INTRODUCTION

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IN MANY CELL TYPES, the intracellular Ca²⁺ concentration ([Ca²⁺]_i) regulates cAMP levels through interactions of Ca²⁺ on cAMP synthesis and/or cAMP hydrolysis. These effects of [Ca²⁺]_i are linked to the presence of Ca²⁺-sensitive adenylyl cyclases (ACs) and/or Ca²⁺/calmodulin-dependent phosphodiesterases (7, 13, 17). Among AC isoforms, the enzymatic activity of type 5 and type 6 AC is directly inhibited by submicromolar concentrations of Ca²⁺ (25, 31, 33). Ca²⁺-inhibitable AC isoforms are sensitive to [Ca²⁺]_i, typically achieved in intact cells as a consequence of phospholipase C activation and/or Ca²⁺ channel activation (4).

Two cell types have been described in kidney collecting duct: principal cells, involved in water homeostasis (30), are the predominant cell type in the collecting duct. In rat kidney, type A intercalated cells, involved in proton secretion and bicarbonate reabsorption, are found in cortical and outer medullary collecting ducts, whereas type B intercalated cells, mostly found in cortical collecting ducts, function in the other direction (5). Type 5 and type 6 AC mRNAs are both expressed in the rat kidney outer medullary collecting duct (OMCD) (6), where principal and type A intercalated cells are the two major cell types. In microdissected OMCDs, AC activity is stimulated by arginine vasopressin (AVP) and glucagon (8, 22). Physiological and biochemical results support the conclusion that AVP stimulates AC activity in principal cells, whereas glucagon is active in intercalated cells ( 6, 8, 21).

An inhibitory effect of Ca²⁺ on the cAMP pathway in the rat OMCD was first suggested by the negative regulation of a Ca²⁺ ionophore on AVP-dependent cAMP accumulation (19). PGE₂ and carbachol, a muscarinic agonist of the acetylcholine receptor, both induce a Ca²⁺-dependent inhibition of hormone-stimulated intracellular cAMP accumulation (1, 6), which is probably linked to the [Ca²⁺]_i increases elicited by PGE₂ and carbachol in rat OMCD (1, 20). However, the effect of these agents on cAMP accumulation is cell type and agonist dependent. Indeed, PGE₂ induces only a small inhibition of AVP-stimulated cAMP synthesis, and its Ca²⁺-dependent inhibitory effect on AVP-dependent cAMP accumulation is due mainly to an increase in cAMP hydrolysis (1, 9). In contrast, carbachol, which has no effect on the response to AVP (2, 6), induces a marked Ca²⁺-dependent inhibition of glucagon-stimulated AC activity (6). In addition, glucagon-stimulated cAMP synthesis is inhibited by extracellular Ca²⁺, whereas AVP-dependent cAMP synthesis is not (6). These observations therefore show that the inhibitory effect of either extracellular Ca²⁺ or agonist-mediated [Ca²⁺]_i changes on intracellular cAMP are cell type dependent. These differences could be accounted for, at least partly, by the cellular localization of type 5 and/or type 6 functional AC proteins in the OMCD.

The purpose of the present experiments was to study the potential role of Ca²⁺-inhibitable AC isoforms in the regulation of cAMP synthesis in the rat OMCD cells.

The localization of type 5 and type 6 AC mRNAs at the cellular level was performed by in situ hybridization. Only type 6 mRNA was detected in rat kidney collecting duct principal cells, whereas both type 5 and type 6 Ca²⁺-inhibitable AC isoforms were found in intercalated cells. In superfused OMCDs, the effects of PGE₂ and carbachol on [Ca²⁺]_i were additive, suggesting that the Ca²⁺ increase elicited by these agents occurs in different cell types. One fundamental property of Ca²⁺-inhibitable AC isoforms is to be regulated by independent Ca²⁺- and G_i-mediated processes, leading to a cumulative inhibition of AC activity (7, 14, 13, 29). This property cannot be verified accurately in principal cells because the Ca²⁺-dependent inhibition of PGE₂ on AVP-stimulated cAMP synthesis is of very low magnitude (1, 7, 9). In contrast, such a dual regulation can be studied in OMCD intercalated cells, where about one-half of glucagon-dependent AC activity is inhibited by either a Ca²⁺-dependent carbachol pathway (6) or a G_i-sensitive PGE₂ pathway (3). In our study, the simultaneous addition of carbachol and PGE₂ induced a cumulative inhibition of glucagon-dependent cAMP synthesis, demonstrating that these agents inhibit the same AC catalytic units. The results therefore localize Ca²⁺-inhibitable AC mRNAs in OMCD and demonstrate the functional expression of Ca²⁺-inhibitable AC isoforms in intercalated cells.

