Together, CP-868596 price these data demonstrate that K+-induced HCO3− entry through NBC activates sAC and leads to the generation of physiologically significant levels of cAMP in cultured astrocytes.

We examined whether HCO3−-sensitive sAC was functionally active in astrocytes in brain slices by directly measuring the sAC-dependent production of cAMP using ELISA. We first used two-photon microscopy to image the pH-sensitive dye 2′,7’-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)/AM to confirm previous reports that high [K+]ext causes widespread astrocyte alkalinization by HCO3− entry (Bevensee et al., 2000; Boyarsky et al., 1993; Pappas and Ransom, 1994; Schmitt et al., 2000) (Figure S4). To provide definitive evidence that the high K+-induced increase in cAMP in the brain was due to activation of sAC, we compared cAMP responses between wild-type and sAC-C1 KO mice. The cAMP levels were significantly increased by raising [K+]ext to 10 mM only in brain slices from wild-type mice (2.5 K+: 6.03 ± 0.26 pmol/ml, n = 7; 10 mM K+: 8.94 ± 0.29 pmol/ml, n = 7, p < 0.001; Figure 3A); in brain slices

FDA approved Drug Library from KO mice, there was no change in cAMP when [K+]ext was raised to 10 mM (2.5 K+: 6.21 ± 0.44 pmol/ml, n = 7; 10 mM K+: 6.03 ± 0.59 pmol/ml, n = 7, p > 0.05; Figure 3A). Next, we examined whether the increase in cAMP in high [K+]ext required HCO3− by comparing the increase when NaHCO3 was removed and brain slices were maintained in a HEPES buffer. In control rat brain slices, raising [K+]ext to 10 mM for 20 min significantly increased the cAMP level (2.5 mM K+: 4.3 ± 0.5 pmol/ml, n = 4; 10 mM K+: 7.5 ± 0.2 pmol/ml, n = 4, p < 0.001; Figure 3B). Similar to our observations in cultured astrocytes, this increase in cAMP was dependent upon extracellular HCO3− and was not observed in matched brain slices in HEPES (2.5 K+: 4.4 ± 0.4 pmol/ml, n = 4; 10 K+: 4.5 ± 0.2 pmol/ml, n = 4, p > 0.05; Figure 3B). The high K+-induced increase in cAMP was significantly Non-specific serine/threonine protein kinase reduced by the sAC-specific inhibitors 2-OH (4.6 ± 0.4 pmol/ml, n = 5, p < 0.001; Figure 3C)

and KH7 (10 μM) (Hess et al., 2005) (4.5 ± 0.6 pmol/ml, n = 5, p < 0.001; Figure 3C) but not by the tmAC inhibitor DDA (9.2 ± 0.6 pmol/ml, n = 5, p > 0.05; Figure 3C). As a negative control for 2-OH, we also determined that 17β-estradiol, an estrogen parent compound that is ineffective on sAC (Hallows et al., 2009), did not reduce the high K+-induced increase in cAMP (17β-estradiol, 20 μM, 9.1 ± 1.3 pmol/ml, n = 5, p > 0.05; Figure 3C). Furthermore, 2-OH had no effect on cAMP production mediated by the activation of beta-adrenoceptors using isoproterenol (100 μM) or norepinephrine (NE, 10 μM) (Figure 3D), receptors that signal via tmACs, confirming that under these conditions, 2-OH is specific for sAC.