Different rate-limiting activities of intracellular pH regulators for HCO3- secretion stimulated by forskolin and carbachol in rat parotid intralobular ducts

Abstract

Intracellular pH (pH_i) regulation fundamentally participates in maintaining HCO₃^- release from HCO₃^--secreting epithelia. We used parotid intralobular ducts loaded with BCECF to investigate the contributions of a carbonic anhydrase (CA), anion channels and a Na⁺-H⁺ exchanger (NHE) to pH_i regulation for HCO₃^- secretion by cAMP and Ca²⁺ signals. Resting pH_i was dispersed between 7.4 and 7.9. Forskolin consistently decreased pH_i showing the dominance of pH_i-lowering activities, but carbachol gathered pH_i around 7.6. CA inhibition suppressed the forskolin-induced decrease in pH_i, while it allowed carbachol to consistently increase pH_i by revealing that carbachol prominently activated NHE via Ca²⁺-calmodulin. Under NHE inhibition, forskolin and carbachol induced the remarkable decreases in pH_i, which were slowed predominantly by CA inhibition and by CA or anion channel inhibition, respectively. Our results suggest that forskolin and carbachol primarily activate the pH_i-lowering CA and pH_i-raising NHE, respectively, to regulate pH_i for HCO₃^- secretion.

Keywords: Bicarbonate secretion; Carbonic anhydorase; Cl− channels; Intracellular pH; Na+–H+ exchanger; Rat parotid intralobular ducts.

Fig. 1

Forskolin + IBMX-induced decrease in the pH_i. a Time courses of the pH_i changes in rat parotid intralobular ducts by the addition of 10 μM forskolin + 100 μM IBMX (F+I). Each point indicates the mean ± SE of pH_i in 19–28 regions of the ducts. The pH_i level was reduced by the addition of F+I in all the ducts tested. b Relationships between the F+I-induced pH_i changes averaged during 4–5 min after F+I addition (ΔpH) and the resting pH_i levels averaged for 1 min just before F+I addition. Each point was obtained from the data in a (n = 9). c The pH_i averaged for 1 min just before F+I addition (control) and 4–5 min after F+I application (F+I). Data shown are mean ± SE calculated from the traces in a (n = 9). d A time course of pH_i change in the duct by F+I addition of under the inhibition of carbonic anhydrases by 1 mM methazolamide (Met). Each point indicates the mean ± SE of pH_i in 20 regions of the duct. e The effect of 1 mM methazolamide on the F+I-induced pH_i change. The pH_i averages for 1 min just before (control, pH_i; 7.57–7.83) and 4–5 min after (Met) the addition of methazolamide, and 4–5 min after the subsequent addition of F+I (Met + F+I) are denoted. Data shown are mean ± SE (n = 10). f A time course of pH_i change in the duct by F+I addition under the blocking of CFTR Cl⁻ channels by 10 μM CFTR inhibitor 172 (CFTR172). Each point indicates the mean ± SE of pH_i in 17 regions of the duct. g The pH_i averages for 1 min just before (control, pH_i; 7.39–7.91) and 4–5 min after the addition of the CFTR inhibitor (CFTR172), and 4–5 min after the subsequent addition of F+I (CFTR172 + F+I) are shown. Data shown are mean ± SE (n = 5). h Addition of 10 μM CFTR inhibitor 172 (CFTR172) almost completely blocked the inward current induced by 10 μM forskolin + 100 μM IBMX (F+I). The current was measured by using the gramicidin-perforated patch techniques in a single ductal cell. Small and brief voltage pulses (5 mV, 0.2 s) were superimposed on the holding potential (−80 mV) in every 10 s. The broken line indicates the zero level of the current. Similar results were obtained in the other 4 experiments

