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[HCO3-]-regulated expression and activity of soluble adenylyl cyclase in corneal endothelial and Calu-3 cells.

Sun XC, Cui M, Bonanno JA - BMC Physiol. (2004)

Bottom Line: Interestingly, BCECs pre-treated with10 microM adenosine or 10 microM forskolin, which increase cAMP levels, showed decreased sAC mRNA expression by 20% and 30%, respectively.HCO3- not only directly activates sAC, but also up-regulates the expression of sAC.These results suggest that active cellular uptake of HCO3- can contribute to the basal level of cellular cAMP in tissues that express sAC.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Optometry, Indiana University, 800 E, Atwater Ave, Bloomington, IN 47405, USA. sxingcai@indiana.edu

ABSTRACT

Background: Bicarbonate activated Soluble Adenylyl Cyclase (sAC) is a unique cytoplasmic and nuclear signaling mechanism for the generation of cAMP. HCO3- activates sAC in bovine corneal endothelial cells (BCECs), increasing [cAMP] and stimulating PKA, leading to phosphorylation of the cystic fibrosis transmembrane-conductance regulator (CFTR) and increased apical Cl- permeability. Here, we examined whether HCO3- may also regulate the expression of sAC and thereby affect the production of cAMP upon activation by HCO3- and the stimulation of CFTR in BCECs.

Results: RT-competitive PCR indicated that sAC mRNA expression in BCECs is dependent on [HCO3-] and incubation time in HCO3-. Immunoblots showed that 10 and 40 mM HCO3- increased sAC protein expression by 45% and 87%, respectively, relative to cells cultured in the absence of HCO3-. Furthermore, 40 mM HCO3- up-regulated sAC protein expression in Calu-3 cells by 93%. On the other hand, sAC expression in BCECs and Calu-3 cells was unaffected by changes in bath pH or osmolarity. Interestingly, BCECs pre-treated with10 microM adenosine or 10 microM forskolin, which increase cAMP levels, showed decreased sAC mRNA expression by 20% and 30%, respectively. Intracellular cAMP production by sAC paralleled the time and [HCO3-]-dependent expression of sAC. Bicarbonate-induced apical Cl- permeability increased by 78% (P < 0.01) in BCECs cultured in HCO3-. However for cells cultured in the absence of HCO3-, apical Cl- permeability increased by only 10.3% (P > 0.05).

Conclusion: HCO3- not only directly activates sAC, but also up-regulates the expression of sAC. These results suggest that active cellular uptake of HCO3- can contribute to the basal level of cellular cAMP in tissues that express sAC.

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The effects of HCO3--regulated sAC expression on intracellular cAMP accumulation. Culture medium was removed from confluent cultured BCECs and replaced with HCO3--free DMEM for 48 hours at 37°C. Cells were then incubated either for different time in 40 mM HCO3- DMEM or for 24 hours in different [HCO3-] at 37°C. Cells were lysed in 0.1 N HCl. [cAMP] was measured by enzyme immunoassay. A: changes in intracellular [cAMP] from cells exposed to 40 mM HCO3- with 0, 3, 6, 12, 24 and 48 hour incubation. B: Intracellular [cAMP] from cells exposed to 0, 5, 10, 20, 40, 60 and 80 mM HCO3- for 24 hour incubation. Error bars indicate ± SE (n = 6)
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Figure 5: The effects of HCO3--regulated sAC expression on intracellular cAMP accumulation. Culture medium was removed from confluent cultured BCECs and replaced with HCO3--free DMEM for 48 hours at 37°C. Cells were then incubated either for different time in 40 mM HCO3- DMEM or for 24 hours in different [HCO3-] at 37°C. Cells were lysed in 0.1 N HCl. [cAMP] was measured by enzyme immunoassay. A: changes in intracellular [cAMP] from cells exposed to 40 mM HCO3- with 0, 3, 6, 12, 24 and 48 hour incubation. B: Intracellular [cAMP] from cells exposed to 0, 5, 10, 20, 40, 60 and 80 mM HCO3- for 24 hour incubation. Error bars indicate ± SE (n = 6)

Mentions: If bicarbonate induces the up-regulation of sAC expression in BCECs, [cAMP]i should also increase with time in bicarbonate culture medium. BCECs were HCO3--starved for 48 hours and then placed in 40 mM HCO3-. As shown in figure 5A, At 0 hour, the [cAMP]i was 19.96 ± 1.22 pmol/mg protein (n = 6). At 12 hours, [cAMP]i was significantly increased to 34.79 ± 3.06 pmol/mg protein (n = 6). At 24 and 48 hours, however, [cAMP]i was 33.87 ± 3.73 (n = 6) and 36.47 ± 4.50 (n = 6) pmol/mg protein respectively, which was not significantly different relative to 12 hour incubation. Therefore, these results indicate that sAC activity reached a maximal level at 12 hours in 40 mM HCO3-, which is consistent with the maximal increase in sAC mRNA (see figure 1C). In figure 5B, BCECs were HCO3--starved for 48 hours and then placed in different [HCO3-] for 24 hours. At 0 HCO3-, the [cAMP]i was 20.40 ± 1.03 pmol/mg protein (n = 6). [cAMP]i increased with increasing [HCO3-] (K1/2 ≈ 10 mM HCO3-) and was maximal between 40 and 60 mM. Higher [HCO3-] did not produce any further significant increase in [cAMP]. Taken together the results from fig. 5A and 5B are consistent with the results from HCO3--induced sAC protein and mRNA expression.


