The effect of angiotensin II (ANG II) on H⁺-ATPase activity is controversial. In proximal [1] and distal [2] rat kidney tubules and in intercalated cells of connecting tubule [3], cortical [3,4] and medullary [5] collecting duct ANG II (10⁻¹² - 10⁻⁹ M) stimulates H⁺-ATPase. However, in the rat cortical [6] and outer medullary [7] collecting duct, ANG II (10⁻¹⁰ - 10⁻⁵ M) causes a dosedependent decrease in H⁺-ATPase activity. In addition, our studies in MDCK cells, a permanent cell line originated from the renal collecting duct, indicate that after intracellular pH (pHi) acidification using an NH₄Cl pulse, ANG II (10⁻¹², 10⁻⁹ or 10⁻⁷ M) stimulates H⁺-ATPase in a dose-dependent manner by increasing the cytosolic free calcium ([Ca²⁺]i). In agreement with these results, atrial natriuretic peptide (ANP) or dimethyl-BAPTA/AM (BAPTA, chelates intracellular calcium) alone decreases the [Ca²⁺]i levels but does not affect the H⁺-ATPase activity; however, these compounds interfere with the pathway responsible for the increase in [Ca²⁺]i, blocking the stimulatory effect of ANG II on H⁺-ATPase [8].

The effect of arginine vasopressin (AVP) on H⁺-ATPase activity is unclear. In an in vivo microperfusion study, we demonstrated that in the late distal tubule of rat kidney, luminal AVP (10⁻⁹ M) stimulates H⁺-ATPase via the activation of V1 receptors [9]. In principal and intercalated cell of rabbit cortical collecting duct, AVP increases cAMP accumulation [10]; however, it has been suggested that in this duct, luminal AVP (10⁻⁹ M) impairs electrogenic H⁺ secretion [11] and in rat medullary thick ascending limb cells, AVP does not affect H⁺-ATPase directly [12]. In addition, our data with MDCK cells suggest that the increase in [Ca²⁺]i and cAMP plays a role in regulating the dose-dependent stimulatory effect of AVP on H⁺-ATPase after pHi acidification, via V1 and V2 receptor mediated pathways; in agreement with these data, ANP or BAPTA inhibits the increase in [Ca²⁺]i in response to AVP and blocks the stimulatory effect of AVP on H⁺-ATPase [13].

Moreover, it is known that: 1) acute cellular acidification stimulates exocytosis and elicits a rapid increase in proton secretion that is mediated by an H⁺-ATPase [14]. 2) an increase in [Ca²⁺]i might reflect a physiological mechanism to stimulate H⁺-ATPase-mediated protein export under acidic conditions [15,16]. 3) cAMP stimulates V-ATPase accumulation, microvillar elongation, and proton extrusion in kidney collecting duct A-intercalated cells [17] and 4) vesicle trafficking and exocytosis play a role in the regulation of H⁺ transport in MDCK cells [18,19].

Based on these findings, in the present study we investigated the effect of ANG II (10⁻¹² and 10⁻⁷ M) or AVP (10⁻¹² and 10⁻⁶ M) alone or plus ANP (10⁻⁶ M) on the subcellular acidic vesicle trafficking in MDCK cells following intracellular acidification using NH₄Cl. The experiments were performed in a Na⁺-free solution containing Schering 28080 (specifically inhibits H⁺/K⁺-ATPase), experimental conditions under which H⁺-ATPase is the only mechanism for pHi recovery in the MDCK cells [13]. To determine if blocking the increase in [Ca²⁺]i affects the ANG II or AVP stimulatory effect on acidic vesicle trafficking, BAPTA (5 × 10⁻⁵ M) was added to the cells. Additionally, we investigated the interaction of ANG II or AVP plus colchicine (10⁻⁵ M, 2-h preincubation; inhibits microtubule-dependent vesicular trafficking [20]) on the acidic vesicle movement. Our data indicate that there is a dose-dependent stimulatory effect of ANG II or AVP on H⁺-ATPase subcellular vesicle trafficking, which is impaired by ANP or BAPTA. The results suggest a role for the [Ca²⁺]i in the regulation of these hormonal effects; whether [Ca²⁺]i is a direct mechanism or is a side effect of other signaling pathways which will require additional studies. Colchicine also abolishes these hormonal effects, suggesting that micro-tubuledependent H⁺-ATPase vesicular trafficking is involved in the stimulatory effect of ANG II or AVP on the pHi recovery mediated by H⁺-ATPase and impaired by ANP.

