INTRODUCTION

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THE DISTAL TUBULE REABSORBS significant amounts of magnesium and plays an important role in determining the final urinary excretion rate (34). In contrast to more proximal segments of the nephron, distal Mg²⁺ transport processes are postulated to be active and transcellular in nature (34). It is likely that hormonal control in this segment provides the fine tuning of renal magnesium conservation contributing to whole body magnesium balance. Knowledge of hormonal actions and interactions within the distal tubule is important to the understanding of overall renal magnesium conservation.

Glucagon has clearly been shown to increase magnesium conservation within both the loop of Henle and the distal tubule (2, 15). Bailly and colleagues (4, 5) have shown that glucagon administration markedly enhances magnesium reabsorption in the loop of Henle and the superficial distal tubule of hormone-deprived rats lacking circulating parathyroid hormone (PTH), calcitonin, antidiuretic hormone, and glucagon (4, 5). Furthermore, using hormone-deprived rats, Elalouf et al. (14) demonstrated that glucagon may play an additive role with arginine vasopressin (AVP) in control of renal magnesium conservation (14). Although it is clear from these micropuncture studies that glucagon acts within the superficial distal tubule, the segment of the distal tubule remains unknown. The mammalian distal tubule is composed of a short post macula densa segment of the thick ascending limb, the distal convoluted tubule, the connecting tubule, and the initial collecting tubule (24). Also unknown are which cellular mechanisms are involved with glucagon stimulation of Mg²⁺ transport. Butlen and Jard (7) have shown that glucagon receptors are present in the rat distal tubule, and Bailly et al. (3) reported that glucagon stimulates adenylate cyclase in isolated rat distal convoluted tubules, so glucagon may act, in part, through release of the second messenger, cAMP.

On the other hand, the actions of AVP within the distal convoluted tubule are poorly understood (23). Morel and colleagues (8, 26, 27) reported that AVP receptors are present in the rat and mouse distal convoluted tubule. However, using hormone-deprived rats, Elalouf et al. (13) reported that AVP enhanced distal sodium and calcium reabsorption but did not significantly alter Mg²⁺ transport. Although it is clear that AVP enhances renal magnesium conservation, the micropuncture data suggest that the major effects appear to be within the thick ascending limb of the loop of Henle, with little effect on Mg²⁺ transport in the distal tubule (13, 33, 35).

The cellular mechanisms of magnesium absorption in the distal convoluted tubule are poorly understood. In vitro microperfusion studies have not been performed because of the difficulty in isolating intact tubule segments. In vivo micropuncture and microperfusion studies of superficial distal tubules do not allow for investigation of cellular mechanisms of Mg²⁺ transport (34). In the present studies, we use an immortalized mouse distal convoluted tubule (MDCT) cell line to determine the effects of glucagon and AVP on Mg²⁺ uptake within this segment (28). The MDCT cell line possesses many of the properties of the intact distal convoluted tubule. The MDCT cells exhibit amiloride-inhibitable sodium transport and chlorothiazide-sensitive NaCl cotransport (17, 18). Amiloride and chlorothiazide also stimulates Ca²⁺ and Mg²⁺ entry in these cells (10, 11, 17, 18). Furthermore, PTH and calcitonin stimulate calcium uptake in MDCT cells (20, 21). Accordingly, we used this cell line to investigate the actions of glucagon and AVP on Mg²⁺ uptake in the distal convoluted tubule. Because there is not an available isotope for magnesium, we determined Mg²⁺ entry into MDCT cells in the present studies by first depleting the cells of intracellular Mg²⁺ by culturing in Mg²⁺-free media for 16 h. The Mg²⁺-depleted MDCT cells were then placed in medium containing 1.5 mM Mg²⁺, and the refill rate, d([Mg²⁺]_i)/dt (where [Mg²⁺]_i is intracellular free Mg²⁺ concentration), was measured with microfluorescent studies using mag-fura 2 (29). Mg²⁺ uptake rate is concentration dependent and selective for magnesium (10). Moreover, the influx rate is rapid and reproducible so that it is possible to determine the effects of extracellular influences on transport rates. In the present study, we show that both glucagon and AVP stimulate Mg²⁺ entry in MDCT, possibly through cAMP-dependent mechanisms. Further studies are required to define the intracellular pathways involved in hormone stimulation of Mg²⁺ absorption in the distal tubule.