	METHODS

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Unless otherwise specified, reagents used were purchased from Merck (Damstardt, Germany), Sigma Chemical (St. Louis, MO), and Calbiochem (San Diego, CA). Experiments were performed in male Sprague-Dawley rats (140-180 g body wt, Iffa-Credo), maintained on a standard diet with free access to water.

Northern blots. Hybridization probes for type 5 and type 6 AC were prepared by random primed labeling of the regions described for in situ hybridization experiments using [-³²P]dCTP. A multiple rat tissue Northern blot (Clontech Laboratories) was hybridized, as per the manufacturer's instructions, in 5 ml of ExpressHyb solution at 68°C for 30 min. The probe was added to 5 ml of fresh ExpressHyb solution and incubated at 68°C for 1 h. Washes were performed as follows: 40 min (with 3 changes of the solution) in solution 1 (2× SSC-0.05% SDS) at room temperature, followed by an incubation of 40 min at 50°C in solution 2 (0.1× SSC-0.1% SDS). The blot was then wrapped in plastic and exposed to X-ray film with an intensifier screen at 80°C for 3 days.

In situ hybridization and immunostaining. Probes specific for type 5 and 6 ACs were designed in the most divergent regions of AC cDNA regions. A 376-bp fragment (Pvu II-Sph I, nucleotides 3965-4342) of type 6 AC cDNA, located in the 3'-untranslated region, was subcloned in pGEM3Zf(+) (Promega Biotech, Madison, WI). A 1,080-bp fragment corresponding to nucleotides 1205-2285 (EcoR I, Pvu II) of type 5 AC cDNA coding region was subcloned in BSSK+ (Stratagene). Sense and antisense cRNA probes were in vitro transcribed with T3, T7, or SP6 RNA polymerases (Promega Biotech) according to the manufacturer's instructions, in the presence of [³⁵S]UTPS (>1,000 Ci/mmol, Amersham, Les Ulis, France).

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1

Isolation of rat kidney OMCDs. The experimental procedure used to microdissect intact segments from collagenase-treated rat kidney has previously been detailed (8, 9). After the rats were anesthetized (pentobarbital, 6 mg/100 g body wt), the left kidney was perfused with microdissection medium containing 0.16% collagenase (Serva, Boehringer Mannheim). After hydrolysis of the kidney (20 min at 30°C in 0.12% collagenase solution), single pieces (0.3-1.5 mm length) of collecting duct were microdissected at 4°C from the outer medulla. The standard microdissection medium was composed of (in mM) 137 NaCl; 5 KCl; 0.8 MgSO_4; 0.33 Na₂HPO₄; 0.44 KH₂PO₄; 1 MgCl₂; 4 NaHCO₃; 10 CH₃COONa; 1.0 or 2.0 CaCl₂ (see below); 5 glucose; and 20 HEPES, pH 7.4, and 0.1% (wt/vol) BSA (fraction V, Pentex, Miles, Kankakee, IL).

Measurement of [Ca²+]i. [Ca²⁺]i was measured in single OMCD samples by using the calcium-sensitive fluorescence probe acetoxymethyl ester of fura 2 (fura 2-AM, Molecular Probes, Eugene, OR) as previously described (1, 10, 27). Briefly, the samples were loaded for 60 min with 10 µM fura 2-AM. Each tubule was then transferred to a superfusion chamber fixed on an inverted fluorescence microscope (Zeiss IM 35, Oberkochen, Germany). Tubules were superfused at 37°C at a rate of 10-12 ml/min, corresponding to ~10 exchanges/min. The superfusion medium (microdissection medium without serum albumin) contained either 2 mM Ca²⁺ or no Ca²⁺ (nominally Ca²⁺-free medium without CaCl₂ and containing 0.1 mM EGTA). After a 5- to 10-min equilibration period, agonists were added to the medium and superfused over tubules. Because of the dead space of the superfusion setup, the time necessary to achieve a full equilibration was 15-20 s. A circular area of 60-µM diameter was selected over the tubule (×400 magnification). The fluorescence intensity emitted from this area (during brief excitation periods at 340 and 380 nm alternatively, at a maximal rate of 30 cycles/min), was recorded every 2 s.

