INTRODUCTION

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IT HAS BEEN RECENTLY ESTABLISHED that the extracellular calcium ion (Ca²⁺_e) behaves as a ligand for specific G protein-coupled receptors (1). The mRNAs coding for a Ca²⁺-sensing receptor, and more recently, the corresponding protein, have been found in the rat kidney where they are predominantly located in the two main sites of renal Ca²⁺ reabsorption, namely, the cortical part (C) of the thick ascending limb (TAL) and the distal convoluted tubule (18, 19, 23).

In addition to the activation of phospholipase C reported in oocytes, the Ca²⁺-sensing receptor seems to couple several transduction pathways, as described in parathyroid cells and transfected cell lines (12). In TAL, Ca²⁺-sensing receptor activation was shown 1) to trigger intracellular Ca²⁺ mobilization (3, 17), 2) to induce production of arachidonic acid metabolites via the cytochrome P-450 pathway (21, 22), and 3) to decrease the antidiuretic hormone (ADH)-stimulated cAMP accumulation (7, 13, 20). This last observation is in agreement with the recent characterization of the Ca²⁺-inhibitable (type 6) adenylyl cyclase in the rat TAL (2).

We undertook the present work to characterize the effect of activating the Ca²⁺-sensing receptor on the reabsorptive function of CTAL and the implied transduction pathways. Since it has been well established that Ca²⁺ and Mg²⁺ are passively reabsorbed in CTAL, driven by a transepithelial voltage that is tightly associated to Cl reabsorption (8), only this last parameter has been determined in the present study. The main results indicate that activation of the Ca²⁺-sensing receptor decreases Cl reabsorption similarly under basal as well as ADH-stimulated conditions by inhibiting cAMP pathway. This effect does not involve protein kinase C or phospholipase A₂ metabolic derivatives.

	METHODS

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Net Cl fluxes (J_Cl) have been determined on in vitro microperfused tubules, as usually performed in our laboratory (6). Briefly, male Sprague-Dawley rats weighing 80-100 g were killed and exsanguinated. Coronal slices from both kidneys were immediately immersed in a cold bathing solution containing 1 mM Ca²⁺ (for composition see below) and with 0.1% bovine serum albumin added. CTAL and medullary TAL (MTAL) were dissected from the medullary rays of the cortex and from the inner stripe of the outer medulla, respectively. Each tubule was then transferred to a Lucite chamber thermostatically maintained at 36.0 ± 0.1°C with a flow rate of ~5 ml/min.

Each perfused tubule was allowed to equilibrate for 1.5 h for CTAL and 0.5 h for MTAL, in the bathing solution containing 1 mM Ca²⁺. At the beginning of the experiment, the bath Ca²⁺ concentration was changed for 0.5 mM. Two or three 30-min periods were performed, as mentioned in the text, and 10 min elapsed between the periods for equilibration. The luminal fluid was collected every 10 min so as to obtain three tubular samples per period. The tubular perfusion rate was not different between two consecutive periods: 4.04 ± 0.17 and 4.01 ± 0.17 nl/min when two periods were performed, and 3.82 ± 0.19, 3.79 ± 0.18, and 3.67 ± 0.15 nl/min when three periods were performed. The composition of the perfusion solution was as follows (mM): 140 NaCl, 2.4 K₂HPO₄, 0.6 NaH₂PO₄, 1 MgCl₂, 1 CaCl₂, 10 HEPES, and 10 urea. For the bathing solution, glucose (5 mM) was added and the Ca²⁺ concentration (0.5 or 3 mM) was modified as described in the text.

Chloride concentrations in collected fluid (C_c) and perfusate (C_p) were determined by microelectrometric titration. The tubular flow rate (V) was calculated from the volume of the collected sample, assuming that water reabsorption was negligible. The length (L) of the perfused tubule was measured with an eyepiece micrometer at ×400 magnification. The mean length was 426 ± 14 µm (n = 69) for CTAL and 460 ± 47 (n = 11) for MTAL. The net chloride flux was calculated as J_Cl = (C_c  C_p) × V/L, expressed in picomoles per minute per millimeter tubular length.