Figure 3 shows the fluorescent cytoplasmic vesicles in an MDCK cell preincubated under the preliminary experimental conditions, i.e., in the Na⁺-free solution containing Schering 28080 and in the absence of acid loading. The data indicate that acridine orange is taken up rapidly by the cell, concentrates in cytoplasmic vesicles and is not lost during the initial 12 min of the experiment (upper) and that ANG II (10⁻¹² M) increases the number of vesicles that can be detected in the cytoplasm (lower).

Figures 1 and 2 demonstrate the movement of the fluorescent vesicles inside the cells during the pHi recovery period following the NH₄Cl loading. As the pHi recovery proceeded, under the control conditions the largest concentration of fluorescence was observed at the basolateral side of the cells over time, and with ANG II (10⁻¹² and 10⁻⁷ M) the fluorescence density at the apical pole of the cells exhibited a dose-dependent increase

with time (Figure 1(a)). However, an increase in the fluorescence density at the apical pole was not observed with ANP alone or in combination with ANG II (10⁻¹² and 10⁻⁷ M) (Figure 1(b)). As the pHi recovery proceeded in the presence of AVP (10⁻¹² and 10⁻⁶ M) the fluorescence density at the apical pole of the cells exhibited a dose-dependent increase with time (Figure 2(a)); nevertheless, it did not increase in the presence of AVP (10⁻¹² and 10⁻⁶ M) plus ANP (Figure 2(b)).

In addition, the results also indicate that, similar to what occurs with ANP, in the presence of BAPTA or colchicine alone or plus ANG II or AVP, as the pHi recovery proceeded, the largest concentration of fluorescence was observed at the basolateral side of the cells, suggesting that the movement of the vesicles toward the apical pole was inhibited (Figures not shown).

Figure 4 ((a) upper and (b) upper) shows that, as the pHi recovery proceeded, under the control conditions the apical/basolateral fluorescence density ratio (FDap/ FDbl) remained almost constant over time. Already with ANG II (10⁻¹² and 10⁻⁷ M) the FDap/FDbl increased markedly, particularly at 10⁻⁷ M ANG II (Figure 4(a) upper); however, in the presence of ANG II (10⁻⁷ M) in combination with ANP, BAPTA or colchicine the (FDap/FDbl) demonstrated only minor changes over time (Figure 4(a) lower). In the presence of AVP (10⁻¹² and 10⁻⁶ M), the FDap/FDbl exhibited a dose-dependent increase with time (Figure 4(b) upper); nevertheless, with AVP (10⁻⁶ M) plus ANP, BAPTA or colchicine the FDap/FDbl did not change significantly with time (Figure 4(b) lower). The mean slopes (changes in FDap/FDbl over time) of the lines shown in Figure 4 were calculated during the first 5.30 min of the pHi recovery period. Table 1 indicates that under the control conditions the mean slope was 0.079 ± 0.0033 min⁻¹ (n = 14), and that it increased significantly in the presence of ANG II, in a dose-dependent manner. However, this increase was not observed with ANP, BAPTA or colchicine alone or in combination with ANG II. Table 1 also demonstrates that in the presence of AVP the mean slopes increased markedly, in a dose-dependent manner. Yet, similar to ANG II, this increase was not observed with AVP plus ANP, BAPTA or colchicine.