Measurement of glucagon-dependent cAMP synthesis. Hormone-dependent cAMP synthesis in an intact single segment was measured as previously reported (1, 9). Microdissection medium (1 mM Ca²⁺) was supplemented with 5 µM indomethacin and 0.5 unit/ml adenosine deaminase (Boehringer Mannheim) to prevent the endogenous synthesis of prostaglandins and the release of adenosine, which interfere with the regulation of cAMP levels in rat OMCD (9). The incubation medium similar to the microdissection medium, included 0.1% (wt/vol) bacitracin (to inhibit peptidase activity) and 1 mM IBMX, an inhibitor of all phosphodiesterases in rat kidney (16). Microdissected tubules were transferred in 2 µl of incubation medium on glass slides (1 or 2 pieces/slide) and photographed to measure their length. Each sample was preincubated for 10 min at 30°C. After the addition of 2 µl incubation medium containing 1 µM glucagon (Neosystem Laboratoire, Strasbourg, France), with or without other agonists, samples were incubated for 4 min at 35°C. All agents were used at concentrations inducing maximal effects (1, 9, 20). The reaction was stopped by rapidly transferring the tubule together with 1 µl incubation medium into a polypropylene tube containing a 20 µl mixture of formic acid in absolute ethanol (5% vol/vol). Samples were evaporated to dryness overnight at 40°C and then kept at 20°C until cAMP assay. The amounts of cAMP were measured in acetylated samples by radioimmunoassay (Sanofi Diagnostics Pasteur, Marnes-La-Coquette, France, or NEN Life Sciences Products, Le Blanc Mesnil, France). Under our conditions, the basal level of cAMP present in one single piece of tubule was similar to, or below, the sensitivity threshold of the assay (1, 9). Thus only hormone-induced cAMP synthesis could be measured. Results were expressed in femtomoles of cAMP accumulated per millimeter of segment per 4-min incubation time (fmol · mm¹ · 4 min¹). In each experiment, all experimental conditions were tested in six to nine tubule samples from the same rat kidney. The mean cAMP value from each condition was expressed in absolute value or in a percentage of inhibition calculated from the mean value obtained with glucagon alone. Results are given as means ± SE calculated from n experiments. Statistical analysis by the one-way analysis of variance was followed by Fisher's least significant difference test.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Northern blotting. The specificity of probes used for in situ hybridization was checked by Northern blotting of several rat tissues (Fig. 1). The probe for type 5 AC produced a strong hybridization signal in heart and brain (Fig. 1A), a weaker signal in kidney and lung and a very weak signal in spleen, liver, skeletal muscle, and testis, consistent with previous reports for type 5 AC (23). Similarly, type 6 AC was detected in nearly all tissues (Fig. 1B), consistent with previous localization (18, 23).

View larger version (69K): [in this window] [in a new window]   Fig. 1.   Northern blotting of rat tissues. A: type 5 adenylyl cyclase (AC) probe strongly stained heart and brain. A signal was also observed in lung and kidney, whereas the staining of spleen, liver, skeletal muscle, and testis was weak. B: type 6 AC probe stained all tissues, including kidney. The strongest signal was observed in heart and brain. Staining was distinct for type 5 and type 6 AC probes in all tissues, demonstrating the specificity of each probe.

In situ hybridization. By in situ hybridization on 4-µm-thick rat kidney sections, both type 5 (Fig. 2a) and type 6 AC (Fig. 2b) mRNAs were relatively abundant in glomeruli. The labeling for type 5 AC mRNA was strongest in small arteries and blood vessels (Fig. 2a, arrows), where little labeling was observed for type 6 AC (not shown). Significant labeling for type 5 and type 6 AC was also observed in the interstitium, between kidney tubules. No significant labeling was observed in kidney with either type 5 or type 6 sense probes (Fig. 2, c and d, respectively). In kidney tubules, as previously reported (14), type 6 AC mRNA was abundant in thick ascending limbs and collecting ducts (Fig. 3, c and d), but proximal tubules were weakly labeled (Fig. 3c). Consistent with previous reports by quantitative RT-PCR (6), type 5 AC was not detected in proximal tubules or thick ascending limbs (Fig. 3, a and b).

View larger version (77K): [in this window] [in a new window]   Fig. 2.   In situ hybridization in kidney cortex. By in situ hybridization, type 5 AC mRNA (a) was abundant in glomeruli (G), small arteries, veins, and capillaries (arrows). Type 6 AC mRNA was abundant in glomeruli (b) but was absent from arteries. No labeling was observed with either type 5 (c) or type 6 (d) sense mRNA probes. Bars = 20 µm.

