Extracellular HCO3- is sensed by mouse cerebral arteries: Regulation of tone by receptor protein tyrosine phosphatase γ

Abstract

We investigate sensing and signaling mechanisms for H(+), [Formula: see text] and CO2 in basilar arteries using out-of-equilibrium solutions. Selectively varying pHo, [[Formula: see text]]o, or pCO2, we find: (a) lowering pHo attenuates vasoconstriction and vascular smooth muscle cell (VSMC) Ca(2+)-responses whereas raising pHo augments vasoconstriction independently of VSMC [Ca(2+)]i, (b) lowering [[Formula: see text]]o increases arterial agonist-sensitivity of tone development without affecting VSMC [Ca(2+)]i but c) no evidence that CO2 has direct net vasomotor effects. Receptor protein tyrosine phosphatase (RPTP)γ is transcribed in endothelial cells, and direct vasomotor effects of [Formula: see text] are absent in arteries from RPTPγ-knockout mice. At pHo 7.4, selective changes in [[Formula: see text]]o or pCO2 have little effect on pHi At pHo 7.1, decreased [[Formula: see text]]o or increased pCO2 causes intracellular acidification, which attenuates vasoconstriction. Under equilibrated conditions, anti-contractile effects of CO2/[Formula: see text] are endothelium-dependent and absent in arteries from RPTPγ-knockout mice. With CO2/[Formula: see text] present, contractile responses to agonist-stimulation are potentiated in arteries from RPTPγ-knockout compared to wild-type mice, and this difference is larger for respiratory than metabolic acidosis. In conclusion, decreased pHo and pHi inhibit vasoconstriction, whereas decreased [[Formula: see text]]o promotes vasoconstriction through RPTPγ-dependent changes in VSMC Ca(2+)-sensitivity. [Formula: see text] serves dual roles, providing substrate for pHi-regulating membrane transporters and modulating arterial responses to acid-base disturbances.

Keywords: Vascular biology; basic science; calcium imaging; confocal microscopy; electrophysiology; endothelium; experimental; pH; physiology; receptors; smooth muscle.

Figure 1.

Application of OOE CO₂/${\text{HCO}}_3^{-}$ solutions to small-artery myography. (a) Schematic showing generation of OOE solutions by rapid mixing and delivery of differently composed CO₂/${\text{HCO}}_3^{-}$ solutions. The time delay from the point of mixing to the artery is <100 ms. See Supplemental Methods for details. (b) Force recordings from a mesenteric artery exposed to increasing concentrations of norepinephrine (NE) under OOE conditions (pH_o=7.7, [${\text{HCO}}_3^{-}$]_o=22 mM, CO₂=5%).

Figure 2.

Selective changes in [${\text{HCO}}_3^{-}$]_o or pH_o—but not pCO₂—directly modulate basilar artery tone under OOE conditions. (a) Effects of selectively varying pH_o, [${\text{HCO}}_3^{-}$]_o or pCO₂ (maintaining other two at control levels) on VSMC pH_i in resting basilar arteries (left panel) or basilar arteries contracted by 10 µM U46619 (right panel). Under “Control” conditions, CO₂ is 5%, pH_o 7.4, and [${\text{HCO}}_3^{-}$]_o 22 mM. Compared to “Control”, “Low” refers to selective changes in CO₂ to 2.5%, [${\text{HCO}}_3^{-}$]_o to 11 mM or pH_o to 7.1, and “High” refers to selective changes in CO₂ to 10%, [${\text{HCO}}_3^{-}$]_o to 44 mM or pH_o to 7.7. Arteries are from wild-type mice (n = 6). (b) Effects of selectively varying pH_o, [${\text{HCO}}_3^{-}$]_o or pCO₂ (maintaining other two at control levels) on VSMC calculated [${\text{HCO}}_3^{-}$]_i in resting (Continued) Figure 2. Continue. basilar arteries (left panel) or basilar arteries contracted by 10 µM U46619 (right panel). Arteries are from wild-type mice (n = 6). (c-e) Effects of selectively varying pH_o, [${\text{HCO}}_3^{-}$]_o, or pCO₂ (maintaining other two at control levels) on U46619-induced tension development in basilar arteries from wild-type mice (n = 12 or 13). (f, g) Effects of selectively varying pH_o or [${\text{HCO}}_3^{-}$]_o (maintaining unvaried parameters at control levels) on U46619-induced VSMC Ca²⁺-responses in basilar arteries from wild-type mice (n = 6 for both). (h) Effects of selective changes in pH_o ([${\text{HCO}}_3^{-}$]_o=22 mM, CO₂=5%) on depolarization-induced tension development of basilar arteries from wild-type mice (n = 8). We induce depolarization by raising [K⁺]_o. (i) Effects of selective changes in pH_o ([${\text{HCO}}_3^{-}$]_o=22 mM, CO₂=5%) on depolarization-induced VSMC Ca²⁺ responses in basilar arteries from wild-type mice (n = 7). The curves in panels (c) through (i) are the results of least-squares fits to sigmoidal functions, and we compare them using extra sum-of-squares F-tests. *P < 0.05, **P < 0.01, ***P < 0.001, NS: not significantly different vs. control conditions (pH_o=7.4, [${\text{HCO}}_3^{-}$]_o=22 mM, CO₂=5%).