Fig. 2

Effects of 5(N,N)-dimethyl amiloride (DMA) and its combination with CFTR inhibitor 172 (CFTR172) or methazolamide on forskolin + IBMX (F+I)-induced pH_i changes. a A time course of pH_i change during the applications of DMA and DMA + forskolin + IBMX. The pH_i was decreased by the addition of 20 μM DMA and further decreased by the subsequent addition of 10 μM forskolin + 100 μM IBMX (F+I). Each point indicates the mean ± SE of pH_i in 19 regions of the duct. b The pH_i averages for 1 min just before (control) and 9–10 min after DMA addition (DMA), and 4–5 min after the subsequent addition of F+I (DMA + F+I). Data shown are mean ± SE (n = 6). c A representative trace of pH_i change during the applications of DMA + CFTR172 (10 μM) and DMA + CFTR172 + F+I. Each point indicates the mean ± SE of pH_i in 13 regions of the duct. d The pH_i averages for 1 min just before (control) and 9–10 min after (DMA + CFTR172) the addition of DMA + CFTR inhibitor 172, and 4–5 min after the subsequent F+I addition (DMA + CFTR172 + F+I). Data shown are mean ± SE (n = 7). e A time course of pH_i change during the applications of DMA + 1 mM methazolamide (DMA + Met) and DMA + Met + F+I. Each point indicates the mean ± SE of pH_i in 14 regions of the duct. f The pH_i averages for 1 min just before (control) and 9–10 min after (DMA + Met) the addition of DMA + methazolamide, and 4–5 min after the subsequent F+I addition (DMA + Met + F+I). Data shown are mean ± SE (n = 6). g The maximal rates of F+I-induced decreases in pH_i (−ΔpH per 10 s) in the presence of DMA. Simultaneous addition of CFTR172 with DMA significantly reduced the rate, and the addition of methazolamide reduced the rate more effectively. *P < 0.05, ***P < 0.001 in the unpaired Student’s t test. Data shown are mean ± SE (n = 6, 7, and 6, respectively)

Fig. 3

Evaluation of the contribution of carbonic anhydrases and CFTR Cl⁻ channels to forskolin + IBMX-induced pH_i changes. a–c Relationships between the averaged rates of pH_i decreases (−ΔpH per 10 s) versus the corresponding, averaged pH_i during the pH_i-decreasing phase by 10 μM forskolin + 100 μM IBMX (F+I) stimulation in the presence of 5(N,N)-dimethyl amiloride (DMA; 20 μM), and its combination with CFTR inhibitor 172 (CFTR172; 10 μM) or methazolamide (Met; 1 mM). The averaged rates of pH_i decreases were plotted at 10 s intervals as the pH_i was decreased by F+I stimulation with the starting point indicated as F+I added. Data shown are mean ± SE (n = 6, 7, and 6, respectively). d The rates of F+I-induced decreases in pH_i (−ΔpH per 10 s) at pH_i of 7.27 in the presence of DMA. Simultaneous addition of CFTR172 with DMA reduced the rate, and the addition of methazolamide reduced the rate more effectively (n = 6, 7, and 6, respectively). The rate at pH_i 7.27 was obtained by using linear interpolation between two data points of the means, as indicated by an arrow, in (a–c) of each condition

Fig. 4

pH_i changes during the stimulation by 10 μM carbachol (CCh) in rat parotid ducts. a Time courses of pH_i changes induced by CCh addition. pH_i dispersion in the resting state became smaller after the addition of CCh. pH_i traces for 30 ducts are shown. b Relationships between the CCh-induced pH_i changes (ΔpH) and the resting pH_i levels. Each point was obtained from the data in (a) (n = 30)

Fig. 5

Effects of blocking H⁺ generation by methazolamide, Ca²⁺ chelation and calmodulin inhibition on 10 μM carbachol (CCh)-induced pH_i changes. a A time course of CCh-induced pH_i change in the presence of 1 mM methazolamide (Met). CCh evoked an increase in pH_i in all the ducts tested in the presence of methazolamide. Each point indicates the mean ± SE of pH_i in 20 regions of the duct. b The pH_i averaged for 1 min just before (control) and 4–5 min after (Met) the addition of 1 mM methazolamide and 4–5 min after 10 μM CCh addition (Met + CCh). The pH_i was increased clearly by CCh in the presence of methazolamide. Data shown are mean ± SE (n = 5). c A representative trace of CCh-induced pH_i change in the absence of the external Ca²⁺ (Ca free) and the presence of 1 mM methazolamide (Met) in the duct loaded with the Ca²⁺-chelating agent, BAPTA (50 μM BAPTA-AM for 15 min at 37 °C). Each point indicates the mean ± SE of pH_i in 18 regions of the duct. d Loading BAPTA into the ducts and external Ca²⁺ removal significantly reduced the CCh-induced increase in pH_i, comparing the data shown in b (Met + CCh) and d (Ca free + Met + CCh) (P < 0.05 in the unpaired Student’s t test, n = 5 and 7). The data in d are shown as mean ± SE. e A time course of CCh-induced pH_i change in the presence of the calmodulin inhibitor, W-7 (W7, 100 μM). Each point indicates the mean ± SE in 24 regions of the ducts. f W-7 suppressed the CCh-induced increase in pH_i in the presence of 1 mM methazolamide (Met). Data shown are mean ± SE (n = 5)

