I. INTRODUCTION: IMPORTANCE OF THE DISTAL CONVOLUTED TUBULE IN RENAL MAGNESIUM BALANCE

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Control of total body magnesium homeostasis principally resides within the nephron segments of the kidney. The proximal tubule reabsorbs 5-15%, the thick ascending limb of the loop of Henle absorbs 70-80%, and the distal tubule reclaims some 5-10% of the filtered magnesium. Although the distal tubule reabsorbs only 10% of the magnesium filtered through the glomerulus, this amount is significant because it represents 60-70% of the magnesium delivered to this segment from the loop of Henle. Because there is little magnesium reabsorption beyond the distal tubule in the collecting ducts, the tubule segments comprising this portion of the nephron play an important role in determining the final urinary excretion of magnesium. The purpose of this review is threefold: 1) to convince the reader that the distal tubule plays an important role in controlling renal magnesium conservation (as important as the thick ascending limb); 2) to discuss recent observations on how magnesium is reabsorbed and what controls magnesium transport within the distal tubule; and 3) to indicate some of the unresolved issues that require further investigation. Most of the recent studies involve the use of isolated cell lines which, on balance, represent the intact distal convoluted tubule (DCT).

	II. GENERAL CHARACTERISTICS OF MAGNESIUM ABSORPTION IN THE DISTAL TUBULE

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Most of our early knowledge concerning magnesium transport in the distal tubule has come from micropuncture and microperfusion studies of the superficial nephron (48, 131, 249, 251, 256, 288, 289, 344).¹ Micropuncture studies showed that significant amounts of magnesium are absorbed in the distal tubule (19, 92, 93, 248, 252, 254). The mammalian distal tubule, located between the macula densa and the cortical collecting duct (CCD), comprises a short post-macula densa segment of thick ascending limb, the DCT, the connecting tubule (CNT), and the initial collecting tubule (333). The micropuncture studies describing distal magnesium absorption may well have included portions of the superficial CNT as well as the DCT. R. J. M. Bindels failed to detect any apical-to-basolateral (absorption) or basolateral-to-apical (secretion) magnesium movement in a mixture of rabbit CNT and CCD cells (personal communication). However, there are significant functional differences among the species and segments studied. There is a gradual transition between the DCT and CNT in human and rats, whereas there is a sharp transition in the rabbit (216). In the rat and human, chlorothiazide-sensitive NaCl cotransport and parathyroid hormone (PTH)-stimulated calcium transport occurs within DCT segments and PTH-responsive Na⁺/Ca²⁺ exchange within the CNT (226). This is in contrast to the rabbit, where thiazide-sensitive NaCl cotransport is found in the DCT but PTH-responsive Na⁺/Ca²⁺ exchange is expressed only by CNT cells (10, 32, 264, 329, 330). The mouse appears to be more like the rat and human because the two transporters are colocalized to DCT cells (108). Again, the species and segmental differences of magnesium transport have not been studied.

Although significant magnesium reabsorption takes place along the distal tubule, this is normally achieved with little change in tubular magnesium concentration. Luminal magnesium concentration may increase along the distal tubule with increased magnesium delivery, or it may decrease with magnesium restriction (92, 131, 248, 298). Normally, distal magnesium absorption is fractionally less than that of sodium or calcium (254). This is commensurate with the fractional urinary excretion of magnesium, normally ~3%, whereas the fractional excretion of sodium and calcium is <1% (250). The evidence is that net distal magnesium reabsorption is essentially unidirectional because no secretion of magnesium into the lumen has been reported (254). Because the distal tubule is characterized by a negative transepithelial voltage and a high epithelial resistance, it is concluded that magnesium transport is active and transcellular in nature (165, 289). This is unlike magnesium transport within the thick ascending limb that occurs passively through the paracellular pathway (249, 289). Although the micropuncture and microperfusion studies have clearly shown that magnesium is reabsorbed in the superficial distal tubule, little knowledge has been gained concerning the cellular mechanisms of magnesium transport.

	III. MAGNESIUM UPTAKE IN ISOLATED DISTAL CELLS

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In vivo micropuncture and microperfusion approaches do not allow for study of cellular mechanisms, and in vitro microperfusion studies have not been performed on DCT because of the difficulty in isolating intact segments. Accordingly, isolated immortalized cell lines have been used to study segmental responses.

We have used an established cell line representing the DCT to study Mg²⁺ transport. This cell line (designated MDCT for mouse distal convoluted tubules) was originally isolated from mouse distal tubules and immortalized by Pizzonia et al. (238). Friedman and Gesek (111, 115-118) have shown that this cell line exhibits many of the functional properties characteristic of the intact DCT studied in vivo, such as amiloride-inhibitable Na⁺ transport, chlorothiazide-sensitive NaCl cotransport, and PTH- and calcitonin-stimulated Ca²⁺ transport. Accordingly, MDCT cells appear to have many of the properties of the convoluted segment of the intact distal tubule.

Electrolyte transport is usually quantitated by isotopic flux measurements, but an appropriate isotope for Mg²⁺ is not available (²⁸Mg has a half-life of 21 h). Accordingly, we developed a cell model to assess Mg²⁺ transport using fluorescent determinations of intracellular free Mg²⁺ concentration ([Mg²⁺]_i) (75, 253). Cytosolic free Mg²⁺ concentration of epithelial cells is on the order of 0.5 mM. This is ~1-2% of the total magnesium, the remainder being complexed to various organic and inorganic ligands and chelated within the mitochondria. Using isolated distal cell lines, we have shown that magnesium entry is through specific and regulated magnesium pathways (76, 254). To determine Mg²⁺ transport, the epithelial cells were first depleted of Mg²⁺ by incubating in magnesium-free culture media. Subsequently, the cells were placed in solutions containing magnesium and [Mg²⁺]_i measured as a function of time (Fig. 1). [Mg²⁺]_i increased until it had reached normal levels. The rate of concentration change {d([Mg²⁺]_i)/dt} is an estimate of Mg²⁺ uptake rate. Influx of Mg²⁺ is concentration dependent so that the rate of Mg²⁺ transport increases with external Mg²⁺ until saturation is attained (70, 76, 253). Mg²⁺ influx into Mg²⁺-depleted cells was inhibited by Mn²⁺ and La³⁺ and by dihydropyridine channel blockers such as nifedipine. Ca²⁺ neither blocked Mg²⁺ entry nor was ⁴⁵Ca uptake changed in the presence of Mg²⁺ depletion or the Mg²⁺-refill process. These observations suggest that the influx pathway is specific for Mg²⁺ and not shared by Ca²⁺ (76, 253). We used this approach to characterize the cellular mechanisms of Mg²⁺ uptake in MDCT cells that may shed light on magnesium transport in distal tubule cells.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Intracellular free Mg2+ concentration ([Mg2+]i) in normal and Mg2+-depleted immortalized mouse distal convoluted tubule (MDCT) cells. Confluent MDCT cells were cultured in either normal (0.6 mM Mg2+) or Mg2+-free media (<0.01 mM) for 16 h. Fluorescence studies were performed in buffer solutions in the absence of extracellular Mg2+, and where indicated, MgCl2 (5.0 mM final concentration) was added to observe changes in [Mg2+]i. The 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 5.0 mM MgCl2. The rate of rise in intracellular Mg2+ concentration, d([Mg2+]i)/dt, is a reflection of the entry rate of Mg2+ into the cell. Fluorescence was measured at 1 data point/s with 25-point signal averaging, and the tracing was smoothed according to methods previously described. [Data from Dai et al. (76).]