	METHODS

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Materials. Basal Dulbecco's minimal essential medium (DMEM) and Ham's F-12 media were purchased from GIBCO. Customized Mg²⁺-free media were purchased from Stem Cell Technologies (Vancouver, BC, Canada). Fetal calf serum was from Flow Laboratories (McLean, VA). Mag-fura 2-AM was obtained from Molecular Probes (Eugene, OR). Glucagon, AVP, 8-bromo-cAMP, phorbol 12-myristate 13-acetate (PMA), and other materials were from Sigma Chemical (St. Louis, MO). The protein kinase A inhibitor, Rp-cAMPS, and protein kinase C inhibitor, R031-8220, were purchased from Calbiochem (San Diego, CA).

Cell culture. Distal convoluted tubule cells were isolated from mice by Pizzonia et al. (28). These cells were immortalized and functionally characterized as previously described by Friedman and Gesek (17, 20). The MDCT cell line was grown on 60-mm plastic culture dishes (Corning Glass Works, Corning Medical and Scientific, Corning, NY) in DMEM-F12 (1:1) media supplemented with 10% fetal calf serum, 1 mM glucose, 5 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin in a humidified environment of 5% CO₂-95% air at 37°C. For the fluorescence studies, confluent cells were washed three times with phosphate-buffered saline containing 5 mM ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid, trypsinized, and seeded on glass coverslips. Aliquots of harvested cells were allowed to settle onto sterile glass coverslips in 100-mm Corning tissue culture dishes, and the cells were grown to subconfluence over 1-2 days in supplemented media as described above. The normal media contained 0.6 mM Mg²⁺ and 1.0 mM Ca²⁺. In the experiments indicated, MDCT cells were cultured in Mg²⁺-free media (<0.01 mM), where indicated for 16-24 h prior to study. Other constituents of the Mg²⁺-free culture media were similar to the complete media. These media contained 0.2% bovine serum albumin (BSA) rather than the fetal calf serum.

Determination of cAMP concentration. cAMP was determined in confluent MDCT cell monolayers cultured in 24-well plates in DMEM-F12 media without serum but with 0.1% BSA. The media contained 0.6 mM Mg²⁺ or zero Mg²⁺ where indicated. After addition of either glucagon or AVP, MDCT cells were incubated at 37°C for 5 min in the presence of 0.1 mM 3-isobutyl-1-methylxanthine. The cAMP was extracted with 5% trichloroacetic acid, which was removed with ether, and then the extract was acidified with 0.1 N HCl. The aqueous phase was dried, dissolved in tris(hydroxymethyl)aminomethane (Tris)-EDTA buffer, and cAMP was measured with a radioimmunoassay kit (Diagnostic Products, Los Angeles, CA).

Cytoplasmic Mg²⁺ measurements. Coverslips were mounted into a perfusion chamber, and intracellular free Mg²⁺ concentration ([Mg²⁺]_i) was determined with the use of the Mg²⁺-sensitive fluorescent dye, mag-fura 2. The cell-permeant acetoxymethyl ester (AM) form of the dye was dissolved in dimethyl sulfoxide to a stock concentration of 5 mM and then diluted to 5 µM mag-fura 2-AM in media for 20 min at 23°C. Cells were subsequently washed three times with buffered salt solution containing (in mM) 145 NaCl, 4.0 KCl, 0.8 K₂HPO₄, 0.2 KH₂PO₄, 1.0 CaCl₂, 5.0 glucose, and 20 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-Tris, at pH 7.4. The MDCT cells were incubated for a further 20 min to allow for complete deesterfication and washed once with this buffer solution before measurement of fluorescence.

