Stimulation of calcium transport by amiloride in mouse distal convoluted tubule cells

Stimulation of calcium transport by amiloride in mouse distal convoluted tubule cells

Kidney International, Vol. 48 (1995), pp. 1427—1434 Stimulation of calcium transport by amiloride in mouse distal convoluted tubule cells PETER A. FR...

811KB Sizes 0 Downloads 7 Views

Kidney International, Vol. 48 (1995), pp. 1427—1434

Stimulation of calcium transport by amiloride in mouse distal convoluted tubule cells PETER A. FRIEDMAN and FRANK A. GESEK Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire, USA

Stimulation of calcium transport by amiloride in mouse distal convoluted tubule cells. This study examined the mechanism by which amiloride dissociates Na and Ca transport in distal convoluted tubules. Control rates of Na uptake averaged 288 nmol/(min mg protein) and were inhibited 39%

by jsM amiloride. Amiloride had no effect on Cl uptake. Resting membrane voltage, measured with the voltage-sensitive dye DiOC6 (3), averaged —70 mV. Amiloride hyperpolarized cells in a reversible manner by 18

mV. Control rates of Ca uptake averaged 2.8 nmol/(min mg protein) and

increased by 39% in the presence of amiloride. Alterations of intracellular Ca activity were measured in single cells loaded with Fura2-AM. Control intracellular Ca activity averaged 100 ni. Amiloride increased intracellular Ca activity in a concentration-dependent manner to a maxi-

mum of 330 n at ILM amiloride. Amiloride analogues ethylisopropyl amiloride (EIPA) and dimethytbenzamil (DMB), which preferentially

block Na/H and Na/Ca exchange, respectively, had no effect on Na or Ca influx or on intracellular Ca activity. The dihydropyridine Ca channel blocker nifedipine inhibited amiloride-stimulated Ca uptake and the rise of intracellular Ca activity but had no effect on membrane voltage. It is

concluded that amiloride blocks Na entry mediated by Na channels. Inhibition of Na entry results in membrane hyperpolarization, which

reduction of the transepithelial voltage [15, 16]. The mechanism whereby amiloride augments calcium transport is unknown. We recently reported that, in primary cell cultures, freshly isolated tubules, and in immortalized distal convoluted tubule cells, thiazide diuretics [7, 17] and parathyroid hormone [18, 19] hyperpolarize distal convoluted tubule cells and increase free intracellular calcium by activating a calcium entry mechanism that is sensitive to dihydropyridine-type calcium channel blockers [17, 20]. We postulated that since amiloride hyperpolarizes distal convoluted tubule cells, it would enhance calcium entry by the same mechanism stimulated by parathyroid hormone and thiazide diuretics. The results show that, in distal convoluted tubule cells, amiloride inhibits Na entry and hyperpolarizes the membrane voltage. Calcium influx was stimulated and produced a sustained rise of [Ca2]. Calcium influx, and the rise of [Ca2J1, were inhibited with dihydropyridine-type calcium channel blockers.

activates Ca entry by dihydropyridine-sensitive Ca channels.

Methods

Preparation and culture of distal convoluted tubule cells Immortalized distal convoluted tubule cells were prepared from Renal sodium and calcium absorption normally proceed in primary cultures of mouse cortical thick ascending limb and distal parallel [1, 2]. This relation can be conspicuously dissociated by convoluted tubule cells isolated by a double antibody isolation

administration of certain drugs such as thiazide diuretics [3] or amiloride [4]. Thiazide diuretics, either alone or in combination with amiloride, reduce renal calcium excretion secondary to enhanced absorption [5]. This action is generally attributed to the dual effect of thiazides to inhibit sodium transport while increasing calcium absorption [6, 7]. Such stimulation of calcium transport proceeds in distal convoluted tubules [8], where amiloride suppresses sodium transport while enhancing calcium absorption [4, 6, 9—11]. Moreover, there is a high degree of correlation between the magnitude of inhibition of sodium transport and stimulation of calcium absorption [6]. These findings suggest that sodium and calcium transport are functionally linked in distal convoluted tubules. In the rabbit, similar functions are found in connecting tubules [12]. Amiloride inhibits sodium transport by distal convoluted tubules and in other epithelial cells by blocking Na channels [10, 13, 14], which results in apical membrane hyperpolarization and a

Received for publication April 3, 1995 and in revised form June 14, 1995 Accepted for publication June 15, 1995

© 1995 by the International Society of Nephrology

technique [21]. The preparation, subcloning, characterization, and culture conditions of immortalized mouse distal convoluted tubule cells have been described [7, 19, 21, 22]. These cells exhibit, in part, the following characteristics: (1) inhibition of sodium and

chloride uptake by chlorothiazide [23, 24]; (2) activation of adenylyl cyclase by PTH [12, 22]; 1,25(OH)2D3 receptors [25] and 1,25(OH)2D3-induced 24OHase activity [26]. Cell passages 7 to 28

were used in the present study.