Many hormonal and nonhormonal factors influence Mg²⁺ uptake into MDCT cells. Our observations using MDCT cells closely resemble earlier results using micropuncture and microperfusion techniques in intact rat and dog distal tubules (Table 1). However, little is known about the cellular mechanisms of electrolyte absorption in the DCT from in vivo micropuncture and microperfusion experiments. Accordingly, the use of isolated cells has allowed a greater in-depth study of cellular mechanisms of these hormonal and nonhormonal influences. The similarity of results between the MDCT cell studies and the micropuncture experiments support the use of this cell line in characterizing cellular mechanisms of Mg²⁺ transport.

Our initial studies looked at the changes in Mg²⁺ entry into MDCT cells following alterations in membrane voltage (76). In these studies, with the same starting [Mg²⁺]_i and the same external Mg²⁺ concentration, the more negative the transmembrane voltage, i.e., more hyperpolarized, the higher was the magnesium influx rate (Fig. 2). Conversely, depolarization of membrane voltage diminishes Mg²⁺ uptake (70). The dependence of magnesium entry on the driving force induced by the electrochemical gradient indicates that Mg²⁺ entry may be mediated by an ion channel.

View larger version (12K): [in this window] [in a new window]   Fig. 2. Membrane voltage influences Mg2+ uptake in MDCT cells. Mg2+ uptake was measured with microfluorescence according to the methods given in the legend to Fig. 1. Transmembrane voltage was measured with a voltage-sensitive fluorescent dye. Hyperpolarization was achieved with addition of various concentrations of the membrane- permeable anion SCN and depolarization with addition of various concentrations of K+ to the bathing solution. The rate of Mg2+ influx, d([Mg2+]i)/dt, was calculated in the first 500 s of the Mg2+ refill in the presence of 1.5 mM MgCl2. Values are means ± SE for 3-6 cells. [Data from Dai et al. (76).]

With the above information, we are able to speculate on the mechanisms involved in magnesium absorption within the DCT cell (Fig. 3). Because the transepithelial voltage is normally in the range of 0 to 30 mV lumen negative, magnesium absorption is active in nature. Distal magnesium absorption is entirely transcellular moving across the DCT cell. Magnesium may move passively into the cell across the luminal membrane driven by a favorable transmembrane voltage. The luminal magnesium concentration is on the order of 0.2-0.7 mM depending on the condition studied, and intracellular free Mg²⁺ is 0.5 mM so that under some circumstances Mg²⁺ entry is against an appreciable concentration gradient (76, 253). We speculate that Mg²⁺ entry is through a unique channel, and this transport is dependent on the transmembrane voltage. The active step in transcellular movement is predicted to be at the basolateral membrane where Mg²⁺ leaves the cell against both electrical and concentration gradients. The means by which Mg²⁺ actively moves across the basolateral membrane is unknown. Evidence taken from studies using nonepithelial cells suggest that Na⁺/Mg²⁺ exchange may occur, Na⁺ moving back into the cell coupled with Mg²⁺ exit from the cell into the interstitium (126). Alternatively, a specific energy-dependent Mg²⁺ pump may be present analogous to that reported for calcium. Intracellular Mg²⁺ plays an important role in enzyme functions including the transport ATPases (102, 250). The ionic transport pumps such as Na⁺-K⁺-ATPase, Ca²⁺-ATPase, and H⁺-ATPase require Mg²⁺ for activity; accordingly, they are Mg²⁺-dependent ATPases (82). If an energy-dependent Mg²⁺ transporter were to exist, it would appear that Mg²⁺ would act as a cofactor, an energy source Mg²⁺-ATP², and a substrate (250). Finally, it is unlikely that intracellular Mg²⁺ concentration would normally fall to levels that would compromise ATPase activity or Mg²⁺-ATP² levels (182, 183). Because there is no evidence for an enzyme-dependent magnesium pump at the present time, we have elected not to include it in Figure 3. In this schematic view, the factors that influence transcellular magnesium absorption include alterations of Mg²⁺ entry across the luminal membrane and changes in Mg²⁺ exit across the basolateral membrane; both of these steps may be modulated by the transmembrane voltage and concentration gradients across the respective membrane.

View larger version (30K): [in this window] [in a new window]   Fig. 3. Schematic model of magnesium absorption in the distal convoluted tubule. Conductive pathways and carrier-mediated transport are denoted by solid arrows. Peptide hormones such as parathyroid hormone (PTH), calcitonin, glucagon, and arginine vasopressin (AVP) enhance magnesium reabsorption in the distal convoluted tubule (DCT). The cellular mechanisms of these hormones are unknown but appear to involve, in part, stimulation of cAMP release and activation of protein kinase A, phospholipase C, and protein kinase C. The extracellular Ca2+/Mg2+-sensing receptor modulates hormone-stimulated Mg2+ transport through Gi protein coupling (20). Steroid hormones increase Mg2+ entry by transcriptional/translational mechanisms. Aldosterone increases hormone receptor-mediated Mg2+ entry, as indicated by the broken arrows, and vitamin D metabolites by distinct pathways as yet unknown (77; G. Ritchie, L.-j. Dai, D. Kerstan, H. S. Kang, L. Canaff, G. N. Hendy, and G. A. Quamme, unpublished observations). Ca2+/Mg2+ sensing inhibits aldosterone- and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-mediated transcriptional gene expression. The sites of the transport inhibitors such as dihydropyridines, thiazides, and amiloride are indicated (70, 76). [Modified from Quamme (251).]

The properties of Mg²⁺ transport in the DCT, to some extent, resemble those described for calcium handling. Brunette et al. (46, 47) have described calcium uptake by rabbit distal apical membrane vesicles through nifedipine-sensitive Ca²⁺ channels. Matsunga et al. (206) further claimed that these dihydropyridine-sensitive Ca²⁺ channels are activated by hyperpolarizing voltages. More recently, Hoenderop and co-workers (149, 150) have identified the gene coding the Ca²⁺ channel from rabbit CCD and DCT that is activated by hyperpolarizing voltages and inhibited by dihydropyridines (149, 150). These findings concerning Ca²⁺ entry are in keeping with the earlier ideas of Costanzo (63). On the other side of the cell, two calcium transporters are responsible for Ca²⁺ efflux: an ATP-dependent calcium pump and, at least in the distal tubule, a Na⁺/Ca²⁺ exchanger (192, 264, 330). These properties of cellular Ca²⁺ handling are similar to those postulated here for Mg²⁺ transport, although the presence of a Mg²⁺ pump and Na⁺/Mg²⁺ exchanger remains to be determined. The regulation of distal magnesium reabsorption may share many of the controls identified for calcium conservation but, as reviewed below, Mg²⁺ transport may be specifically altered by influences not shared with calcium, thus affecting magnesium homeostasis independent of calcium balance.

