxygen the tissue requires but controlling its own distribution to meet that need appropriately.

The author thanks Drs. Hans H. Dietrich, Randy S. Sprague, and William McCullough and Ms. Diane Collins for their assistance with these investigations.

The studies reported by the author were supported by grants from the National Institutes of Health (HL39226, HL54629, and HL02602 (RCDA)).

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Keywords:

OXYGEN SUPPLY; BLOOD FLOW; OXYGEN TENSION; OXYGEN SATURATION

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