Statistical analysis. Data at the steady state in each 30-min period were pooled and considered as one point. Values are expressed as means ± SE. Statistical significance was evaluated within each series by the paired Student's t-test and between the different series by the one-way analysis of variance followed by Fisher's least significant difference test. P < 0.05 was considered as significant.

Materials. N-(2-aminoethyl)-5-isoquinolinesulfonamide (i.e., H-9) and bisindolylmaleimide I (GF-109203X) were provided by Research Biochemicals International (Natick, MA); N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (i.e., H-89) was purchased from Calbiochem (La Jolla, CA), and all the other products were from Sigma (St. Louis, MO).

	RESULTS

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Effect of Ca²⁺_e on J_Cl.. Maneuvers conducted to activate a Ca²⁺-sensing receptor were performed on the basolateral face of the tubule. Indeed, preliminary results indicated that no increase in intracellular Ca²⁺ concentration could be observed when the luminal ion concentration was increased from 1 to 5 mM, whereas a response was elicited by 5 mM Ca²⁺_e at the basolateral pole of the same tubules (data not shown). These results suggest, in agreement with Riccardi et al. (18), that the phospholipase C-coupled Ca²⁺ receptor RaKCaR is not present at the luminal face of rat CTAL.

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View larger version (34K): [in this window] [in a new window]   Fig. 1.   Effect of Ca2+-sensing receptor activation on the antidiuretic hormone (ADH)-induced increase in net chloride flux (JCl). Basal Ca2+ concentration was 0.5 mM in the bath. A-C: after a control period (C, stippled columns), 1010 M ADH was added to the bath for two consecutive periods (hatched columns). The Ca2+-sensing receptor was activated either by increasing the bath Ca2+ concentration from 0.5 to 3 mM (3Ca, solid columns) or by adding 200 µM neomycin (Neo, open column). D: after 2-h equilibration in 1 mM Ca2+ (see METHODS), 1010 M ADH was added with 3 mM Ca2+ (solid columns), then the Ca2+ concentration was decreased to 0.5 mM (hatched columns). * Significantly different from the preceding value; number of tubules is in parentheses.

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View larger version (34K): [in this window] [in a new window]   Fig. 2.   Effect of Ca2+-sensing receptor activation on JCl, in absence of ADH. After a control period with 0.5 mM Ca2+ in the bath (C, stippled columns), the Ca2+-sensing receptor was activated either by increasing the Ca2+ concentration to 3 mM (n = 3) or by adding 200 µM neomycin (n = 3) (Ca2+/Neo, solid columns). * Significantly different from the control value in the same group; number of tubules is in parentheses.

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View larger version (19K): [in this window] [in a new window]   Fig. 3.   Role of protein kinase A in the basal and neomycin-inhibited JCl. A: after a control period (C, stippled column), a protein kinase A inhibitor, H-89 (n = 5, 5 × 107 M) or H-9 (n = 3, 106 M), was added to the bath (PKA inh., open columns). B: either H-9 (106 M) or GF-109203X (107 M), a protein kinase C inhibitor, was present in the bath from the control period onwards (C, open columns). To the bath was then added 1010 M ADH (hatched columns), then 200 µM neomycin was also added (Neo, solid columns). * Significantly different from the preceding value; number of tubules is in parentheses.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Effect of activating the Ca2+-sensing receptor in the presence of protein kinase A inhibitors; n = number of tubules. A: H-9 (106 M) was present since the control period onwards (C, stippled columns). Neo, neomycin (200 µM). B: time course of JCl, expressed as the percentage of the first control value (C1). Mean C1 value was 50.5 ± 7.7 pmol · min1 · mm1 (n = 5). At the time indicated by the arrows, H-89 (5 × 107 M) was added, and the Ca2+ concentration in the bath was increased from 0.5 to 3 mM. Note that JCl stabilized 20 min after H-89 administration. * Significantly different from the preceding value.