	IV. LOAD DEPENDENCE OF MAGNESIUM ABSORPTION IN THE DISTAL TUBULE

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Distal magnesium absorption is load dependent in that an increase in magnesium delivery to the DCT is associated with an increase in magnesium absorption (19, 71, 248, 254). In one set of studies, rat distal tubules were perfused with solutions containing variable magnesium concentrations, and early and late collections were performed on the same distal tubule. These collection sites were localized in the first 10-60% of the superficial distal tubule and likely represent DCT rather than connecting tubules or initial collecting ducts (254). The first studies involved perfusing DCT with solutions containing zero magnesium. No magnesium was detected in early or late collections when the nephrons were perfused with magnesium-free Ringer solutions in either normal animals or hypermagnesemic rats in which plasma magnesium was elevated to increase the concentration gradient from plasma to lumen. These data suggested that magnesium absorption in the distal nephron is unidirectional, i.e., there is no magnesium secretion in the DCT. Net magnesium absorption was observed when the distal tubules were perfused with solutions containing magnesium. Absolute magnesium absorption increased from 1.35 ± 0.35 to 6.5 ± 0.9 pmol·min¹·mm¹ as magnesium in the perfusate was increased from 0.5 to 2.5 mM; accordingly, there was about a fivefold increase in magnesium absorption with about a fivefold increase in luminal magnesium concentration (Fig. 4). PTH increased absolute and fractional magnesium absorption independent of delivery. Increased magnesium delivery due to hypermagnesemia or hypercalcemia resulted in an increase in absolute absorption but a decrease in fractional transport (246, 254). Other studies were performed where furosemide was used to inhibit loop magnesium absorption, thereby increasing magnesium delivery to the DCT (248). Absolute magnesium reabsorption increased from 0.6 ± 0.2 to 3.0 ± 1.7 pmol·min¹·mm¹ with increased delivery, whereas fractional absorption remained unchanged in these experiments. The basis for the dependence of magnesium absorption on magnesium delivery and luminal magnesium concentration is also observed at the single-cell level (76). Magnesium reabsorption in the DCT is under the control of a number of hormones and altered by a number of influences. However, the dependence of magnesium absorption with magnesium delivery is maintained in the presence of these factors (246, 254).

View larger version (9K): [in this window] [in a new window]   Fig. 4. Effect of luminal magnesium on net magnesium absorption in the superficial rat distal tubule. Distal tubules were perfused from the proximal tubule at constant flow rates (25 nl/min) with solutions containing variable MgCl2 concentrations (0-4 mM). Magnesium absorption was calculated by comparing the delivery rates at early and late collection sites along the same perfused distal tubule. Magnesium was quantitated by electron microprobe analysis and water absorption with the use of inulin. The results are from thyroparathyroidectomized rats. [Data from Quamme and Dirks (254).]

	V. HORMONAL CONTROL OF MAGNESIUM TRANSPORT IN THE DISTAL CONVOLUTED TUBULE

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A.  Peptide Hormones

1.  PTH

A large number of hormones stimulate magnesium absorption within the distal tubule (Table 1). The first to be described was PTH. Infusion of PTH to thyroparathyroidectomized (TPTXed) animals increased the reabsorption of magnesium and diminished urinary magnesium excretion (204). Micropuncture studies showed that part of this hormonal action occurred within the distal tubule (19, 131, 254). An increase in magnesium conservation was observed even in the face of enhanced magnesium delivery to this segment (248, 252, 254). The largest changes were observed in TPTXed hamsters where the mean tubular fluid-to-ultrafiltrable magnesium ratio (TF/UF_Mg) at the distal sampling site fell from 0.56 ± 0.08 to 0.33 ± 0.08 after administration of PTH (131). This was associated with a fall in fractional magnesium excretion from ~14 to 3%. De Rouffignac et al. (287) and Bailly and Amiel (16) have shown that PTH and other hormones stimulate magnesium absorption in the rat distal tubule. They used Brattleboro rats with hereditary diabetes insipidus, which lack endogenous ADH, and they infused either glucose or somatostatin to inhibit glucagon secretion. Furthermore, they TPTXed the animals to eliminate circulating PTH and calcitonin. Thus a "hormone-deprived" animal model was created to serve as a basis for evaluating the respective actions of each hormone (287). Micropuncture studies were then performed to determine the effects of hormone administration; importantly, these studies were all performed with physiological hormone concentrations. Infusion of PTH-(1---34) to hormone-deprived rats led to diminished magnesium and calcium delivery to early and late distal tubule sampling sites (Fig. 5). These studies clearly demonstrate that PTH enhances magnesium absorption within the distal tubule. Dai and Quamme (75) showed that PTH also stimulates Mg²⁺ entry in isolated MDCT cells in excess of 30% above control uptake rates. The change in transport was associated with increases in hormone-mediated cAMP formation, suggesting that PTH acts, in part, through protein kinase A signaling pathways (Fig. 6).

View larger version (15K): [in this window] [in a new window]   Fig. 5. Fractional reabsorption of magnesium in the superficial distal tubule. Studies were performed in "hormone-deprived" rats lacking vasopressin, PTH, calcitonin, and glucagon. Tubular fluid was collected from the early and late sites of the same distal tubule, and net magnesium reabsorption is expressed as a fraction of that delivered to the segment. The PTH (5 mU/min) and glucagon (5 ng/min) studies were from Bailly et al. (19), and those of calcitonin (1.0 mU/min) and AVP (20 pg/min) were from Elalouf et al. (92) and Rouffignac et al. (286), respectively. Values are means ± SE. *Significance from the respective control values.

View larger version (20K): [in this window] [in a new window]   Fig. 6. Peptide hormones stimulate intracellular cAMP formation and 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. Measurement of cAMP was performed with radioimmunoassay. Fluorescence studies were performed in buffer solutions in the absence of Mg2+, and where indicated, MgCl2 (1.5 mM final concentration) was added to observe changes in intracellular Mg2+ concentration. PTH, calcitonin, glucagon, and AVP were added to the buffer solution where indicated. Values are means ± SE. *Changes in significance (P < 0.01) of Mg2+ uptake. and +Significance (P < 0.001) of cAMP release from the respective control values. [Data from Dai et al. (67, 69) and G. Ritchie, L.-j. Dai, D. Kerstan, H. S. Kang, L. Canaff, G. N. Hendy, and G. A. Quamme, unpublished data.]

Calcitonin infusions have clearly been shown to enhance renal magnesium conservation in the rat (243). Poujeol et al. (243) infused calcitonin to TPTXed rats and observed a fall in fractional magnesium excretion from 4.1 ± 0.4 to 1.0 ± 0.3%, which they attributed to an increase in magnesium reabsorption in the loop of Henle. However, subsequent studies with the hormone-deprived rat showed that calcitonin markedly stimulated fractional magnesium absorption in the superficial distal tubule as well as the loop (81). Fractional absorption in the distal tubule increased by 27 ± 6% in this study (Fig. 5). These micropuncture studies indicate that calcitonin enhances magnesium conservation, in part, by actions within the distal tubule. Calcitonin has been shown to stimulate intracellular cAMP formation and Mg²⁺ entry in MDCT cells, indicating that the DCT segment of the distal tubule is a target for this hormone (Fig. 6). Maximal concentrations of calcitonin increased Mg²⁺ uptake by 49 ± 5%, which is greater than that observed for PTH. Again, hormone-stimulated Mg²⁺ entry rates were associated with increases in intracellular cAMP levels.