Figure 3.

Increasing pCO₂ at low pH_o (pH 7.1) attenuates tension production of basilar arteries under OOE conditions. (a) Effects of selectively varying [${\text{HCO}}_3^{-}$]_o or pCO₂ (maintaining the other at its control level, with pH_o fixed at 7.1) on VSMC pH_i in resting basilar arteries (left panel) or basilar arteries contracted by 10 µM U46619 (right panel). “Low” and “High” refer to half and twice the concentration, respectively, of CO₂ and ${\text{HCO}}_3^{-}$ compared to “Control” levels ([${\text{HCO}}_3^{-}$]_o=22 mM, CO₂=5%). The reference pH_i value on the y-axis, “(6.99)”, corresponds to control levels of all three parameters (pH_o=7.4, [${\text{HCO}}_3^{-}$]_o=22 mM, CO₂=5%) in the same arteries from wild-type mice (n = 6). (b) Effects of selectively varying [${\text{HCO}}_3^{-}$]_o or pCO₂ (maintaining the other at its control level, with pH_o fixed at 7.1) on VSMC calculated [${\text{HCO}}_3^{-}$]_i in resting basilar arteries (left panel) or basilar arteries contracted by 10 µM U46619 (right panel). The reference [${\text{HCO}}_3^{-}$]_i value on the y-axis, “(8.74 mM)”, corresponds to control levels of all three parameters (pH_o=7.4, [${\text{HCO}}_3^{-}$]_o=22 mM, CO₂=5%) in the same arteries from wild-type mice (n = 6). (c, d) Effects of selectively varying [${\text{HCO}}_3^{-}$]_o or pCO₂ (maintaining the other at its control level, with pH_o fixed at 7.1) on U46619-induced tension development in basilar arteries from wild-type mice (n = 6 in panel (c), n = 11 in panel (d)). For comparison, in panel (c), we show the reference tension response (solid gray curve) in the same arteries, under conditions that correspond to control levels of all three parameters (pH_o=7.4, [${\text{HCO}}_3^{-}$]_o=22 mM, CO₂=5%). (e) Effects of selectively varying pCO₂ (fixed pH_o=7.1, [${\text{HCO}}_3^{-}$]_o=22 mM) on U46619-induced VSMC Ca²⁺ responses in basilar arteries from wild-type mice (n = 5). The curves in panel (c) through (e) are the results of least-squares fits to sigmoidal functions, and we compare them using extra sum-of-squares F-tests. *P < 0.05, **P < 0.01, NS: not significantly different vs. control acidic conditions (pH_o=7.1, [${\text{HCO}}_3^{-}$]_o=22 mM, CO₂=5%).

Figure 4.

Ptprg transcriptional activity is prominent in ECs of basilar arteries. (a, b) Basilar arteries from an RPTPγ-knockout (KO, panel (a)) and a wild-type (WT, panel (b)) mouse, stained histochemically for β-galactosidase activity. Each image is representative of five experiments. (c, d) Histological sections (8 µm thick) of basilar arteries from an RPTPγ-knockout and a wild-type mouse, stained histochemically for β-galactosidase activity. Each image is representative of three experiments. The size bars in panels (a) and (b) represent 100 µm, in (c) and (d) 10 µm. Lu indicates lumen, Adv indicates adventitia and EC indicates endothelial cell.

Figure 5.