	RESULTS

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Glucagon and AVP stimulate cAMP release in Mg²⁺-depleted MDCT cells. Bailly et al. (3) have reported that glucagon stimulates cAMP release in rat distal convoluted tubules but not rabbit distal segments. Morel and colleagues (26, 27) have shown that AVP-sensitive adenylate cyclase is present in the distal tubule, but, as with glucagon, there were some species differences. AVP stimulated cAMP release along the entire length of rat and mouse distal tubules, whereas it was observed only in the late segment of the rabbit distal tubule. In the support of these studies, Friedman and Gesek (18) showed that both glucagon and AVP stimulated cAMP release in MDCT cells. In the present study, we determined the concentration dependence of glucagon- and AVP-stimulated cAMP release in MDCT cells (Fig. 1). Both hormones elicited intracellular cAMP accumulation in a concentration-dependent fashion. Second, we determined whether these hormones elicit cAMP accumulation in the Mg²⁺-depleted MDCT cells used here. Glucagon or AVP at submaximal (10⁹ M) and maximal (10⁷ M) concentrations increased cAMP by similar amounts in normal and Mg²⁺-depleted MDCT cells (Table 1). The concentration dependence of glucagon and AVP are far removed from the concentrations normally observed in vitro. A number of explanations may provide the basis for these discrepancies, including absence of normal bathing plasma, diminished circulation of hormone, or changes in membrane receptors due to cell culture conditions. Nevertheless, these responses indicate that hormone receptors are present in this cell line that activate intracellular signaling processes. These data confirm the observations of Friedman and Gesek (18) that receptors for these hormones are present in the MDCT cells, which, upon stimulation, release intracellular cAMP. Intracellular Mg²⁺ depletion did not alter hormone-responsive cAMP release in these cells (Table 1). Finally, glucagon or AVP did not induce rapid intracellular Ca²⁺ transients (data not shown), indicating that cytosolic Ca²⁺ signaling is not involved in the glucagon- or AVP-mediated pathways in MDCT cells (18).

View larger version (9K): [in this window] [in a new window]   View larger version (8K): [in this window] [in a new window]   Fig. 1.   Glucagon and arginine vasopressin (AVP) stimulate cAMP accumulation in MDCT cells. Glucagon (A) or AVP (B) were added, at the concentrations indicated, 5 min prior to measurement of cAMP. Values are means ± SE for 3-9 observations, for 2 separate preparations. * P < 0.05, significantly different compared with control values.

Mg²⁺ uptake in MDCT cells. To determine Mg²⁺ uptake, subconfluent MDCT monolayers were cultured in Mg²⁺-free medium for 16 h. These cells possessed a significantly lower [Mg²⁺]_i ( 0.22 ± 0.01 mM) than cells cultured in normal media (0.53 ± 0.02 mM). When the Mg²⁺-depleted MDCT cells were placed in a bathing solution containing 1.5 mM MgCl₂, [Mg²⁺]_i increased with time and leveled at a [Mg²⁺]_i value of 0.52 ± 0.06 mM (n = 9), which was similar to basal levels observed in normal cells (Fig. 2). The average rate of refill, d([Mg²⁺]_i)/dt, measured as the change in [Mg²⁺]_i with time, was 164 ± 5 nM/s (n = 6 cells), as determined over the first 500 s following addition of 1.5 mM MgCl₂.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Glucagon and AVP stimulate Mg2+ uptake in Mg2+-depleted mouse distal convoluted tubule (MDCT) cells. Confluent MDCT cells were cultured in Mg2+-free media (<0.01 mM) for 16-20 h. Fluorescence studies were performed in buffer solutions in absence of Mg2+, and where indicated MgCl2 (1.5 mM final concentration) was added to observe changes in intracellular Mg2+ concentration ([Mg2+]i). Buffer solutions contained (in mM) 145 NaCl, 4.0 KCl, 0.8 K2HPO4, 0.2 KH2PO4, 1.0 CaCl2, 5.0 glucose, and 10 HEPES-Tris, pH 7.4, with and without 1.5 mM MgCl2. Glucagon, 107 M, or AVP, 3 × 107 M, were added with this buffer solution. Fluorescence was measured at 1 data point/s with 25-point signal averaging, and the tracing was smoothed according to methods previously described (29).