Glucagon is a potent renal magnesium-conserving hormone (16). Bailly and Amiel (16) reported that the acute infusion of pharmacological concentrations of glucagon in parathyroid gland-intact rats leads to a rapid fall in fractional magnesium excretion from 16 ± 1 to 9 ± 2% (16). The response to glucagon is even greater in hormone-deprived animals. Bailly et al. (19) showed that fractional magnesium excretion markedly decreased by ~50% (from 71.5 ± 8.0 to 39.6 ± 5.7 nmol/min) with glucagon administration in rats deficient in endogenous PTH, calcitonin, glucagon, and antidiuretic hormone. This was attributed to a doubling of absolute reabsorption within both the loop of Henle (increase from 6.5 ± 0.7 to 11.7 ± 0.7 nmol/min) and the distal tubule (increase from 0.85 ± 0.1 to 1.75 ± 0.3 nmol/min). Accordingly, glucagon acts within the loop and distal tubule of the rat.

We have performed studies in isolated MDCT cells to determine the cellular mechanisms of hormonal stimulation of magnesium transport (67). Glucagon maximally increased Mg²⁺ uptake by ~20% (Fig. 6). This stimulation was concentration dependent, associated with intracellular cAMP generation, and inhibited by the channel blocker nifedipine. Clearly, of the segments comprising the distal tubule, the evidence from this study indicates that the convoluted portion is involved with glucagon-induced magnesium conservation.

The actions of arginine vasopressin (AVP) within the DCT are poorly understood (165). AVP has been shown to be an effective magnesium-conserving hormone in anesthetized and conscious hormone-deprived rats (37, 93, 286). Micropuncture studies of these animals have shown that AVP actions occur principally within Henle's loop (93). Elalouf et al. (93) failed to discern any change in fractional magnesium absorption in the superficial distal tubule after physiological administration of AVP (93). In these studies, Elalouf et al. (93) reported that fractional calcium absorption significantly increased from 42.0 ± 5.8 to 62.8 ± 7.1%, whereas the change in fractional magnesium transport with AVP was not significant although it increased from 45.5 ± 7.8 to 55.3 ± 15.5% (Fig. 5). These changes may have been significant if a greater number of tubules were sampled. Costanzo and Windhager (65) did not observe any change in calcium absorption in the microperfused rat distal tubule with administration of AVP. These animals were TPTXed but not hormone deprived as were those used by Elalouf et al. (93). In both studies, AVP enhanced sodium absorption in the distal tubule.

AVP also stimulates Mg²⁺ entry into MDCT cells in a concentration-dependent fashion that is sensitive to nifedipine (Fig. 6). These observations suggest that AVP plays a role in control of magnesium conservation within the DCT (67). Many of the hormones that have been shown to increase magnesium reabsorption in the distal tubule have additive effects (91, 285). AVP and glucagon are additive, below maximal concentrations, in stimulating Mg²⁺ uptake into MDCT cells (67). Accordingly, these hormones, and probably others, together orchestrate magnesium reabsorption in the distal tubule. It would be difficult to predict the effects of one hormone on a background of many. This was the rationale for De Rouffignac (285) using hormone-deprived animals in their studies investigating individual hormone effects. The advantage of using isolated MDCT cells is that hormone-mediated signaling pathways may be investigated, which is difficult or impossible to perform with in vivo studies.

The cellular mechanisms underlying the hormonal actions on distal magnesium absorption are becoming clearer. Morel and co-workers (17, 215) have shown that PTH, calcitonin, and glucagon stimulate receptor-mediated cAMP release in the DCT (45). They also reported that AVP receptors may be present in the DCT, but there were marked species differences in adenylate cyclase responsiveness (217). There was little AVP-stimulated cAMP release in the DCT of the rabbit and human, intermediate response in the mouse, and greatest in the rat (215). We have shown that PTH, calcitonin, glucagon, and AVP stimulate Mg²⁺ uptake into MDCT cells (21, 67, 69; Fig. 6). Friedman and Gesek (108, 113, 117) reported that these hormones also stimulate cellular cAMP accumulation. We have demonstrated that an increase in exogenous intracellular cAMP, with 8-bromo-cAMP, or endogenous cAMP formation, with forskolin, increased Mg²⁺ uptake, whereas inhibition of protein kinase A with R_p-cAMPS prevented hormone-stimulated uptake (67). Accordingly, receptor-mediated cAMP release and activation of protein kinase A plays a role in hormone-stimulated Mg²⁺ uptake in MDCT cells. However, it is apparent that other signaling pathways are present for hormone-mediated Mg²⁺ uptake in MDCT cells. Hormone-stimulated Mg²⁺ uptake rates do not correlate with the measured intracellular cAMP levels in MDCT cells (38, 107, 138). Furthermore, phospholipase C inhibition with U-73122 and protein kinase C inhibition with Ro31-8220 abolished PTH- and calcitonin-stimulated Mg²⁺ uptake (Fig. 7). This was true for all of the hormones tested: PTH, calcitonin, glucagon, and AVP (67-69; L.-j. Dai, G. Ritchie, D. Kerstan, H. S. Kang, and G. A. Quamme, unpublished observations). We have previously reported that chelerythrine, a putative protein kinase C inhibitor, did not alter hormone-mediated responses (67, 69). However, this agent has recently been shown to have no inhibitory actions on protein kinase C (176). Our evidence indicates that protein kinase C is involved in hormone signaling responses in MDCT cells; however, the isotype(s) of protein kinase C is not known. These hormones do not elicit receptor-mediated intracellular Ca²⁺ transients, suggesting that Ca²⁺ signaling is not involved with the responses (110, 116). It is well known that multiple receptors can converge on a single G protein, and in many cases a single receptor can activate more than one G protein and thereby modulate multiple intracellular signals (128). It is evident that cAMP-dependent protein kinase A, phospholipase C, and protein kinase C pathways are necessary for hormone-stimulated Mg²⁺ entry into MDCT cells. The details of how peptide hormones act on Mg²⁺ transport in MDCT cells and intact distal tubules are unknown.

View larger version (14K): [in this window] [in a new window]   Fig. 7. PTH stimulates Mg2+ uptake through cAMP- and phospholipase C-mediated pathways. The inhibitors for protein kinase A, Rp-cAMPS, phospholipase C, U-73122, protein kinase C, and Ro31-822, were added at concentrations of 0.05, 15, and 0.1 µM, respectively, 10 min before the determination of Mg2+ uptake with and without PTH (107 M). Values are means ± SE. *Significance (P < 0.01) of Mg2+ uptake in the presence of Rp-cAMPS versus control for PTH, respectively. +Significance (P < 0.01) of cAMP and Mg2+ entry rates with PTH vs. the respective control values. [Data from Dai et al. (74) and G. Ritchie, L.-j. Dai, D. Kerstan, H. S. Kang, L. Canaff, G. N. Hendy, and G. A. Quamme, unpublished data.]