Basilar arteries from RPTPγ-knockout mice are insensitive to changes in [${\text{HCO}}_3^{-}$]_o. (a, b) Effects of selectively varying pH_o or [${\text{HCO}}_3^{-}$]_o (maintaining the other at its control level, with CO₂ fixed at 5%) on U46619-induced tension development in basilar arteries from RPTPγ-knockout mice (n = 4 in panel (a), n = 8 in panel (b)). (c) Effects of selectively varying [${\text{HCO}}_3^{-}$]_o (maintaining pH_o at 7.4, CO₂ at 5%) on U46619-induced VSMC Ca²⁺-responses in basilar arteries from RPTPγ-knockout mice (n = 8). (d) Effects of selectively varying [${\text{HCO}}_3^{-}$]_o at low pH_o (maintaining pH_o at 7.1, CO₂ at 5%) on U46619-induced tension development of basilar arteries from RPTPγ-knockout mice (n = 6). (e) Effects of selectively varying [${\text{HCO}}_3^{-}$]_o at low pH_o (maintaining pH_o at 7.1, CO₂ at 5%) on U46619-induced VSMC Ca²⁺-responses in basilar arteries from RPTPγ-knockout mice (n = 4). (f-i). Effects of omitting CO₂/${\text{HCO}}_3^{-}$ on U46619-induced tension development at constant pH_o of 7.4. We perform experiments with basilar arteries from wild-type (WT, panels (f) and (h)) and RPTPγ-knockout (KO, panels (g) and (i)) mice. In panel (h) and (i), we inhibit vasorelaxant effects of the endothelium by incubation with 100 µM L-NAME, 3 µM indomethacin, 50 nM apamin and 1 µM TRAM-34. To avoid development of resting tone after inhibition of endothelium-dependent vasorelaxation, we add 600 nM SNAP to achieve an equal [NO] in the tested arteries. The curves are the results of least-squares fits to sigmoidal functions, and we compare them using extra sum-of-squares F-tests. *P < 0.05, **P < 0.01, ***P < 0.001, NS: not significantly different vs. control conditions (panels (a–c) and (f–i): pH_o=7.4, [${\text{HCO}}_3^{-}$]_o=22 mM, CO₂=5%) or standard acidic conditions (panels (d) and (e): pH_o=7.1, [${\text{HCO}}_3^{-}$]_o=22 mM, CO₂=5%).

Figure 6.

Under equilibrated conditions, RPTPγ is required for ${\text{HCO}}_{3\text{o}}^{-}$ sensing—such that basilar arteries from RPTPγ-knockout mice are hypercontractile—and vasorelaxation in response to metabolic acidosis is explained by a combination of attenuated Ca²⁺ uptake and reduced Ca²⁺-sensitivity. (a–e) Comparison of U46619-induced contractile responses of basilar arteries from RPTPγ-knockout (KO, n=8) vs. wild-type (WT, n = 10) mice. Panel (a) summarizes experiments under control conditions (pH_o=7.4, [${\text{HCO}}_3^{-}$]_o=22 mM, CO₂=5%); panel (b), under respiratory acidosis (pH_o=7.1, [${\text{HCO}}_3^{-}$]_o=22 mM, CO₂=10%); and panel (d), under metabolic acidosis (pH_o=7.1, [${\text{HCO}}_3^{-}$]_o=11 mM, CO₂=5%). Panels (c) and (e) summarize the average differences in U46619-induced contractile responses in basilar arteries from RPTPγ-knockout and wild-type mice during respiratory (panel c) and metabolic (panel e) acidosis compared to control conditions. (f) Representative time courses of arterial tension development and VSMC Ca²⁺-dependent fluorescence signal in Ca²⁺-depleted basilar arteries from RPTPγ-knockout mice in response to step-increases of [Ca²⁺]_o during constant exposure to 10 µM U46619. We perform experiments under control conditions (pH_o=7.4, [${\text{HCO}}_3^{-}$]_o=22 mM, CO₂=5%) and during metabolic acidosis (pH_o=7.1, [${\text{HCO}}_3^{-}$]_o=11 mM, CO₂=5%). The dots indicate step-wise increments in [Ca²⁺]_o from 0 to 0.05, 0.1, 0.2, 0.4, 0.8 and 1.6 mM. (g) Average ${\text{Ca}}_{\text{i}}^{2+}$-responses from experiments (n = 5) like that in panel (f). (h) Average tension development of basilar arteries from RPTPγ-knockout mice (n = 5) as a function of the VSMC ${\text{Ca}}_{\text{i}}^{2+}$-response under control (Continued) Figure 6. Continue. conditions (pH_o=7.4, [${\text{HCO}}_3^{-}$]_o=22 mM, CO₂=5%) and during metabolic acidosis (pH_o=7.1, [${\text{HCO}}_3^{-}$]_o=11 mM, CO₂=5%). The curves in panels (a), (b), (d) and (g) are the results of least-squares fits to sigmoidal functions; in panels (a) and (g), we compare them using extra sum-of-squares F-tests. The data points in panels (c), (e) and (h) are compared by repeated-measures two-way ANOVA; in panel (h), we report the significance level based on Bonferroni post-tests. *P < 0.05, **P < 0.01, ***P < 0.001, NS: not significantly different vs. wild-type (panels (a), (c) and (e)) or control conditions (panels (g) and (h)).

Figure 7.

Schematic of proposed signaling pathways affecting cerebral vascular tone in response to acid-base disturbances. Note that in the cases of endothelium-mediated RPTPγ-dependent actions of ${\text{HCO}}_{3\text{o}}^{-}$ and the effect of ${\text{H}}_{\text{o}}^{+}$ acting on Ca²⁺-sensitivity (which could occur via a parallel change in pH_i), we have only seen effects with reducing the concentrations. RPTPγ in the vascular smooth muscle cell is shown in gray because we have evidence for expression but no function has yet been demonstrated. VDCC, voltage-dependent Ca²⁺-channels; VSMC, vascular smooth muscle cell.