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.
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.
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.
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)).
1. Berg, B. R., K. D. Cohen, and I. H. Sarelius. Direct coupling between blood flow
and metabolism at the capillary level in striated muscle. Am. J. Physiol. 272: H2693–H2700, 1997.
2. Bergfeld, G. R., and T. Forrester. Release of ATP from human erythrocytes in response to a brief period of hypoxia and hypercapnia. Cardiovasc. Res. 26: 40–47, 1992.
3. Chen, H., J. Liao, L. Kuo, and S. Ho. Centrogenic pulmonary hemorrhagic edema induced by cerebral compression in rats: mechanism of volume and pressure loading in the pulmonary circulation. Circ. Res. 47: 366–373, 1980.
4. Cohen, K. D., R. R. Berg, and I. H. Sarelius. Remote arteriolar dilations in response to muscle contraction under capillaries. Am. J. Physiol. 278: H1916–H1923, 2000.
5. Collins, D. M., W. T. Mccullough, and M. L. Ellsworth. Conducted vascular responses: Communication across the capillary bed. Microvasc. Res. 56: 43–53, 1998.
6. Dietrich, H. H., M. L. Ellsworth, and R. G. Dacey, Jr. The red blood cell, ATP and integrated vascular responses to neuronal stimulation. Int. Congress Series 1235: 277–287, 2002.
7. Dietrich, H. H., M. L. Ellsworth, R. S. Sprague, and R. G. Dacey, Jr. Red blood cell regulation of microvascular tone through adenosine triphosphate. Am. J. Physiol. 278: H1294–H1298, 2000.
8. Duling, B. Oxygen sensitivity of vascular smooth muscle. II. In vivo studies. Am. J. Physiol. 227: 42–49, 1974.
9. Ellis, C. G., M. L. Ellsworth, and R. N. Pittman. Determination of red blood cell oxygenation in vivo by dual video densitometric image analysis. Am. J. Physiol. 258: H1216–H1223, 1990.
10. Ellsworth, M. L. The red blood cell as an oxygen sensor: what is the evidence? Acta Physiol. Scand. 168: 551–559, 2000.
11. Ellsworth, M. L., C. G. Ellis, A. S. Popel, and R. N. Pittman. Role of microvessels in oxygen supply
to tissue. NIPS 9: 119–123, 1994.
12. Ellsworth, M. L., T. Forrester, C. G. Ellis, and H. H. Dietrich. The erythrocyte as a regulator of vascular tone. Am. J. Physiol. 269: H2155–H2161, 1995.
13. Ellsworth, M. L., and R. N. Pittman. Arterioles supply oxygen to capillaries by diffusion as well as by convection. Am. J. Physiol. 258: H1240–H1243, 1990.
14. Ellsworth, M. L., A. S. Popel, and R. N. Pittman. Assessment and impact of heterogeneities of convective oxygen transport parameters in capillaries of striated muscle. Microvasc. Res. 35: 341–362, 1988.
15. Faber, J. In situ analysis of alpha-adrenoceptors on arteriolar and venular smooth muscle in rat skeletal muscle microcirculation. Circ. Res. 62: 37–50, 1988.
16. Forrester, T. An estimate of adenosine triphosphate release into the venous effluent from exercising human forearm muscle. J. Physiol. 224: 611–628, 1972.
17. Forrester, T., and A. R. Lind. Identification of adenosine triphosphate in human plasma and the concentration in the venous effluent of forearm muscle before, during and after sustained contractions. J. Physiol. 204: 347–364, 1969.
18. González-Alonso, J., D. B. Olsen, and B. Saltin. Erythrocyte and the Regulation of Human Skeletal Muscle Blood Flow
and Oxygen Delivery: Role of Circulating ATP. Circ. Res. 91: 1046–1055, 2002.
19. Haddy, F. J., and J. B. Scott. Metabolic factors in peripheral circulatory regulation. Fed. Proc. 34: 2006–2011, 1975.
20. Hammer, L., A. Ligon, and R. L. Hester. ATP-mediated release of arachidonic acid metabolites from venular endothelium causes arteriolar dilation. Am. J. Physiol. 280: H2616–H2622, 2001.
21. Hester, R. L. Venular-arteriolar diffusion of adenosine in hamster cremaster microcirculation. Am. J. Physiol. 258: H1918–H1924, 1990.
22. Hester, R. L. Uptake of metabolites by postcapillary venules: Mechanism for the control of arteriolar diameter. Microvasc. Res. 46: 254–261, 1993.
23. Hester, R. L., and L. Hammer. Venular-arteriolar communication in the regulation of blood flow
. Am. J. Physiol. 282: R1280–R1285, 2002.
24. Hoffman, J. F. ATP compartmentation in human erythrocytes. Curr. Opin. Hematol. 4: 112–115, 1997.
25. Jackson, W. F. Arteriolar oxygen reactivity: where is the sensor. Am. J. Physiol. 253: H1120–H1126, 1987.
26. Jagger, J. E., R. M. Bateman, M. L. Ellsworth, and C. G. Ellis. Role of erythrocyte in regulating local O2
delivery mediated by hemoglobin oxygenation. Am. J. Physiol. 280: H2833–H2839, 2001.
27. Johnson, P. C. The myogenic response. In: Handbook of Physiology: The Cardiovascular System II: Vascular Smooth Muscle, D. F. Bohr, A. P. Somlyo, and H. V. Sparks, Jr. (Eds.). Bethesda, MD: American Physiological Society, 1980, pp. 409–442.