These observations of hormone-stimulated Mg²⁺ entry are somewhat different from those reported for Ca²⁺ entry into MDCT cells. Friedman and Gesek (108, 113, 117) showed that PTH and calcitonin increased calcium uptake in the DCT through changes in membrane voltage. They provided evidence that receptor-mediated increases in cAMP activate basolateral Cl channels resulting in cellular efflux of Cl, diminished intracellular Cl activity, and hyperpolarization of the apical membrane of the MDCT cell through a decrease in the electrochemical Cl gradient (110). Membrane hyperpolarization activates Ca²⁺ entry and increases the driving force for Ca²⁺ movement into the cell which, in turn, is removed across the basolateral membrane into the blood. More recent studies indicate that PTH-mediated stimulation of calcium transport is through intermediary pathways involving activation of both protein kinase A and protein kinase C (107, 144). PTH and other hormones may also directly act on Ca²⁺ entry through apical Ca²⁺ channels and Ca²⁺ exit through Na⁺/Ca²⁺ exchange, again through activation of these kinases (10, 11, 38). Brunette and co-workers (144, 174) have shown that distal luminal vesicles prepared from rabbit tubules pretreated with PTH transport much more calcium than vesicles from untreated tubules, inferring a covalent modification of putative apical Ca²⁺ channels. Accordingly, PTH and possibly other hormones may have direct actions on Ca²⁺ entry in addition to their ability to alter the membrane voltage. Further research is required to determine the cellular mechanisms underlying the action of these hormones on distal divalent cation absorption. Interestingly, Friedman and Gesek (108) failed to demonstrate any effect of glucagon and AVP on Ca²⁺ entry into MDCT cells, although these hormones increased cellular cAMP accumulation (108). It is apparent that hormonal controls of Ca²⁺ entry in MDCT cells are different from those reported for Mg²⁺. In support of this notion, differential controls of renal Ca²⁺ and Mg²⁺ transport are apparent in the clinical presentation of many familial and acquired diseases (see sects. IX and X).

Finally, a comment should be made concerning the differences between hormone-mediated Ca²⁺ within the CCD and hormone-stimulated Mg²⁺ uptake in MDCT cells because it emphasizes different controls for these two divalent cations in the distal tubule. Hoenderop et al. (151) have extensively characterized hormone-stimulated Ca²⁺ transport in primary rabbit CCD cells. The rabbit CCD does not reabsorb Mg²⁺ even with chronic magnesium depletion (R. J. M. Bindels, personal communication). However, the comparisons of hormonal controls between the two segments are informative. The rate of transepithelial Ca²⁺ reabsorption within the rabbit CCD is determined by an apical 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]-responsive calcium channel (149). This Ca²⁺ channel (ECaC) is activated by hyperpolarization but does not transport Mg²⁺, nor does external Mg²⁺ inhibit Ca²⁺ transport, clearly different from the Mg²⁺ transporter (150). Ca²⁺ reabsorption is stimulated by peptide hormones such as PTH and AVP (32). It has been reported that Ca²⁺ transport is stimulated by hormone-mediated signaling pathways that are independent of cAMP but involve a chelerythrine-inhibitable protein kinase C that is not downregulated after chronic phorbol ester treatment (31, 146, 155, 331). Our studies have shown that both cAMP-protein kinase A and protein kinase C (not sensitive to chelerythrine) are essential in hormonal action within the MDCT cells. Accordingly, hormone signaling within the two segments is qualitatively different. The differential control of calcium reabsorption within the DCT and CCD and magnesium transport in the DCT provides a unique mechanism to differentially regulate renal calcium and magnesium balance.

B.  Steroid Hormones

1.  Mineralocorticoid hormones

Mineralocorticoid receptors are present in DCT cells, which are thought to be involved in expression of NaCl cotransport, Na⁺ conductance, and sodium pump activity (57, 283, 333). The effects of aldosterone on distal tubule magnesium absorption have not been studied with micropuncture techniques. Clearance studies have shown that chronic aldosterone administration results in renal magnesium wasting, but this has been explained by extracellular volume expansion leading to diminished NaCl and magnesium reabsorption within the loop (202, 203, 319). We have studied the effects of aldosterone on Mg²⁺ entry into MDCT cells (77). Incubation of aldosterone, for 16 h before determination of Mg²⁺ uptake, failed to have any effect on basal magnesium transport. However, pretreatment of MDCT cells with aldosterone potentiated hormone-stimulated Mg²⁺ uptake (Fig. 8). This was associated with potentiation of hormone-mediated cAMP release in aldosterone-treated MDCT cells (77). Cycloheximide, an inhibitor of protein synthesis, abolished the potentiation of aldosterone on hormone-stimulated cAMP release and Mg²⁺ uptake (59). Accordingly, aldosterone may enhance hormone-stimulated Mg²⁺ entry by increasing cAMP and its responses. This notion is supported by the observations of others (155). Rajerison et al. (259) demonstrated that adrenalectomy reduced AVP-stimulated adenylate cyclase activity in membrane fractions prepared from rat kidney medulla. Doucet and co-workers (85, 95) have shown that glucagon- and AVP-responsive cAMP generation is diminished in thick ascending limb and collecting tubule segments harvested from adrenalectomized rats compared with animals treated with physiological doses of aldosterone. These investigators postulate that aldosterone induces a protein(s) that stimulates hormone-sensitive adenylate cyclase activity. Studies with kidney membrane fractions and isolated segments in the absence of aldosterone demonstrated an impairment of coupling between hormone receptors and adenylate cyclase catalytic units that was responsible for diminished cAMP generation (95). Steroid hormones have significant effects on expression and posttranslational targeting of heterotrimeric G proteins so that associated channels are covalently modified (218, 281, 293). The mechanism(s) through which steroids control G_S proteins (synthesis and/or degradation vs. activity of each unit) associated with Mg²⁺ uptake in MDCT cells is not known (85). The role of mineralocorticoids in the physiological maintenance of renal magnesium handling also requires further research.

View larger version (17K): [in this window] [in a new window]   Fig. 8. Aldosterone potentiates hormone-stimulated intracellular cAMP formation and Mg2+ entry in MDCT cells. MDCT cells were treated with aldosterone (107 M) where indicated for 16 h before cAMP determinations and fluorescent measurements. PTH (107) was added 5 min before cAMP and immediately with Mg2+ uptake determinations. Values are means ± SE. *Significance (P < 0.01) of Mg2+ uptake values. +Significance (P < 0.001) of cAMP analysis from control values. [Data from Dai et al. (77).]

