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Departments of Anesthesiology and Physiology, Medical College of Wisconsin, and Department of Veterans Affairs Medical Center, Milwaukee, Wisconsin
Submitted 14 June 2004 ; accepted in final form 10 September 2004
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ABSTRACT |
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2-receptors is known to be sensitive to acidic pH, but little is known about the pH sensitivity of P2X receptors. ATP is a cotransmitter released with norepinephrine from the sympathetic nerves and causes vasoconstriction via P2X purinergic receptors on vascular smooth muscle. We hypothesized that reductions in pH would attenuate P2X-mediated vasoconstriction in iliofemoral artery rings. Twenty-five rats were killed, and the iliac and femoral arteries were dissected out and placed in modified Krebs-Henseleit buffer. The arteries were cut into 2-mm sections and mounted in an organ tissue bath. Tension (g) was measured during a potassium chloride and norepinephrine challenge (maximal tension). The arteries were then exposed to ,
-methylene ATP (10–7-10–3 M; n = 13) or phenylephrine (10–7-10–4 M; n = 6) with a tissue bath pH of 7.8, 7.4, and 7.0. Dose-response curves were fit with nonlinear regression analysis to calculate the EC50 and slope. The peak tension with ,-methylene ATP was lower during pH 7.0 (1.37 ± 0.09 g) compared with pH 7.8 (1.90 ± 0.12 g). EC50 was highest with pH 7.4 (–5.38 ± 0.18 log M ,-methylene ATP) and lowest with pH 7.0 (–4.9 ± 0.10 log M ,-methylene ATP). The slopes of the dose-response curves were not different. Pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) abolished contraction caused by the addition of ,-methylene ATP (n = 6). There was no effect of pH on phenylephrine dose-response curves. These data indicate that the vasoconstrictor response to ,-methylene ATP is sensitive to pH and that lower pH attenuates the response of P2X purinergic receptors.
hydrogen ion; vascular smooth muscle; pH
1- and 2-postjunctional receptors on vascular smooth muscle. However, there is growing evidence that P2X purinergic receptors on vascular smooth muscle play an important role in vasoconstriction at rest and during exercise (4, 5). These receptors are activated by ATP, which is a cotransmitter released with norepinephrine from the sympathetic nerves (7, 9, 11, 19).
In the skeletal muscle vascular bed, physiological conditions (e.g., exercise) and pathological conditions (e.g., peripheral arterial insufficiency) cause a decrease in pH that may influence the regulation of vascular control (2, 13, 21). The pH sensitivity of adrenergic receptors in conduit arteries varies widely across vascular beds, but data suggest that the sensitivity of 2-receptors is decreased with acidic pH (12, 15, 17). The sensitivity of 1-receptors to pH is dependent on the vascular bed studied (12, 16). However, there is relatively little research regarding the response of P2X receptors to changes in pH.
Because there is attenuation of 2-mediated vasoconstriction with acidosis, it seems plausible that P2X receptors may also be sensitive to pH. The purpose of this investigation was to determine whether P2X receptor-mediated vasoconstriction is pH sensitive. We hypothesized that reductions in pH would inhibit P2X-mediated vasoconstriction in skeletal muscle conduit artery rings.
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METHODS |
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Protocols.
The response to the P2X agonist ,-methylene ATP (10–7-10–3 M) was measured with tissue bath pH adjusted to 7.8, 7.4, and 7.0 with sodium hydroxide or hydrochloric acid (n = 13). Baths were rinsed five times over 15 min between each concentration to avoid P2X receptor desensitization by ,-methylene ATP. The order of ,-methylene ATP concentrations was randomized within each pH level. To determine that the vasoconstriction to ,-methylene ATP was P2X mediated, in another group of vessels the P2X antagonist pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS; Sigma) was added 1 min before the solutions of ,-methylene ATP in pH 7.4 bath solution (n = 6). Additionally, cumulative phenylephrine (1-agonist) curves using bath concentrations of 10–7-10–4 M were performed in six rats at pH 7.8, 7.4, and 7.0. In all experiments the order of pH levels was randomized.
Data analysis.