View larger version (115K): [in this window] [in a new window]   Fig. 3.   In situ hybridization in kidney outer medulla. In kidney tubules, type 5 AC mRNA was only detected in collecting ducts (), where principal cells were stained by anti-aquaporin-3 (AQP3) antibodies (a) and intercalated cells were stained by anti H+-ATPase antibodies (b). In these tubules, labeling for type 5 AC was localized in intercalated cells (arrowheads) but absent from principal cells (arrows). Type 6 AC mRNA (c, d) was abundant in both collecting duct () and thick ascending limbs (T). Weak labeling was also detected in proximal tubules (P). In the collecting duct, both principal cells (arrows) stained with anti-AQP3 antibodies (c) and intercalated cells (arrowheads) stained with anti-H+-ATPase antibodies (d) were labeled by the type 6 AC mRNA probe. Both type 5 and type 6 AC mRNA were detected in the interstitium. Bars = 20 µm.

View larger version (104K): [in this window] [in a new window]   Fig. 4.   In situ hybridization in kidney inner medulla. No labeling was observed in inner medullary collecting ducts () with the type 5 AC mRNA probe (a). In contrast, type 6 AC was still detected in inner medullary collecting duct cells (b). Sections were stained with anti-AQP3 antibodies. Bars = 20 µm.

Quantification of in situ hybridization labeling. The in situ observations above were confirmed by quantification of labeling in cells unambiguously detected as either principal or intercalated cells by antibody (anti-AQP3 or anti-proton pump) staining. Results are reported in Table 1 and show no statistically significant differences of labeling for type 6 AC in OMCD principal and intercalated cells. In contrast, labeling for type 5 AC was much greater in intercalated cells and significantly different from principal cells, where labeling was not significant.

Effect of carbachol and PGE₂ on [Ca²+]_i increases in rat OMCD. As underlined at the beginning of this study, [Ca²⁺]_i increases induced by PGE₂ and carbachol in rat OMCD (1, 20) appear a prerequisite condition to observe the inhibition elicited by PGE₂ on AVP-dependent cAMP accumulation (1) and the inhibition elicited by carbachol on glucagon-dependent cAMP synthesis (6). These observations led to the hypothesis that a PGE₂-mediated [Ca²⁺]_i increase might be mainly effective in the vasopressin-sensitive cells, whereas a carbachol-mediated [Ca²⁺]_i increase might be located in the glucagon-sensitive cells. This hypothesis was tested by comparing [Ca²⁺]_i variations induced by the addition of both agents to the responses obtained with each agent added alone to the superfusion medium.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   PGE2- and carbachol-mediated intracellular Ca2+ concentration ([Ca2+]i) increases. A: representative traces of [Ca2+]i variations elicited by repeated superfusion of the same tubule with 100 µM carbachol, then 0.3 µM PGE2, and finally carbachol. Experiment was performed in 2 mM Ca2+ medium. Solid horizontal bars, superfusion period of each agonist. B: mean values of [Ca2+]i variations at the peak are expressed as maximal increases over basal value. Values are means ± SE obtained in tubules superfused successively with the 2 agonists: either a first superfusion with 0.3 µM PGE2 and then 100 µM carbachol (n = 7 tubules) or conversely (n = 5 tubules). For a given agonist, [Ca2+]i increases are similar whatever the order of superfusion.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Additive effect of PGE2 and carbachol on [Ca2+]i increases in rat outer medullary collecting duct (OMCD). A: representative traces of [Ca2+]i variations elicited by different agonists added in 2 mM Ca2+ medium. The tubule was superfused with 100 µM carbachol, then the combination of both agonists, and finally 0.3 µM PGE2. Horizontal bars, superfusion period of each solution. The magnitude of the responses was independent from the order of the superfusion (see Table 2). B: representative traces of [Ca2+]i variations elicited by different agonists added in Ca2+-free medium. The tubule was superfused with 2 mM Ca2+ and then with Ca2+-free medium. Rapid superfusion of PGE2 (3 µM) and then carbachol (100 µM) elicited successive [Ca2+]i increases (left). The magnitude of carbachol- or PGE2-mediated [Ca2+]i increase was independent from the order of superfusion [carbachol = 48 ± 8 nM (n = 8) and 38 ± 5 (n = 5) with the 1st and 2nd superfusion, respectively; PGE2 = 121 ± 15 nM (n = 5) and 111 ± 11 (n = 8) with the 1st and 2nd superfusion, respectively]. Note that, in contrast to successive superfusions of different agonists, a second superfusion with the same agonist did not elicit a second response (data not shown). The combination of both agonists added in Ca2+-free medium induced a response higher than that elicited by either carbachol or PGE2 (right). Note that, after the treatment with PGE2 followed by the superfusion of carbachol, it was necessary to superfuse the tubule with 2 mM Ca2+ medium to refill the intracellular Ca2+ stores. The magnitude of the different responses was independent from the order of superfusion (see Table 2).