Vitamin D₃ metabolites have important effects on mineral metabolism by 1,25(OH)₂D₃ actions on epithelial transport. Although it is clear that 1,25(OH)₂D₃ increases calcium and magnesium absorption within the intestine, its actions within the kidney are unclear (172, 332). The 1,25(OH)₂D₃-dependent calcium binding proteins, calbindin-D_9K and calbindin-D_28K, have long been thought to be involved in facilitating calcium transport across epithelial cells (100, 172, 332). The mechanisms of calcium stimulation are obscure, but because of its close association with the basolateral membrane, it is postulated that they somehow increase the calcium pump (Ca²⁺-Mg²⁺-ATPase) activity (33, 36, 172). Within the kidney, calbindin-D is localized in the distal tubule where a significant portion of calcium and magnesium is reabsorbed (187, 197, 284, 316, 324) . The distal tubule, including the convoluted segment, also possesses 1,25(OH)₂D₃ receptors (186, 187, 284). Accordingly, 1,25(OH)₂D₃ and perhaps its dependent calbindin-D may have significant actions within the DCT. The effects of 1,25(OH)₂D₃ and calbindins on magnesium transport are unknown. Calbindin-D_9K has a relatively high affinity for Mg²⁺ (6, 49, 185), and it is appropriately altered by changes in magnesium balance (137), suggesting a role for these binding proteins in renal magnesium control.

In contrast to calcium, little information is available concerning the effects of vitamin D₃ metabolites on tubular Mg²⁺ transport. On balance, data from clinical or experimental studies indicate little or no effect of 1,25(OH)₂D₃ on renal magnesium handling as determined by clearance techniques (51, 129, 130, 184, 213, 219, 270). There have been no micropuncture studies performed to localize the actions, if any, of vitamin D₃ on magnesium transport. The effects of 1,25(OH)₂D₃ on renal calcium absorption are more substantial but not clear (32, 39, 40, 64, 166). The most convincing data demonstrating that 1,25(OH)₂D₃ may have some direct effects on Ca²⁺ transport are those using isolated cells. Bindels and co-workers (32, 330) showed that 1,25(OH)₂D₃ increased calbindin-D_28k and stimulated transcellular calcium absorption in primary cultures of the rabbit CCD. The maximal response occurred about 48 h posttreatment, suggesting to these investigators that the response involved initiation of transcriptional processes (32). The responses of 1,25(OH)₂D₃ were independent of PTH and not additive to PTH-stimulated Ca²⁺ transport. In a more recent report, these authors have shown that 1,25(OH)₂D₃ increased calbindin-D_28k RNA and protein content without a change in Na⁺/Ca²⁺ exchanger or Ca²⁺-ATPase RNA and protein (32). The second study to clearly show effects of vitamin D₃ metabolites on calcium transport was performed in MDCT cells by Friedman and Gesek (109). These workers reported that 1,25(OH)₂D₃ did not alter basal Ca²⁺ uptake but accelerated PTH-dependent calcium entry rates. This response was rapid, concentration dependent, significant at 2 h and maximal by 5 h, and mediated by transcriptional processes because it was inhibited by cycloheximide (109). The reasons for the discrepancies between these two reports are not known; it may be the cell type used in the two separate studies or the techniques by which calcium transport was measured. Nevertheless, it is clear that 1,25(OH)₂D₃ increases calcium-binding protein in distal tubules and suggests that it may have significant actions on basal or hormone-mediated calcium transport.

Lui et al. (186) have shown that 1,25(OH)₂D₃ enhances calbindin-D in murine distal cells as it does in other species (8, 59, 66, 330). Friedman and Gesek (110) have reported that the MDCT cell line used here possesses calbindin-D. We have demonstrated that 1,25(OH)₂D₃ increases Mg²⁺ entry rates in MDCT cells (Fig. 9). The response is concentration dependent, involves transcriptional processes involving de novo protein synthesis, and does not appear to be related to cAMP-mediated stimulation of Mg²⁺ uptake (G. Ritchie, L.-j. Dai, D. Kestan, H. S. Kang, L. Canaff, G. N. Hendy, and G. A. Quamme, unpublished observations). Finally, 1,25(OH)₂D₃-stimulated Mg²⁺ transport is additive to PTH-mediated uptake, suggesting that the peptide and steroid hormones regulate magnesium absorption through distinctive intracellular signaling pathways (Fig. 9). These studies show that vitamin D₃ metabolites modulate magnesium transport in the DCT.

View larger version (20K): [in this window] [in a new window]   Fig. 9. 1,25(OH)2D3 and PTH stimulate Mg2+ entry into MDCT cells by separate mechanisms. Maximally effective concentrations of 1,25(OH)2D3 (107 M) and PTH (107 M) were added where indicated. cAMP was measured by radioimmunoassay and d([Mg2+]i)/dt by fluorescence. Values are means ± SE for 4 or 5 preparations. *Significance (P < 0.01) of Mg2+ uptake rates. +Significance of cAMP concentrations following PTH or 1,25(OH)2D3 plus PTH compared with the respective control values. Significance (P < 0.01) of Mg2+ uptake of 1,25(OH)2D3 vs. 1,25(OH)2D3 plus PTH. [Data from Dai et al. (68).]

Vitamin D₃ administration has often been associated with increases in urinary magnesium and calcium excretion. Vitamin D₃ metabolites increase intestinal absorption of magnesium and calcium so that a positive divalent cation balance may lead to hypermagnesemia and hypercalcemia and increased divalent cation urinary excretion over time. Vitamin D₃ induces hypermagnesemia and hypercalcemia that diminishes magnesium absorption in the loop of Henle (254) and distal tubule (20, 254) through the extracellular Ca²⁺/Mg²⁺-sensing receptor (see sect. VI). This may also explain the increase or no change in urinary magnesium excretion after administration of vitamin D₃. The net effect on magnesium balance would thus depend on the relative magnitudes of vitamin D₃ actions at the intestinal and renal levels.