Results are expressed as means ± SE. Data are expressed as absolute tension in grams and as a percentage of the maximal tension produced during the potassium chloride and norepinephrine challenge. Dose-response curves were fit with nonlinear regression analysis to calculate the EC50 of peak tension and the slope. Percent maximal values were used for regression analysis. Statistical analysis of minimum tension, peak tension, EC50, and the slope for ,-methylene ATP and phenylephrine was performed with a one-way repeated-measures analysis of variance followed by Tukey’s post hoc test when appropriate.
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RESULTS |
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,-methylene ATP at pH 7.8 (1.90 ± 0.12 g) and 7.4 (1.83 ± 0.11 g) were significantly greater than peak tension produced at pH 7.0 (1.37 ± 0.09 g; P < 0.05). Figure 1 summarizes the mean tension (%maximal) response to all doses of ,-methylene ATP at pH 7.8, 7.4, and 7.0. EC50 for the ,-methylene ATP curves was significantly lower with pH 7.0 compared with pH 7.4 but not significantly different from pH 7.8 (Fig. 2). The slope of the dose-response curve was not affected by changes in pH (1.36 ± 0.07 %maximal tension/log M ,-methylene ATP; P > 0.05). Addition of the P2 antagonist PPADS to the bath abolished the contractile response to 10–5, 10–4, and 10–3 M ,-methylene ATP (Fig. 3).
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DISCUSSION |
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,-methylene ATP was sensitive to hydrogen ions in the rat iliofemoral artery. In contrast, we found no effect of pH on reactivity to the 1-agonist phenylephrine, suggesting that pH does not have a nonspecific effect on the vascular smooth muscle. To our knowledge, there have been no previous studies investigating the effect of pH on purinergic vasoconstriction in skeletal muscle arteries. These data demonstrate that the vasoconstrictor response to ,-methylene ATP is sensitive to extracellular hydrogen ions and that lower pH attenuates the response to P2X purinergic receptors.
Consistent with previous work in cloned receptors, we found a rightward shift in the ,-methylene ATP dose-response curve, with pH 7.0 resulting in a decrease in EC50 (10, 22). This finding is in contrast to others who have shown an increased sensitivity of P2X receptor response to acidification (6, 10, 14, 15). The apparently conflicting results of these studies may be due to different P2X receptor subtypes in different tissues (22). Other studies have shown that most P2X subunits are sensitive to pH, with P2X1, P2X3, and P2X4 activity being attenuated by low pH, whereas P2X2 and P2X2/3 activity are enhanced by low pH (22). Therefore, our findings are most consistent with P2X receptors with type 1, 3, or 4 subunits. The results from the present study suggest that the response of P2X receptors to pH may be a functional means for distinguishing among P2X receptor subtypes.
Although the exact mechanism is unknown, a decrease in P2X receptor-mediated vasoconstriction may occur through three different mechanisms including the availability of bioactive ATP, altered binding sites for ATP, and altered ion channel permeability. A decrease in available bioactive ATP through an increase in ATPase activity or ionization of ATP may explain these results under certain conditions. However, in the current study we used ,-methylene ATP, which is resistant to breakdown by ATPase (25). In addition, reduction of bioavailable ATP is unlikely because investigations using similar bath solutions have reported that ionization affects <10% of the ATP within the pH range used (8, 22). Although we cannot rule out the possibility that a decrease in bioavailable ATP occurs in vivo, it is not a likely mechanism to explain the present results. Our findings imply that another mechanism is at least partially responsible for our results.
Another mechanism by which vasoconstriction to ,-methylene ATP may be attenuated is by altering the binding sites for ATP. Evidence in other ligand-gated channels suggests that histidine residues may interact with hydrogen ions to modify ATP binding (10, 22). However, Stoop et al. (22) and Li et al. (10) showed that diethyl pyrocarbonate, a histidine carboxylator, did not change the effect of pH on P2X2 or P2X4 receptor activity induced by ATP. It is also important to point out that P2X receptor rates of deactivation by ,-methylene ATP may be altered by protonation. Studies have shown mixed results with respect to deactivation rates such that Ralevic (15) reported no change in P2X receptor deactivation in mesenteric arteries but Li et al. (10) found decreasing deactivation rates of ATP-activated current with decreasing pH in cloned P2X2 receptors.