Multiple combined inhibition of glucagon-dependent AC activity. Glucagon-dependent cAMP synthesis is inhibited by both carbachol through a Ca²⁺-dependent process (6) and PGE₂ through a Ca²⁺-independent, G_i-mediated process (3). If present in a same cell, these regulations suggest that different mechanisms may inhibit the same AC enzymatic activity. This hypothesis was tested in multiple, combined inhibition experiments by using criteria previously defined (1). PGE₂ or carbachol inhibited to a comparable extent, close to 50-60%, the response to glucagon (Table 3). The simultaneous addition of both agents led to a residual cAMP value lower than that obtained with each agent alone, but the response to glucagon was not fully abolished (Table 3). This result establishes that PGE₂ and carbachol were active in the same glucagon-sensitive cells. The results were further analyzed by comparing the values measured to those that could be expected if a different mechanism of inhibition on AC activity accounted for carbachol- and PGE₂-mediated regulation, i.e., if these two agents elicited a cumulative inhibition of cAMP synthesis. The measured value (8.7 ± 0.9 fmol · mm¹ · 4 min¹, Table 3) was not different from the theoretical value calculated assuming an hypothesis of cumulative inhibition (7.5 ± 0.6 fmol · mm¹ · 4 min¹). These results therefore demonstrate that PGE₂ and carbachol inhibit the same pool of glucagon-sensitive AC catalytic units in rat OMCD by different and independent mechanisms.

	DISCUSSION

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In this study, Ca²⁺-inhibitable AC isoforms were localized by in situ hybridization at the cellular level in the rat OMCD to further define the regulation of cAMP content in this segment. Hormone-dependent cAMP accumulation is inhibited by Ca²⁺ in both cell types of the OMCD, but the agonist involved and the mechanism of Ca²⁺ inhibition are cell specific. Indeed, the muscarinic agonist carbachol induces a Ca²⁺-dependent inhibition of glucagon-mediated cAMP synthesis but has no effect on AVP-stimulated cAMP synthesis (6) or cAMP accumulation (2). On the other hand, PGE₂ inhibits AVP-dependent cAMP accumulation, mainly by an increase in cAMP hydrolysis, through a Ca²⁺-mediated process that is insensitive to pertussis toxin (1, 3). In addition, PGE₂ inhibits glucagon-dependent cAMP synthesis through a Ca²⁺-independent, G_i-mediated process (3). These mechanisms are summarized in Fig. 7.

View larger version (31K): [in this window] [in a new window]   Fig. 7.   Schematic summary of intracellular cAMP regulations in rat kidney OMCD. PLC, phospholipase C; PDE, phosphodiesterase; ag, agonist; R, receptor.

In the OMCD, carbachol and PGE₂ increase [Ca²⁺]_i through an interaction with the m1 subtype of the muscarinic receptor (20) and, very likely, with the EP1 subtype of the PGE₂ receptor (3), respectively. These receptor subtypes are usually coupled to the phospholipase C pathway (12, 15). In our experiments, simultaneous superfusion of PGE₂ and carbachol, in either 2 mM Ca²⁺ or Ca²⁺-free medium, produced [Ca²⁺]_i peaks corresponding to a full additivity of the effects of both agonists. This result and the observation of a Ca²⁺-dependent inhibition of cAMP content elicited by either PGE₂ in vasopressin-sensitive cells (1) or carbachol in glucagon-sensitive cells (6) strongly suggest a cell-specific [Ca²⁺]_i increase induced by PGE₂ and carbachol in principal and intercalated cells, respectively (Fig. 7).