Glucagon stimulates Mg²⁺ uptake in MDCT cells. Glucagon, 10⁷ M, added to the refill buffer solution increased the rate of Mg²⁺ entry into Mg²⁺-depleted MDCT cells (Fig. 2). Glucagon, 10⁷ M, increased the mean Mg²⁺ entry rate from 164 ± 5 to 196 ± 11 nM/s (n = 5), which represented a stimulation of 20 ± 6% above control values. In all cases where measured, [Mg²⁺]_i returned to basal levels of 0.52 ± 0.03 mM. The effect of glucagon on Mg²⁺ uptake was concentration dependent with maximal rate of stimulation at 10⁶ M (273 ± 6 nM/s) and half-maximal stimulation at a concentration ~10⁷ M (Fig. 3). We have previously reported that dihydropyridines inhibit Mg²⁺ uptake into Mg²⁺-depleted MDCT cells (10). To determine whether glucagon-induced Mg²⁺ entry is mediated through a dihydropyridine-sensitive pathway, we examined the effect of the channel blocker, nifedipine, on the changes in [Mg²⁺]_i following placement in the refill buffer solution containing 1.5 mM MgCl₂. The presence of 10⁵ M nifedipine inhibited glucagon-stimulated Mg²⁺ uptake (24 ± 2 nM/s), indicating that this pathway is sensitive to the channel blocker and supporting the notion that glucagon-stimulated uptake is the same as the entry pathway observed in control cells (Table 2).

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Concentration dependence of glucagon stimulation of Mg2+ entry in MDCT cells. Rate of Mg2+ influx as determined by d([Mg2+]i)/dt was measured with the given glucagon concentrations, using fluorescence techniques performed as described in legend to Fig. 1. d([Mg2+]i)/dt values were determined over the first 500 s of fluorescence measurements. Values are means ± SE for 3-6 cells. * P < 0.05, significantly different from control values.

AVP stimulates Mg²⁺ entry. Figure 2 illustrates the effect of AVP, 3 × 10⁷ M, on Mg²⁺ uptake in Mg²⁺-depleted MDCT cells. The mean uptake rate, d([Mg²⁺])_i/dt, increased from control levels (164 ± 5 to 189 ± 6 nM/s, n = 6). Unlike the actions of glucagon, AVP responses were not immediate with the addition of the hormone to the refill solution. As shown, the response was initiated within 5-10 min following addition of the hormone to the superfusion solution. The reasons for this delay are not apparent at the present time. The following refill studies were uniformly performed 6 min after AVP was added to the MDCT cells. The refill rate, d([Mg²⁺]_i)/dt, was determined over a standard 500-s time interval following the 6-min delay. AVP stimulates Mg²⁺ entry in a concentration-dependent manner (Fig. 4). The maximal effect was observed at ~3 × 10⁶ M, which resulted in an increase of d([Mg²⁺]_i)/dt to 188 ± 2 nM/s. This change was significantly less than the response to maximal glucagon concentrations. AVP-stimulated Mg²⁺ uptake was inhibited by nifedipine, indicating that the hormonal response is through activation of channels responsible for Mg²⁺ entry in control MDCT cells (Table 2).