A third mechanism by which P2X-mediated vasoconstriction may be attenuated by hydrogen ions is by modifying the P2X ion pore (22). Stoop et al. (22) suggested that a decrease in peak current to ATP application is an indicator of a reduction in ion pore permeability rather than a modification in the ligand or ligand binding. Unique to this study, we found a significant attenuation of peak tension at pH 7.0. These data are in contrast to those of Stoop et al. (22) and Li et al. (10), who found no change in peak ATP-induced activity at low pH. It is possible that the differing results are due to differences in P2X receptor subtypes studied (10, 22). Nevertheless, our data from rodent skeletal muscle arteries suggest that hydrogen ions may reduce ion pore permeability. This possibility needs to be confirmed by further patch-clamp experiments.
One might consider that mechanisms not specific to the P2X receptor may be involved in reduced tension development with acidosis. They include activation of ATP-sensitive potassium channels and hydrogen ion interference with the contractile machinery (1, 18, 23, 24). However, the P2 antagonist PPADS abolished tension produced by ,-methylene ATP, indicating that vasoconstriction to ,-methylene ATP was mediated solely by purinergic receptors. In addition, we found no effect of pH on tension produced by the 1-agonist phenylephrine. These data argue against the possibility of a nonspecific effect of hydrogen ions on modifying channels or proteins not related to the P2X receptor (1).
The physiological significance of these findings is that they provide a potential mechanism to explain observations regarding skeletal muscle vasculature during exercise (4). Recently, our lab demonstrated (5) that P2X receptors mediate tonic vasoconstriction in exercising skeletal muscle. However, the amount of vasoconstriction to exogenously administered ,-methylene ATP was attenuated during heavy exercise. We hypothesized that the mechanism of this attenuation may be related to a change in the chemical environment during exercise. Heavy exercise can result in decreased pH, and, therefore, the current study provides a plausible mechanism for these observations (4, 5, 13, 21).
In conclusion, we found a rightward shift in the dose-response curve and a decrease in peak tension with ,-methylene ATP and acidification. The -agonist dose-response curve was unaffected by pH. To our knowledge, this is the first study to investigate the sensitivity of P2X-mediated vasoconstriction with extracellular acidification in skeletal muscle arteries. Although the mechanism requires further investigation, the results show that lower pH attenuates the vasoconstrictor response to P2X purinergic receptors. These results imply that during conditions such as exercise and peripheral arterial insufficiency where pH is reduced, diminished P2X-mediated responsiveness may influence vascular tone.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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,-methylene ATP and suramin in rat tail artery. Br J Pharmacol 110: 1421–1428, 1993.[ISI][Medline]
1- and 2-adrenoceptors. Implications for neural and metabolic regulation. Circ Res 66: 1643–1657, 1990.This article has been cited by other articles:
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  Z. Gao, J. D. Li, L. I. Sinoway, and J. Li Effect of muscle interstitial pH on P2X and TRPV1 receptor-mediated pressor response J Appl Physiol, June 1, 2007; 102(6): 2288 - 2293. [Abstract] [Full Text] [PDF]
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 A. E. Kindig, S. G. Hayes, and M. P. Kaufman Purinergic 2 receptor blockade prevents the responses of group IV afferents to post-contraction circulatory occlusion J. Physiol., January 1, 2007; 578(1): 301 - 308. [Abstract] [Full Text] [PDF]
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 H. A. Kluess, J. B. Buckwalter, J. J. Hamann, D. S. DeLorey, and P. S. Clifford Frequency and pattern dependence of adrenergic and purinergic vasoconstriction in rat skeletal muscle arteries Exp Physiol, November 1, 2006; 91(6): 1051 - 1058. [Abstract] [Full Text] [PDF]
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H. A. Kluess, J. B. Buckwalter, J. J. Hamann, and P. S. Clifford Elevated temperature decreases sensitivity of P2X purinergic receptors in skeletal muscle arteries J Appl Physiol, September 1, 2005; 99(3): 995 - 998. [Abstract] [Full Text] [PDF]
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