The activities of three adenylyl cyclases (AC1, AC3 and AC8) are stimulated by calcium in a calmodulin-dependent manner, whilst AC5 and AC6 exhibit calmodulin-independent Ca²⁺-inhibition (Guillou et al. 1999). It is possible that physiological shifts in both pH and calcium have significant consequences for AC activity in vivo. We have examined whether Ca²⁺-regulation of AC8 (Ca²⁺-stimulable) and AC6 (Ca²⁺-inhibitable) can be modulated by modest changes in pH.

Adenylyl cyclase activity was assayed in vitro using isolated cell membranes from HEK 293 cells transfected with AC8 and C6 glioma cells expressing endogenous AC6 as previously described (Boyajian et al. 1991). Calcium concentration-response curves for AC8 and AC6 activities were compared at pH 7.14, 7.50 and 7.85. At pH 7.14 basal activities decreased and there was a loss of Ca²⁺-regulation of AC6 with a marked reduction in the Ca²⁺-sensitivity of AC8. In contrast basal activities and Ca²⁺-sensitivities of both cyclases were enhanced at least 2-fold at pH 7.85. To examine the effects of intracellular pH (pH_i) changes on whole-cell measurements of cAMP accumulation a weak acid (10 mM propionate) and weak base (20 mM trimethylamine) were used to induce pH_i shifts of ~0.3 pH units measured using the fluorescent dye BCECF. Both AC8 and AC6 are sensitive to capacitative Ca²⁺ entry (CCE) rather than store Ca²⁺ release in transfected HEK 293 cells (Fagan et al. 1996) and C6 glioma cells respectively (Fagan et al. 1998). Hence cells were pre-incubated for 3 min with 100 nM thapsigargin to empty the calcium stores and cAMP production was assayed for 1 min following addition of extracellular Ca²⁺ to evoke CCE. Whilst Ca²⁺-stimulation of AC8 continued to display a similar (but weaker) dependence on pH, Ca²⁺-inhibition of AC6 was insensitive to changes in pH_i. In contrast AC6 exhibited a clear decrease in Ca²⁺-sensitivity when extracellular pH (pH_o) was reduced to pH 7.15 and potentiation of Ca²⁺-sensitivity at pH_o 7.85.

Our findings suggest that Ca²⁺ dependent stimulation of AC8 and inhibition of AC6 is potentially sensitive to ‘physiological’ changes in pH. AC8 shows enhanced sensitivity to Ca²⁺ under alkaline conditions but this effect may be minimized due to reduced CCE in HEK 293 cells when pH_i is increased. Ca²⁺ regulation of AC6 is more sensitive to shifts in pH_o than in pH_i and this may be particularly relevant in the kidney where AC6 is the major isoform.

This work was funded by the Wellcome Trust