[HCO3-]-regulated expression and activity of soluble adenylyl cyclase in corneal endothelial and Calu-3 cells.

Sun XC, Cui M, Bonanno JA - BMC Physiol. (2004)

The effects of HCO3--regulated sAC expression on intracellular cAMP accumulation. Culture medium was removed from confluent cultured BCECs and replaced with HCO3--free DMEM for 48 hours at 37°C. Cells were then incubated either for different time in 40 mM HCO3- DMEM or for 24 hours in different [HCO3-] at 37°C. Cells were lysed in 0.1 N HCl. [cAMP] was measured by enzyme immunoassay. A: changes in intracellular [cAMP] from cells exposed to 40 mM HCO3- with 0, 3, 6, 12, 24 and 48 hour incubation. B: Intracellular [cAMP] from cells exposed to 0, 5, 10, 20, 40, 60 and 80 mM HCO3- for 24 hour incubation. Error bars indicate ± SE (n = 6)
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Related In: Results  -  Collection

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Figure 5: The effects of HCO3--regulated sAC expression on intracellular cAMP accumulation. Culture medium was removed from confluent cultured BCECs and replaced with HCO3--free DMEM for 48 hours at 37°C. Cells were then incubated either for different time in 40 mM HCO3- DMEM or for 24 hours in different [HCO3-] at 37°C. Cells were lysed in 0.1 N HCl. [cAMP] was measured by enzyme immunoassay. A: changes in intracellular [cAMP] from cells exposed to 40 mM HCO3- with 0, 3, 6, 12, 24 and 48 hour incubation. B: Intracellular [cAMP] from cells exposed to 0, 5, 10, 20, 40, 60 and 80 mM HCO3- for 24 hour incubation. Error bars indicate ± SE (n = 6)
Mentions: If bicarbonate induces the up-regulation of sAC expression in BCECs, [cAMP]i should also increase with time in bicarbonate culture medium. BCECs were HCO3--starved for 48 hours and then placed in 40 mM HCO3-. As shown in figure 5A, At 0 hour, the [cAMP]i was 19.96 ± 1.22 pmol/mg protein (n = 6). At 12 hours, [cAMP]i was significantly increased to 34.79 ± 3.06 pmol/mg protein (n = 6). At 24 and 48 hours, however, [cAMP]i was 33.87 ± 3.73 (n = 6) and 36.47 ± 4.50 (n = 6) pmol/mg protein respectively, which was not significantly different relative to 12 hour incubation. Therefore, these results indicate that sAC activity reached a maximal level at 12 hours in 40 mM HCO3-, which is consistent with the maximal increase in sAC mRNA (see figure 1C). In figure 5B, BCECs were HCO3--starved for 48 hours and then placed in different [HCO3-] for 24 hours. At 0 HCO3-, the [cAMP]i was 20.40 ± 1.03 pmol/mg protein (n = 6). [cAMP]i increased with increasing [HCO3-] (K1/2 ≈ 10 mM HCO3-) and was maximal between 40 and 60 mM. Higher [HCO3-] did not produce any further significant increase in [cAMP]. Taken together the results from fig. 5A and 5B are consistent with the results from HCO3--induced sAC protein and mRNA expression.

Bottom Line: Interestingly, BCECs pre-treated with10 microM adenosine or 10 microM forskolin, which increase cAMP levels, showed decreased sAC mRNA expression by 20% and 30%, respectively.HCO3- not only directly activates sAC, but also up-regulates the expression of sAC.These results suggest that active cellular uptake of HCO3- can contribute to the basal level of cellular cAMP in tissues that express sAC.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Optometry, Indiana University, 800 E, Atwater Ave, Bloomington, IN 47405, USA. sxingcai@indiana.edu

ABSTRACT

Background: Bicarbonate activated Soluble Adenylyl Cyclase (sAC) is a unique cytoplasmic and nuclear signaling mechanism for the generation of cAMP. HCO3- activates sAC in bovine corneal endothelial cells (BCECs), increasing [cAMP] and stimulating PKA, leading to phosphorylation of the cystic fibrosis transmembrane-conductance regulator (CFTR) and increased apical Cl- permeability. Here, we examined whether HCO3- may also regulate the expression of sAC and thereby affect the production of cAMP upon activation by HCO3- and the stimulation of CFTR in BCECs.

Results: RT-competitive PCR indicated that sAC mRNA expression in BCECs is dependent on [HCO3-] and incubation time in HCO3-. Immunoblots showed that 10 and 40 mM HCO3- increased sAC protein expression by 45% and 87%, respectively, relative to cells cultured in the absence of HCO3-. Furthermore, 40 mM HCO3- up-regulated sAC protein expression in Calu-3 cells by 93%. On the other hand, sAC expression in BCECs and Calu-3 cells was unaffected by changes in bath pH or osmolarity. Interestingly, BCECs pre-treated with10 microM adenosine or 10 microM forskolin, which increase cAMP levels, showed decreased sAC mRNA expression by 20% and 30%, respectively. Intracellular cAMP production by sAC paralleled the time and [HCO3-]-dependent expression of sAC. Bicarbonate-induced apical Cl- permeability increased by 78% (P < 0.01) in BCECs cultured in HCO3-. However for cells cultured in the absence of HCO3-, apical Cl- permeability increased by only 10.3% (P > 0.05).

Conclusion: HCO3- not only directly activates sAC, but also up-regulates the expression of sAC. These results suggest that active cellular uptake of HCO3- can contribute to the basal level of cellular cAMP in tissues that express sAC.

Show MeSH
Related in: MedlinePlus