View larger version (10K): [in this window] [in a new window]   Fig. 4.   Concentration dependence of AVP stimulation of Mg2+ uptake in MDCT cells. Rate of Mg2+ influx as determined by d([Mg2+]i)/dt was measured with the given AVP concentrations using fluorescence techniques performed as described in legend to Fig. 1. Entry rate, d([Mg2+]i)/dt, was determined over 500 s, 5 min after addition of AVP. Values are means ± SE for 3-6 cells. * P < 0.05, significantly different from control values.

Glucagon- and AVP-stimulated Mg²⁺ entry does not require protein synthesis. Gesek and Friedman (20) have reported that PTH-stimulated calcium uptake in MDCT cells is sensitive to cycloheximide (1 µg/min for 30 min), whereas calcitonin actions are insensitive to protein synthesis inhibitors. Accordingly, there may be diverse pathways involved with hormonal regulation of cation transport. Pretreatment of MDCT cells with cycloheximide, 1 µg/ml for 30 min, did not alter glucagon or AVP stimulation of Mg²⁺ entry into MDCT cells (Table 3). It is apparent that de novo protein synthesis is not required for the acute actions of glucagon or AVP on Mg²⁺ uptake.

cAMP stimulates Mg²⁺ uptake in MDCT cells. Next, we determined whether activation of either protein kinase A or protein kinase C may have stimulated Mg²⁺ entry in Mg²⁺-depleted MDCT cells. Friedman et al. (16) and Hilal et al. (22) have shown that activation of both pathways are necessary for PTH-mediated increases in calcium uptake in MDCT cells and distal tubule vesicles, respectively. The activations of these kinases with cAMP and phorbol esters were each without effect alone but significantly stimulated calcium entry when administered together. In the present studies, the addition of 8-bromo-cAMP, 10⁴ M, 6 min prior to the fluorescence determinations stimulated Mg²⁺ uptake by 137 ± 6% above control values (Fig. 5). There was an apparent latent period prior to the effects of 8-bromo-cAMP of ~5-10 min, not unlike that observed for the responses of AVP. The addition of phorbol esters, on the other hand, had no apparent effect on Mg²⁺ entry at any time frame (Fig. 5). Moreover, cAMP, 10⁴ M, stimulated Mg²⁺ uptake in the presence of the phorbol ester. We infer from these studies that hormone-stimulated Mg²⁺ uptake may involve intracellular cAMP accumulation.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   cAMP stimulates Mg2+ entry in MDCT cells. Either 8-bromo-cAMP (104 M) or phorbol 12-myristate 13-acetate (PMA, 107 M) was added 6 min prior to determination of d([Mg2+]i)/dt with microfluorescence according to the techniques illustrated in Fig. 1. Values are means ± SE for 3-6 cells. * P < 0.01, significantly different from control values.

	DISCUSSION

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Glucagon is a potent renal magnesium-conserving hormone (34). Bailly and Amiel (2) reported that the acute infusion of pharmacological concentrations of glucagon into parathyroid gland-intact rats led to a rapid fall in fractional magnesium excretion from 16 ± 1% to 9 ± 2%. The response to glucagon is even greater in hormone-deprived animals (33). In these rats, fractional magnesium excretion markedly decreased by ~50% (from ~28% to basal levels) with glucagon administration (4, 5). This decrease in urinary excretion was attributed to a doubling of absolute magnesium reabsorption within the loop of Henle (increase from 6.5 ± 0.7 to 11.7 ± 0.7 pmol/min) and the distal tubule (increase from 0.85 ± 0.1 to 1.75 ± 0.3 pmol/min). In the loop, the increment in magnesium reabsorption was accompanied by an increase in calcium, sodium, potassium, and chloride, whereas in the distal tubule there was an increase in calcium transport but no change in fractional sodium, potassium, and chloride absorption (4, 32). Interestingly, Friedman and Gesek (18) failed to observe any effect of glucagon on ⁴⁵Ca²⁺ uptake into MDCT cells, even though they observed a significant increase in glucagon-stimulated cAMP formation. The micropuncture studies indicate that glucagon stimulates calcium as well as magnesium absorption in the distal tubule, although the effects on magnesium are relatively greater than those for calcium (5, 33). The results suggest that there may be quantitative differences in hormone action in the control of transport of the two divalent cations. Further studies are required to define the intracellular regulation of these cations within the nephron segments composing the distal tubule.