Fig. 6

Effects of 5(N,N)-dimethyl amiloride (DMA) and its combination with diphenylamine-2-carboxylate (DPC) or methazolamide on carbachol (CCh)-induced pH_i changes. a A time course of pH_i change during the applications of DMA and DMA + CCh. The pH_i was decreased by the addition of 20 μM DMA and further decreased by the subsequent addition of 10 μM CCh. Each point indicates the mean ± SE of pH_i in 16 regions of the duct. b The pH_i averages for 1 min just before (control) and 9–10 min after DMA addition (DMA), and 4–5 min after the subsequent addition of CCh (DMA + CCh). Data shown are mean ± SE (n = 7). c A representative trace of pH_i change during the applications of DMA + DPC (200 μM) and DMA + DPC + CCh. Each point indicates the mean ± SE of pH_i in 17 regions of the duct. d The pH_i averages for 1 min just before (control) and 9–10 min after (DMA + DPC) the addition of DMA + DPC, and 4–5 min after the subsequent CCh addition (DMA + DPC + CCh). Data shown are mean ± SE (n = 6). e A time course of pH_i change during the applications of DMA + 1 mM methazolamide (DMA + Met) and DMA + Met + CCh. Each point indicates the mean ± SE of pH_i in 18 regions of the duct. f The pH_i averages for 1 min just before (control) and 9–10 min after (DMA + Met) the addition of DMA + methazolamide, and 4–5 min after the subsequent CCh addition (DMA + Met + CCh). Data shown are mean ± SE (n = 5). g The maximal rates of CCh-induced decreases in pH_i (−ΔpH per 10 s) in the presence of DMA. Simultaneous addition of either DPC or methazolamide with DMA reduced the rate. ***P < 0.001 in the unpaired Student’s t test. Data shown are mean ± SE (n = 7, 6, and 5, respectively)

Fig. 7

Evaluation of the contribution of carbonic anhydrases and diphenylamine-2-carboxylate (DPC)-sensitive Cl⁻ channels to carbachol (CCh)-induced pH_i changes. a–c Relationships between the averaged rates of pH_i decreases (−ΔpH per 10 s) versus the corresponding, averaged pH_i during the pH_i-decreasing phase by 10 μM CCh stimulation in the presence of 5(N,N)-dimethyl amiloride (DMA; 20 μM), and its combination with 200 μM DPC or methazolamide (Met; 1 mM). The averaged rates of pH_i decreases were plotted at 10-s intervals as the pH_i was decreased by CCh stimulation with the starting point indicated as CCh added. Data shown are mean ± SE (n = 7, 6, and 5, respectively). d The rates of CCh-induced decreases in pH_i (−ΔpH per 10 s) at pH_i of 7.27 in the presence of DMA. Simultaneous addition of either DPC or methazolamide with DMA reduced the rate (n = 7, 6, and 5, respectively). The rate at pH_i of 7.27 was obtained by using linear interpolation between two data points of the means, as is indicated by an arrow, in the figure (a–c) of each condition

Fig. 8

Schematic illustration for pH_i regulation during HCO₃ ⁻ secretion in rat parotid intralobular ducts. Sympathetic β-adrenergic stimulation induces activation of the carbonic anhydrase (CA) rather than the Na⁺–H⁺ exchanger (NHE), whereas parasympathetic muscarinic stimulation activates NHE on the basolateral membrane at least partially via Ca²⁺-calmodulin. Both mechanisms facilitate HCO₃ ⁻ secretion through the Cl⁻ channels activated by the stimulations. Thick arrows indicate the rate-limiting activation