Effect of Ca²⁺_e on cAMP-increased J_Cl. Addition to the bath of both the permeant cAMP analog 8-bromo-cAMP (10⁴ M) and the phosphodiesterase inhibitor IBMX (10⁴ M) induced a significant increase in J_Cl from 89.8 ± 16.1 to 137.4 ± 23.5 pmol · min¹ · mm¹ (P < 0.01, Fig. 5). Under these conditions, increasing Ca²⁺_e concentration was not associated with a significant decrease in J_Cl (128.9 ± 19.0 pmol · min¹ · mm¹, representing an inhibition of 4.2 ± 3.1%). These results indicate that the effect of Ca²⁺_e does not take place at a step beyond nucleotide accumulation.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Individual values of JCl representing the absence of Ca2+ effect in presence of cAMP analog. After a control period (C), 8-bromo-cAMP and the phosphodiesterase inhibitor IBMX were both added to the bath at the concentration of 104 M (8-Br cAMP). Ca2+ concentration in the bath was further increased from 0.5 to 3 mM (8-Br cAMP + 3Ca). * Significantly different from the preceding value.

Role of phospholipase A₂ and cytochrome P-450 pathway. The possibility that the Ca²⁺_e-induced effect on J_Cl was mediated by arachidonic acid derivatives was evaluated by the use of a phospholipase A₂ inhibitor, 4-bromophenacyl bromide (BPB, 10⁶ M) and of a cytochrome P-450 monooxygenase inhibitor, octadecynoic acid (ODYA, 10⁷ M). Each of these agents was added to the bath simultaneously with 3 mM Ca²⁺_e. Following this protocol and in agreement with results reported by Grider et al. (11), these agents dampened significantly the inhibitory effect of 10⁸ M bradykinin on J_Cl in the rat MTAL (9.6 ± 2.0% and 4.6 ± 3.1% in the presence of BPB and ODYA, respectively, vs. 27.6 ± 2.7%, in the absence of these agents, P < 0.001). Increasing Ca²⁺_e concentration reduced J_Cl by 28.5 ± 2.2% and 31.7 ± 3.6%, in the presence of BPB and ODYA, respectively (Table 1), an effect not significantly different from the one obtained in the absence of inhibitor (27.7 ± 5.0%, Fig. 2).

	DISCUSSION

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The results presented in this study show that activation of a Ca²⁺-sensing receptor inhibits J_Cl and, supposedly, Ca²⁺ and Mg²⁺ reabsorption in rat CTAL (8). Moreover, they indicate that this effect occurs mainly through an inhibition of cAMP pathway, since 1) it is totally abolished by protein kinase A inhibitors, 2) it is not altered by inhibitors of protein kinase C, phospholipase A₂, and cytochrome P-450 monooxygenase, and 3) it is not observed in the presence of a nonhydrolyzable cAMP analog. This last result further emphasizes that the effect of Ca²⁺_e on J_Cl does not take place at a step beyond cAMP accumulation, consistent with the demonstration that Ca²⁺_e inhibits adenylyl cyclase activity and stimulates phosphodiesterase activity in the rat MTAL (13) and CTAL (7).

Increasing Ca²⁺_e concentration might have modified the transepithelial resistance per se and thus influenced the reabsorptive function of CTAL. However, the fact that the effect of Ca²⁺_e was reproduced by neomycin argues for the involvement of intracellular mediators coupled to the Ca²⁺-sensing receptor.

That phospholipase A₂ activation or cytochrome P-450 pathway does not account for the Ca²⁺_e effect on J_Cl in rat TAL disagrees with recent observations (21, 22); from these studies, these pathways are involved in the inhibitory effect of Ca²⁺-sensing receptor on the activity of a patched apical K channel, and, thereby, on sodium reabsorption. This discrepancy cannot be due to an artifact resulting from the in vitro microperfusion technique, since this one allowed the display of a modulation of the bradykinin effect on MTAL by phospholipase A₂ metabolic derivatives (Ref. 11, and our results). Three possible explanations may be afforded. 1) Compared with the 3 mM Ca²⁺_e used here, the 5 mM concentration used in the studies from Wang et al. (21, 22) may have activated phospholipase A₂; however, the fact that on other structures this pathway is activated by 2 mM Ca²⁺_e mitigates such a hypothesis (12). 2) The patched K⁺ channels were isolated from undiscriminated CTAL and MTAL so that the reported effects of Ca²⁺_e may have occurred mainly in this latter segment, which is known to exhibit high level of phospholipase A₂ activity; in this same study, moreover, the increase in 20-hydroxyeicosatetraenoic acid (i.e., 20-HETE) production induced by 5 mM Ca²⁺_e was described in MTAL only. 3) The regulation of the patched K⁺ channel (70 pS) may have no influence on the overall Cl reabsorption in TAL. Indeed, other proteins such as a second K⁺ channel (30 pS), apical Na⁺-K⁺-2Cl cotransport(s) and basolateral Cl channels are involved in Cl reabsorption and may be submitted to different regulatory process than the one described for the 70-pS K⁺ channel. Consistent with this last comment, it must be pointed out that nitric oxide via the cGMP pathway would activate locally the 70-pS K⁺ channel (14), albeit this pathway was shown to inhibit J_Cl reabsorption in TAL (15, 16).