The micropuncture studies clearly indicated that glucagon stimulates Mg²⁺ transport in the superficial distal tubule (5). This segment comprises the distal convoluted tubule, connecting tubule, and initial cortical collecting tubule. Prior to the present studies, information was not available concerning which distal segment was involved with hormone-stimulated distal tubular magnesium reabsorption. Of the segments composing the distal tubule, the evidence from the present study clearly indicates that the convoluted portion is involved with glucagon-induced magnesium conservation. Accordingly, it is inferred that glucagon enhances active Mg²⁺ transport in the distal convoluted tubule. Cellular mechanisms of Mg²⁺ transport in the other distal segments, connecting tubule and initial collecting tubule, have not been studied.

AVP has been shown to be an effective magnesium-conserving hormone in anesthetized and conscious hormone-deprived rats (1, 6, 13). Micropuncture studies of these animals have shown that AVP actions occur principally within Henle's loop (13, 30). Using microperfusion studies, Wittner and Di Stefano (35) demonstrated that AVP enhances magnesium absorption in mouse thick ascending limbs through changes in passive transport commensurate with increases in transepithelial voltage. In the micropuncture studies with hormone-deprived rats, Elalouf and colleagues (13) failed to discern any change in fractional magnesium absorption in the superficial distal tubule following physiological administration of AVP. In these studies, Elalouf et al. (13) reported that the fractional calcium absorption increased significantly from 42.0 ± 5.8% to 62.8 ± 7.1%, whereas AVP increased fractional Mg²⁺ transport from 45.5 ± 7.8% to 55.3 ± 15.5%, but this change was not statistically significant. Costanzo and Windhager (9) did not observe any change in calcium absorption in the microperfused rat distal tubule with administration of AVP. The animals used in this latter study were thyroparathyroidectomized but not hormone deprived as were those used by Elalouf et al. (13). In both studies, AVP enhanced sodium absorption in the distal tubule. In the present studies, AVP increased Mg²⁺ entry into an established distal convoluted tubule (MDCT) cell line, showing that this hormone acts within this segment of the distal tubule and is involved with renal magnesium conservation. Mg²⁺ transport in the distal convoluted tubule is probably transcellular and active in nature because of the high resistance and lumen-negative transepithelial voltage of this segment. Accordingly, AVP likely stimulates active Mg²⁺ transport in contrast to enhancing passive Mg²⁺ absorption in the thick ascending limb (35).

Interestingly, AVP stimulates Mg²⁺ entry 5-10 min following addition of the hormone (Fig. 1). This latency period was not observed with glucagon stimulation of Mg²⁺ uptake. The reasons for these differences are not known. Gesek and Friedman (20, 21) observed similar differences in action of PTH and calcitonin stimulation of Ca²⁺ uptake in MDCT cells. There was a latency period of 10-15 min prior to PTH stimulation of Ca²⁺ entry compared with calcitonin actions, which occurred immediately upon addition of the hormone (20, 21). Although the basis for this latency period is not known, it may have something to do with translational processes and protein synthesis, because Gesek and Friedman (20, 21) showed that incubation of MDCT cells with cycloheximide for 15 min inhibited PTH-stimulated Ca²⁺ entry but had no effect on calcitonin actions. Furthermore, treatment of the cells with 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] shortened the latency period associated with PTH-stimulated increases of [Ca²⁺]_i and Ca²⁺ entry but had no effect on Ca²⁺ entry by itself (19). These workers were unable to explain the origin of the latency period but concluded that it had a genomic mechanism that involved transcription because 5-6-dichloro-1--D-ribofuranosyl benzimidazole blocked the stimulatory response of 1,25(OH)₂D₃ without inhibiting PTH actions (20). Friedman and Gesek (19) speculated that vitamin D may increase the number of PTH receptors expressed in the MDCT cells. These investigators were unable to show hormone-stimulated calcium entry with either AVP or glucagon (18). In the present studies, pretreatment of MDCT cells with cycloheximide did not inhibit either AVP- or glucagon-stimulated Mg²⁺ uptake, nor did it affect the latent period associated with AVP. It is apparent from the above-mentioned studies that peptide hormones act through different mechanisms to stimulate divalent cation transport in distal convoluted tubules. Further studies are needed to explain these diverse observations, but the hormonal control of calcium and magnesium in the distal convoluted tubule may involve unique intracellular signaling pathways.