28. Johnson, P. C., and H. A. Henrich. Metabolic and myogenic factors in local regulation of the microcirculation. Fed. Proc. 34: 2020–2024, 1975.
29. Jones, D. P., and F. G. Kennedy. When is a mammalian cell hypoxic? Insights from studies of cell versus mitochondria. Mol. Physiol. 8: 473–482, 1985.
30. Kuo, L., W. Chilian, and M. Davis. Interaction of pressure- and flow-induced responses in porcine coronary resistance vessels. Am. J. Physiol. 261: H1706–H1715, 1991.
31. Kuo, L., and R. N. Pittman. Effect of hemodilution on oxygen transport in arteriolar networks of hamster striated muscle. Am. J. Physiol. 254: H331–H339, 1988.
32. Kuo, L., and R. N. Pittman. Influence of hemoconcentration on arteriolar oxygen transport in hamster striated muscle. Am. J. Physiol. 259: H1694–H1702, 1990.
33. Kurjiaka, D. T., and S. S. Segal. Conducted vasodilation elevates flow in arteriole networks of hamster striated muscle. Am. J. Physiol. 269: H1723–H1728, 1995.
34. Mccullough, W. T., D. M. Collins, and M. L. Ellsworth. Arteriolar responses to extracellular ATP in striated muscle. Am. J. Physiol. 272: H1886–H1891, 1997.
35. Mckay, M. K., A. L. Gardner, D. Boyd, and R. L. Hester. Influence of venular prostaglandin release on arteriolar diameter during functional hyperemia. Hypertension 31 (Suppl.): 213–217, 1998.
36. Meininger, G. A., and M. J. Davis. Cellular mechanisms involved in the vascular myogenic response. Am. J. Physiol. 263: H647–H659, 1992.
37. Messina, E. J., D. Sun, A. Koller, M. S. Wolin, and G. Kaley. Increases in oxygen tension
evoke arteriolar constriction by inhibiting endothelial prostaglandin synthesis. Microvasc. Res. 48: 151–160, 1994.
38. Miseta, A., P. Bogner, E. Berenyi, et al. Relationship between cellular ATP, potassium, sodium and magnesium concentrations in mammalian and avian erythrocytes. Biochim. Biophys. Acta 1175: 133–139, 1993.
39. Ohyanagi, M., K. Nishigaki, and J. E. Faber. Interaction between microvascular α1
- and α2
-adrenoceptors and endothelium-derived relaxing factor. Circ. Res. 71: 188–200, 1992.
40. Pittman, R. N., and B. R. Duling. Oxygen sensitivity of vascular smooth muscle. Microvasc. Res. 6: 202–211, 1973.
41. Ralevic, V., and G. Burnstock. Receptors for purines and pyrimidines. Pharmcol. Rev. 50: 413–492, 1998.
42. Saito, Y., A. Eraslan, and R. L. Hester. Importance of venular flow in control of arteriolar diameter in hamster cremaster muscle. Am. J. Physiol. 265: H1294–H1300, 1993.
43. Saito, Y., A. Eraslan, V. Lockard, and R. L. Hester. Role of venular endothelium in control of arteriolar diameter during functional hyperemia. Am. J. Physiol. 267: H1227–H1231, 1994.
44. Sarelius, I. H., and B. R. Duling. Direct measurement of microvessel Hct, red cell flux, velocity, and transit time. Am. J. Physiol. 243: H1018–H1026, 1982.
45. Sauls, B. A., and M. A. Boegehold. Adenosine linking reduced O2
to arteriolar NO release in intestine is not formed from extracellular ATP. Am. J. Physiol. 281: H1193–H1200, 2001.
46. Segal, S. S., and B. R. Duling. Propagation of vasodilation in resistance vessels of the hamster: development and review of a working hypothesis. Circ. Res. 61 (Suppl. II): II20–II25, 1987.
47. Sparks, H. Effect of local metabolic factors on vascular smooth muscle. In: The Handbook of Physiology, The Cardiovascular System, D. Bohr, A. Somlyo, and H. Sparks, (Eds.). Baltimore: Waverly Press, 1980, pp. 475–513.
48. Sprague, R. S., M. L. Ellsworth, A. H. Stephenson, M. E. Kleinhenz, and A. J. Lonigro. Deformation-induced ATP release from red blood cells requires CFTR activity. Am. J. Physiol. 275: H1726–H1732, 1998.
49. Sprague, R. S., M. L. Ellsworth, A. H. Stephenson, and A. J. Lonigro. Participation of cAMP in a signal-transduction pathway relating erythrocyte deformation to ATP release. Am. J. Physiol. 281: C1158–C1164, 2001.
50. Stein, J. C., and M. L. Ellsworth. Capillary oxygen transport during severe hypoxia role of hemoglobin oxygen affinity. J. Appl. Physiol. 75: 1601–1607, 1993.
51. Strehler, B. L., and W. D. Mcelroy. Assay of adenosine triphosphate. In: Methods in Enzymology, Vol 3, S. P. Colowickand N. O. Kaplan (Eds.). New York: Academic Press, 1957, pp. 871–873.
52. Sullivan, S. M., and R. N. Pittman. Hamster retractor muscle: a new preparation for intravital microscopy. Microvasc. Res. 23: 329–335, 1982.
53. Swain, D. P., and R. N. Pittman. Oxygen exchange in the microcirculation of hamster retractor muscle. Am. J. Physiol. 256: H247–H255, 1989.