Activation of the Ca²⁺-sensing receptor inhibits cAMP pathway under ADH-stimulated as well as basal conditions, providing three comments. 1) This result emphasizes that the basal protein kinase A activity must be elevated enough to activate chloride reabsorption in the absence of adenylyl cyclase-stimulating hormones; this is actually the case, since protein kinase A inhibitors can decrease both the basal J_Cl, as shown here in CTAL, and the basal protein kinase A activity, as reported in MTAL (9). This compelling evidence supports the hypothesis that Ca²⁺-sensing receptor can modulate physiological Ca²⁺ and Mg²⁺ reabsorption in CTAL under basal status. 2) That activation of Ca²⁺-sensing receptor inhibits cAMP pathway when the nucleotide synthesis is not stimulated differs from the report that prostaglandin E₂ inhibits J_Cl in the presence of ADH only (5). Such a difference may be explained by the mechanisms underlying the effects of the two agonists in CTAL. That is, on the one hand, prostaglandin E₂ decreases solely cAMP synthesis, through an _i-mediated inhibition of the ADH-induced stimulation of adenylyl cyclase (10); on the other hand, recent results from our laboratory (7) indicate that Ca²⁺-sensing receptor activation decreases adenylyl cyclase activity through an _i-independent mechanism and, in addition, increases nucleotide degradation. Moreover, the fact that Ca²⁺_e may not interact with ADH action, as prostaglandin E₂ does, but directly inhibits adenylyl cyclase (4) is in accordance with our observation that the absolute decrease of J_Cl induced by this ion is similar under basal or ADH-stimulated conditions. 3) Extracellular Ca²⁺ does not decrease J_Cl below the control values in the presence of ADH (see Fig. 1), i.e., the effects observed under stimulated and basal conditions are not additive. Such an absence of additivity affords further evidence for precluding mechanisms other than inhibition of cAMP pathway. So long as the Ca²⁺_e inhibitory effect is constant regardless of the J_Cl (J_Cl of ~20 pmol · min¹ · mm¹), it may be proposed that the similarity of the mean values observed in control period and in the presence of ADH plus 3 mM Ca²⁺_e is circumstantial; as shown in Fig. 6, when the ADH-induced increase in J_Cl exceeds 20 pmol · min¹ · mm¹, then Ca²⁺_e fails to restore J_Cl to the control value. Conversely, when the ADH-induced increase in J_Cl is lower than 20 pmol · min¹ · mm¹, then Ca²⁺_e leads to a fall in J_Cl below the control value. In the present experiments, it may be coincidental if the mean of the net ADH-induced increases in J_Cl is close to 20 pmol · min¹ · mm¹ so that activation of the Ca²⁺-sensing receptor appears to compensate this effect, as shown in Fig. 1.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Individual variations in JCl induced by ADH and extracellular Ca2+. Data represent the experiments performed with ADH in absence (Fig. 1, A and B) and in presence of GF-109203X (Fig. 3B, right). Ordinates represent, for the same tubule, the absolute differences in JCl between ADH and the control period (ADH-C), for the one part, and between Ca2+-sensing receptor activation and control period (ADH/Ca2+-C) for the other part. The dotted line at zero value indicates JCl not different from the control value.

In summary, the present study indicates that activation of Ca²⁺-sensing receptor decreases chloride reabsorption in rat CTAL, the site of passive Ca²⁺ and Mg²⁺ reabsorption. This effect occurs by inhibiting cAMP pathway, regardless of whether ADH is present, and does not involve protein kinase C and phospholipase A₂ metabolic derivatives.