Recent studies by Friedman et al. (16) indicate that PTH-mediated stimulation of calcium transport is through intermediary pathways involving activation of both protein kinase A and protein kinase C, as cAMP or phorbol esters had no effect on Ca²⁺ entry alone, but together they enhanced uptake. These studies are commensurate with the observations of Hilal et al. (22) using isolated membrane vesicles from rabbit distal tubules. Moreover, Lajeunesse et al. (25) have shown that ⁴⁵Ca²⁺ uptake in apical membrane vesicles harvested from rabbits pretreated with PTH is greater than those from control animals. Accordingly, PTH may have additional effects on the apical membrane to effect an increase in Ca²⁺ uptake. In the present studies, we show that addition of 8-bromo-cAMP stimulates Mg²⁺ uptake, but PMA does not increase entry rates (Fig. 4). We infer that activation of protein kinase A is involved in glucagon and/or AVP actions. However, it is not known how activation of protein kinase A stimulates Mg²⁺ uptake, nor is it known what other pathways may be involved. Glucagon appears to stimulate Mg²⁺ uptake to a greater degree than AVP, even though AVP generates similar amounts of cAMP (Figs. 1, 3, 4). On balance, the differences in time frame of hormone action and the disparity of cAMP release suggest that glucagon and AVP may operate through separate but interactive intracellular signaling pathways. More research is required to sort out these intracellular controlling mechanisms.

Although it is clear that glucagon and AVP stimulate Mg²⁺ transport in the distal tubule, it is not known what role these hormones play in orchestrating magnesium homeostasis. A protein meal markedly enhances glomerular filtration rate (GFR) and is a potent stimulus for glucagon secretion (1). Elevated circulating glucagon concentrations contribute to the disposal of nitrogen metabolites of amino acids and excretion of phosphate and potassium (1). Conversely, glucagon enhances sodium, calcium, and magnesium reabsorption within the loop of Henle and distal tubule (4, 5, 12). Glucagon may be important in magnesium conservation following a protein ingestion with its associated increase in GFR and filtered magnesium (1). Stimulation of loop and distal magnesium conservation by AVP, on the other hand, may be necessary to maintain normal magnesium balance when salt and water flow to the distal tubule are reduced during antidiuresis. Evidence has been provided by de Rouffignac (31) that glucagon and AVP may regulate electrolyte homeostasis in concert with other hormones such as PTH, calcitonin, insulin and -adrenergic agonists. These hormones act within the thick ascending limb and distal tubule to effect the complex regulation of magnesium balance.

In summary, glucagon and AVP stimulate Mg²⁺ uptake into MDCT cells, supporting the results of micropuncture reports that these hormones may act within the convoluted segment of the distal tubule. The cellular mechanisms by which glucagon and AVP stimulate uptake are unclear. The notable differences in hormone responses include the time frame of action and the disparity in maximally induced Mg²⁺ uptake, suggesting different intracellular pathways are involved with stimulation of Mg²⁺ uptake. Further studies are required to determine the intracellular signaling pathway mediated by these hormones.