PGE₂ is the major arachidonate metabolite synthesized by cyclooxygenase in the mammalian kidney. PGE₂ has a number of diverse actions on the kidney in addition to its ability to influence renal hemodynamics. PGE₂ inhibits NaCl absorption within the thick ascending limb (313) and modulates sodium and water transport in the CCD (134, 135). On balance, prostaglandins are thought to be natriuretic by way of their actions on the thick ascending limb and CCD (18, 313). Three clearance studies concluded that arachidonic acid metabolites inhibit tubular reabsorption of calcium and magnesium resulting in increased urinary excretion (106, 282, 295). Schneider et al. (295) infused PGE₂ into dog renal arteries and showed that calcium and magnesium excretion increased in association with a rise in urinary sodium excretion. Roman et al. (282) and Friedlander and Amiel (106) reported that meclofenamate or indomethacin infusion in rats decreased fractional magnesium excretion by ~40%. Again, the changes in urinary magnesium and calcium were associated with similar changes in sodium excretion. Because PGE₂ inhibits NaCl absorption in the thick ascending limb, it may be expected that prostaglandins would increase calcium and magnesium excretion through diminished reabsorption in the loop (342). However, van Baal et al. (330) have shown that PGE₂ stimulated calcium reabsorption in the rabbit CCD segment of the distal tubule. They reported that PGE₂ stimulated net apical-to-basolateral calcium transport in CCD cells grown to confluence on permeable supports. PGE₂ also stimulated cAMP formation in these cells, initially suggesting that protein kinase A-dependent pathways were involved (249). However, in a later preliminary report, these investigators reported that the changes in PGE₂-stimulated calcium transport were not directly associated with cAMP formation so that other signaling pathways may be present in rabbit CCD cells (146). Finally, van Baal et al. (329) have shown that primary CCD cells produce endogenous prostaglandins that affect basal calcium transport. Like the CCD, the DCT synthesizes prostaglandins, principally PGE₂ (35, 99). The above functions are mediated by four different prostaglandin receptors (EP₁, EP₂, EP₃, and EP₄) that are selectively located to the apical and/or basolateral epithelial membranes (42, 61, 135, 291, 317, 329). EP₁ and EP₃ subtypes mediate intracellular Ca²⁺ signaling and inhibition of adenylate cyclase, respectively, that result in inhibition of NaCl absorption within the thick ascending limb (313) and CCD (107, 155, 244) and AVP-stimulated water transport in the CCD (134). EP₂ and EP₄ subtypes are coupled to adenylate cyclase that upon stimulation enhances transepithelial calcium transport in the rabbit CCD (329). Moreover, these receptors may be colocalized to the same cell type but polarized to apical or basolateral membranes (135, 213, 329). Van Baal et al. (329) have shown that apical and basolateral PGE₂ stimulate calcium absorption through EP₂ and/or EP₄ receptors, whereas activation of basolateral EP₃ receptors inhibits basal and hormone-stimulated calcium transport. We have shown that PGE₂ is a potent stimulator of Mg²⁺ uptake into MDCT cells (69). These actions are, in part, through cAMP-mediated mechanisms, but we were unable to determine the polarization of receptors because the immortalized MDCT cells used do not form tight junctions and are unlikely to be polarized (110). Accordingly, it is not known if the PGE₂ effects in the DCT are due to luminal or basolateral prostaglandin. We infer from these results with MDCT cells that prostaglandins may modulate distal tubule magnesium transport and together with peptide and steroid hormones orchestrate renal magnesium conservation.

Insulin has clearly been shown to have antinatriuretic and antimagnesiuric effects by its actions on the thick ascending limb of the loop of Henle (78, 79, 153, 163, 195). Among the tubular segments studied, insulin receptor binding is highest along the thick ascending limb and DCT so that by inference insulin may affect electrolyte transport within the DCT as well as the loop (52, 101, 105, 221). Insulin also stimulates sodium transport in A₆ cells, which are a distal cell line from Xenopus laevis that have properties similar to the mammalian distal nephron (143, 205, 262). Insulin-stimulated sodium transport was partly inhibited by genistein, indicating tyrosine kinase was important but was independent on cAMP levels (262). Insulin did not increase cAMP formation in A₆ cells, nor did adenylate cyclase inhibition diminish transport (205, 276). Because genistein did not completely inhibit insulin actions, Rodriguez-Commes et al. (276) suggested that parallel non-tyrosine kinase-dependent pathways were also involved. They showed that insulin actions in A₆ cells required intracellular Ca²⁺ signaling, suggesting to these investigators that protein kinase C is needed for some of the responses. Clearly, the insulin-receptor signaling pathways are different in the various cell types studied. We have recently shown that insulin stimulates Mg²⁺ uptake in MDCT cells (77). Insulin stimulated Mg²⁺ entry in a concentration-dependent manner with maximal response of 214 ± 12 nM/s, which represented a 30 ± 5% increase in the mean uptake rate above control values, d([Mg²⁺]_i)/dt, of 164 ± 5 nM/s. This was associated with a 2.5-fold increase in insulin-mediated cAMP generation, 52 ± 3 pmol·mg protein¹·5 min¹. Genistein, a tyrosine kinase inhibitor, diminished insulin-stimulated Mg²⁺ uptake, back to control values, 169 ± 11 nM/s, but did not change insulin-mediated cAMP formation, 47 ± 5 pmol·mg protein¹·5 min¹. The evidence is that insulin stimulates Mg²⁺ entry into MDCT cells through genistein-sensitive tyrosine phosphorylation. Additionally, insulin stimulates cAMP formation in MDCT cells and presumably activates protein kinase A (77). Maximal concentrations of PTH plus insulin increased cAMP levels and Mg²⁺ entry rates to a greater extent than each of the hormones alone (Fig. 10). Mandon et al. (195) have explained this potentiation of insulin with other peptide hormones as interactions at different sites along the established hormone-adenylate cyclase signaling system. The actions of insulin-mediated effects on peptides hormone responses are complicated because aldosterone had no effect on insulin actions but potentiated PTH-stimulated cAMP and Mg²⁺ uptake (77). Although the intracellular mechanisms are unclear, we infer from these studies that insulin plays a singular role in control of magnesium conservation and modulates hormonal regulation of magnesium transport within the distal tubule.

View larger version (21K): [in this window] [in a new window]   Fig. 10. Insulin potentiates PTH-stimulated Mg2+ entry into MDCT cells. Maximal concentrations of insulin (107 M) and PTH (107 M) were added where indicated. cAMP was measured by radioimmunoassay and d([Mg2+]i)/dt by fluorescence. Values are means ± SE for 3-5 preparations. * and +Significance (P < 0.01) of Mg2+ entry rates and cAMP concentrations, respectively, compared with the respective control values. Significance (P < 0.01) of these data for insulin plus PTH compared with either insulin or PTH alone. [Data from Dai et al. (68).]

E.  Other Hormones and Factors

1.  - and -adrenergic agonists

The nephron is richly innervated along its length from the glomerulus to the collecting tubule (22). Renal nerve stimulation significantly increases NaCl and water reabsorption in the proximal tubule, loop of Henle, and distal tubule (83, 113). Renal nerves also mediate calcium reabsorption through -adrenergic receptors (14). Gesek (113) has reported that MDCT cells possess ₂-adrenergic receptors, and epinephrine or B-HT 933, an ₂-agonist, stimulates Na⁺ uptake and Na⁺-K⁺-ATPase activity in this cell line. This response was dependent on protein kinase C activity and was associated with increases in phospholipase C and inositol 1,4,5-trisphosphate and diacylglycerol, but ₂-agonists had no effect on basal or hormone-stimulated cAMP accumulation (113, 114). These findings are consistent with a pertussis toxin-insensitive mechanism. Interestingly, Gesek (113) was unable to observe an effect on Ca²⁺ uptake in MDCT cells. The actions of -adrenergic agonists on Mg²⁺ transport in the DCT have not been studied.

-Adrenergic agonists also mediate direct effects on tubular transport (22, 211). Although -adrenergic agents have been shown to increase magnesium absorption in the thick ascending limb, no experiments have been directed at the distal tubule (18). Gesek and White (119) have demonstrated that MDCT cells possess ₁- and ₂-receptor subtypes that upon activation with isoproterenol elicited marked increases in cAMP formation (119). Recently, we tested the effects of -adrenergic receptor activities on Mg²⁺ uptake in Mg²⁺-depleted MDCT cells. Isoproterenol increased Mg²⁺ entry by 18 ± 4% and cAMP formation 3.2-fold above control values. Although not tested, -adrenergic activation may stimulate Mg²⁺ uptake through cAMP-mediated pathways (119). We infer from these studies with MDCT cells that renal nerves and circulating catecholamines may play a role in control of magnesium transport in the distal tubule.