Type 5 and type 6 AC mRNAs have been detected by quantitative RT-PCR in the OMCD, and functional data have suggested that this localization corresponds to the expression of functional proteins (6). Consistently with previous RT-PCR (6), type 6 AC mRNA was highly expressed in thick ascending limbs and collecting ducts. Colocalization of type 6 AC mRNA by in situ hybridization and cells positive for AQP3 as well as cells positive for vacuolar H⁺-ATPase by immunocytochemistry demonstrated that type 6 AC mRNA is present in both collecting duct principal and intercalated cells. In contrast, type 5 AC mRNA labeling was mostly detected in intercalated cells, where the proton pump was found, but where no staining for AQP3 could be detected. Because mRNA for type 5 and type 6 AC was detected in every type A (proton secreting) intercalated cell, these observations suggest that principal cells express only type 6 AC, whereas both type 5 and type 6 AC mRNAs are expressed in type A intercalated cells.

A property of Ca²⁺-inhibitable ACs is their sensitivity to both submicromolar concentrations of Ca²⁺ and to a G_i-mediated pathway (7, 13, 29). In the presence of an inhibitor of phosphodiesterases, the simultaneous addition of carbachol and PGE₂ elicited a cumulative inhibition of glucagon-mediated cAMP synthesis. This dual negative regulation demonstrates the functional expression of Ca²⁺-inhibitable AC in intercalated cells. In addition, although two distinct Ca²⁺-inhibitable AC isoforms were detected by in situ hybridization in intercalated cells, the cumulative inhibition demonstrates that carbachol and PGE₂ regulate a single pool of AC catalytic subunits in this cell type, either type 5 AC, type 6 AC, or both. The enzymatic properties of type 5 AC and type 6 AC isoforms and the regulation of their activities are closely similar (7, 13). Thus at the present time it appears difficult to further define the functional isoform(s) involved in cAMP synthesis in intercalated cells. Figure 7 summarizes previous and present data on the regulation of cAMP accumulation in the OMCD.

Extracellular Ca²⁺, or carbachol-mediated [Ca²⁺]_i increases, inhibit glucagon-dependent AC activity by ~50% (6). By comparison, in OMCD principal cells or in the cortical thick ascending limb (cTAL), where the Ca²⁺-inhibitable type 6 AC is also expressed, PGE₂ or angiotensin II induces a Ca²⁺-mediated inhibition of cAMP synthesis of only 10-20% (7, 9, 14). Ca²⁺-inhibitable AC can be inhibited by either [Ca²⁺]_i peaks or Ca²⁺ entry (11, 14). The following two major hypotheses can be discussed to explain the high sensitivity of OMCD glucagon-sensitive cells AC activity to Ca²⁺.

Role of Ca²+ in intercalated cell AC activity. The activation of the Ca²⁺-sensing receptor RaKCaR can inhibit cAMP synthesis by up to 90% in the cTAL, in contrast to the small inhibitory effect of angiotensin II in this segment. This high inhibition involves both a [Ca²⁺]_i peak and a capacitive Ca²⁺ entry (14). Although there is no evidence for the expression of RaKCaR in OMCD basolateral plasma membranes (10, 24, 32), the presence of a yet unknown Ca²⁺-sensing receptor in intercalated cells could account for the great sensitivity of intercalated cell AC to extracellular Ca²⁺. The inhibition of glucagon-dependent, but not AVP-dependent, AC activity (6) by extracellular Ca²⁺ supports also the hypothesis that Ca²⁺ channels are specifically expressed in intercalated cells. Accordingly, the presence of non-voltage-gated Ca²⁺ channels has been demonstrated in rat OMCD (10). In addition, the [Ca²⁺]_i increase elicited by carbachol in glucagon-sensitive cells is characterized by a plateau phase of markedly larger amplitude than that observed with PGE₂ in AVP-sensitive cells (Refs. 1 and 20 and this study) or with angiotensin II in cTAL (14). This plateau reflects Ca²⁺ entry triggered by a [Ca²⁺]_i release (1, 20) and could also result from the activation of Ca²⁺ channels. It can be noted that in some cell types, carbachol was described to induce a direct activation of Ca²⁺ channels (15, 28). A carbachol-induced Ca²⁺ entry could therefore account for the high inhibition of glucagon-dependent AC activity by Ca²⁺.

Role of AC isoforms in intercalated cell sensitivity to Ca²+. Type 5 AC is expressed only in intercalated cells. The rabbit type 5 AC isoform was previously reported to be more sensitive to Ca²⁺ than type 6 AC (31). However, recent results with the canine type 5 AC isoform did not confirm this property (25). Additional experiments are therefore necessary to demonstrate a different sensitivity to Ca²⁺ of type 5 and type 6 AC that might account for the high Ca²⁺-dependent inhibition of AC activity observed in intercalated cells.