In addition to the hormones indicated above, others may be involved in regulation of magnesium reabsorption within the DCT including kinins and growth factors (239). Hoenderop et al. (147) have reported that adenosine increases Ca²⁺ reabsorption in rabbit CNT and CCD cells. Adenosine responses were through A₁ receptor-mediated pathways involving activation of phospholipase C. Stimulation of Ca²⁺ transport was independent of cAMP and protein kinase C activity, suggesting that an additional unidentified signaling pathway may be involved in adenosine responses. Based on these observations, we tested whether adenosine stimulates Mg²⁺ uptake in MDCT cells. Adenosine did not have any effect on Mg²⁺ increase in MDCT cells but stimulated cAMP formation, indicating the presence of A₂ receptors acting through G_s coupling. There was no evidence for A₁ receptors in this cell line. Additionally, DCT cells, including MDCT cells, have P₂ purinogenic receptors that modulate distal hormone-sensitive calcium transport (167). Finally, ANP receptors are present in the DCT and MDCT cells, but the effects on magnesium transport have not been studied (Dai et al., unpublished observations). ANP stimulates calcium reabsorption in rabbit CCD through cGMP-dependent protein kinase type III (148).

Micropuncture and microperfusion studies and experiments with isolated cells have shown that many hormones regulate magnesium absorption in the distal tubule. Interestingly, with the exception of prostaglandins, all of these hormones and factors also increase magnesium transport in the thick ascending limb of the loop of Henle (285). Accordingly, these hormone-mediated responses are serially organized to similarly regulate magnesium absorption in both the loop and distal tubule. This organization may be of benefit because influences acting in either the loop or distal tubule may be modified by changes in the other segment.

The interactions of the various peptide and steroid hormones, prostaglandins, and renal innervations are complex (251, 285). It can be inferred that overall distal magnesium absorption is controlled by all of these influences initiated individually but coming together through shared intracellular signaling pathways. Few studies have been directed at describing these interactions. Clearly, control of renal magnesium handling is important enough to warrant multiple hormonal control.

	VI. EXTRACELLULAR CALCIUM/MAGNESIUM-SENSING RECEPTORS IN THE DISTAL CONVOLUTED TUBULE

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An extracellular Ca²⁺/Mg²⁺-sensing receptor has recently been shown to be expressed along the entire length of the nephron, particularly the loop of Henle, DCT, and inner medullary collecting duct (267, 268, 349). This receptor is very similar to the one expressed in the parathyroid gland (44, 45). It comprises three major domains: 1) a large extracellular amino-terminal domain consisting of 613 amino acids, which is thought to possess cationic binding sites; 2) a 250-amino acid domain with 7 predicted membrane-spanning segments characteristic of the G protein-coupled receptor family; and 3) a carboxy-terminal domain of 222 amino acids that likely resides in the cytoplasm and is involved with intracellular signaling processes (44). The evidence is that elevated plasma Ca²⁺ or Mg²⁺ binds to the extracellular domain of the receptor and initiates a number of intracellular signals. Among these is stimulation of G_i proteins that modulate adenylate cyclase activity and cAMP levels and G_q proteins that activate phospholipase C releasing inositol 1,4,5-trisphosphate and cytosolic Ca²⁺ (44). Hebert and co-workers (136, 292, 336) postulate that elevated plasma calcium or magnesium and activation of the Ca²⁺/Mg²⁺-sensing receptor leads to diminished salt (sodium, calcium and magnesium) transport within the loop and water absorption within the inner medullary collecting duct. This results in an increase in urinary water flow in addition to calcium and magnesium excretion, minimizing the opportunity of stone formation. Riccardi et al. (266) and Yang et al. (349) have shown that the Ca²⁺/Mg²⁺-sensing receptors are also present on the basolateral membrane of the DCT. Ca²⁺/Mg²⁺-mediated intracellular signaling may have important effects on distal cellular function including sodium, potassium, and divalent cation transport (136, 228).

We have recently shown that MDCT cells possess a Ca²⁺/Mg²⁺-sensing mechanism(s) that is equally sensitive to extracellular Ca²⁺ and Mg²⁺ concentrations normally found in the plasma (21). Figure 11 summarizes the comparative effects of extracellular Mg²⁺ and Ca²⁺ on glucagon-mediated intracellular cAMP accumulation. The half-maximal effective concentrations were 0.5 mM Mg²⁺ and 1.5 mM Ca²⁺, which are consistent with normal plasma concentrations. This is unlike the observations with other cells where extracellular Ca²⁺ has been found to be a more potent stimulator of Ca²⁺/Mg²⁺-sensing receptor-induced intracellular signaling than external Mg²⁺. The threshold value for extracellular Ca²⁺ has been reported to be on the order of 1-5 mM for renal cells, whereas a similar cytosolic Ca²⁺ response requires as much as 5-20 mM Mg²⁺ (241, 261). These relative potencies of extracellular Ca²⁺ and Mg²⁺ recapitulate their actions in bovine parathyroid cells and in Xenopus oocytes injected with cRNA and HEK 293 cells transfected with DNA of the cloned Ca²⁺/Mg²⁺-sensing receptor (43, 55, 222). Thus it was of interest that the polyvalent cation-sensitive mechanism of MDCT cells was apparently as sensitive to extracellular Mg²⁺ as it was to Ca²⁺. This is particularly noteworthy because the Mg²⁺ studies were performed in the presence of normal or elevated Ca²⁺ (21). The functional consequences of changes in the amino acid sequence of the Ca²⁺/Mg²⁺-sensing receptor have been investigated by expressing a variety of mutated receptors in HEK 293 cells (13). Some of the mutations diminish Ca²⁺/Mg²⁺-sensing receptor signaling, others enhance the sensitivity to external Ca²⁺, and still others are completely nonfunctional with no intracellular signaling as determined by changes in intracellular Ca²⁺ or inositol phosphate (12, 133, 241). Bräuner-Osborne et al. (41) observed that the EC₅₀ for Mg²⁺ significantly decreased (4.7 ± 0.1 to 2.6 ± 0.4 mM), whereas that for Ca²⁺ increased (3.2 ± 0.1 to 3.3 ± 0.2 mM) after mutations of Ser¹⁴⁷ and Ser¹⁷⁰, which are located in the amino-terminal domain and are involved in the agonist binding (41). From these transfection studies of the mutated Ca²⁺/Mg²⁺-sensing receptor, it has been suggested that discrete but interrelated cation binding sites may be a feature of this receptor (12). These discrete binding sites remain to be identified, but it is apparent from these studies that changes in the extracellular domain of the Ca²⁺/Mg²⁺-sensing receptor may alter its sensitivity to the various ligands (12). Subtle changes in the Ca²⁺/Mg²⁺-sensing receptor may significantly affect its selectivity. The present studies with the endogenous polyvalent cation-sensitive ^<