Na, C1, and Ca2 uptake A rapid filtration technique, detailed in previous reports [7, 19],

was used to measure uptake of 22Na, 36Cl and 45Ca2. Briefly, 5 to 6 x 106 cells/60 mm dish were removed by brief ( 5 mm) treatment with 0.125% trypsin and rinsed with a modified KrebsRinger (KR) solution containing (in mM): 140 NaCl, 5 KCI, 1 CaCl2, I MgCl2, buffered with 18 Tris base and 28 Hepes to pH 7.40 0.01 and 295 1 mOsm/kg H20. The buffer, with or without amiloride or other inhibitors, (total volume = 100 xl) was placed in 12 x 75 mm polystyrene tubes. A 50 1.d aliquot of cells was added for one minute before addition and rapid mixing of a 50 jxl aliquot of 22Na, 36C1, or 45Ca2 to initiate isotope uptake. All sample tubes were maintained at 37°C in a shaking incubator. Control experiments indicated that tracer uptake was linear for

1427

1428

Friedman and Gesek: Amiloride-stimulated Ca2 transport

two minutes. In all experiments tracer uptake was terminated after one minute by rapid addition of ice-cold isosmotic, Li2SO4-Hepes rinse buffer (140 mivi Li2SO4, 10 mrvi Hepes; pH 7.40, 295 2 mOsmlkg H20) and filtered onto Whatman GF/C filters using a Millipore 12-port manifold followed by two additional rinses with Li2SO4-Hepes buffer. Nonspecific binding of isotopes to the filters and cells was determined in the presence of Li2SO4-Hepes buffer added to the cells before addition of 22Na, 36CF, or 45Ca2t All cellular uptake experiments were normalized for protein content, which was measured by the Lowly procedure on 50 1.d aliquots of cells [27]. Tracer uptakes are expressed as nanomoles per minute

a)

400 300

E

200 £

100

a) Ca

0

x mg protein (nmol/min' X mg proteint).

Na Fig. 1. Effects

I

A

Control Amiloride

Measurement of free intracellular Ca2 activity ([Ca2]) Cells were grown to near confluence on 25 mm glass coverslips. Coverslips were rinsed three times with the Krebs-Ringer buffer and incubated with iO— M Fura-2 AM in 2.5 ml Krebs-Ringer buffer for 60 minutes at 37°C on a rotating incubator. Coverslips were then rinsed twice with Krebs-Ringer buffer and placed in a

I

Control Amiloride C1

of 106 M amiloride on 22Na (solid bars) and 36C1

(hatched bars) uptake by immortalized mouse distal convoluted tubule cells.

Bars represent the means

SE of triplicate determinations in 5 experi< 0.01, compared to control levels ments with 22Na and 4 with 36C1. of uptake.

temperature-controlled chamber of a microincubation system (37°C; MS-C, Narishige, Greenvale, NY, USA) affixed to the stage

of a Nikon Diaphot inverted microscope. A Nikon Photoscan-2 (Nikon, Inc., Natick, MA, USA) was used to measure fluores- Fura2-AM and DiOC6 [3] (Molecular Probes, Inc., Eugene, OR, cence intensity. Interference excitation filters of 340 nm and 380 USA). DMB was a gift from Merck, Sharp & Dohme (West Point, PA, USA). Carrier-free 22 Na (10 j.tCi/ml) was from Amersham nm (Omega Optical; Brattleboro, VT, USA) and a 510 nm dichroic mirror (DM510, Nikon) in series with a 510 5 nm (Arlington Heights, IL, USA); 36CY (58 1.tCi/ml) from ICN Radiochemicals (Costa Mesa, CA, USA); and carrier-free 45Ca2 interference filter (Omega Optical) were employed. Fields of 1 to 3 cells on the cover slip were selected by using a shutter assembly (1 Ci/m1) from New England Nuclear (Boston, MA, USA). mounted in front of the photomultiplier tube. Background Chemicals and other reagents were of the highest grade commerautofluorescence in each experiment was measured on the cover- cially available. Solutions containing drugs were prepared fresh slip in an area devoid of cells or on cells not incubated with Fura-2 AM. Background fluorescence was typically observed to be <5%

daily.

Statistical analysis

of the total fluorescence of cells loaded with Fura-2. A baseline Isotope uptake measurements were performed in triplicate recording of [Ca2]1 was obtained prior to addition of amiloride within an individual experiment. The data are presented as means or other agents. Absolute values of [Ca2]1 for each experiment su, where N indicates the number of independent experiments. were determined as described previously [7, 18, 19] using an Effects of drugs on [Ca2] and membrane voltage were assessed internal calibration procedure and the equation derived by Grynby paired comparisons within experiments and reported as the kieiwcz, Poenie and Tsien [28] with an assumed Kd of 224 ns. mean SE of N individual experiments. Comparisons between control and experimental treatment groups were evaluated by Membrane voltage Fluorescence measurements of membrane voltage were per- analysis of variance (ANOVA) and post-hoc analysis of multiple formed with the voltage sensitive probe 3,3'dihexyloxacarbocya- comparisons using the Bonferroni method (Instat; Graph-Pad Software, CA, USA) or with Newman-Keuls tests using ANCOM nine iodide (DiOC6 [3]) as previously described [7, 19]. Distal 0.05 were convoluted tubule cells grown on coverslips were incubated for 60 (SciLab; Guilderland, NY, USA). Values of P assumed to be significant. minutes with 10—6 M DiOC6 [3]. An excitation wavelength of 490

nm and emission wavelength of 510 nm were utilized. Fluorescence intensity was sampled at a rate of 1 point/second. At the conclusion of each experiment a calibration was performed by a null-point procedure using graded changes of external potassium in the presence of 106 M valinomycin [29]. Membrane voltage was normalized for the average fluorescence intensity during the control observation period and reported as the ratio F1/F0, where F1 are the readings during the experimental periods and F0 is the control observation period. Materials

Specific reagents were obtained from the following sources: amiloride (Sigma Chemical Co., St. Louis, MO, USA); EIPA and nifedipine (Research Biochemicals, Inc., Natick, MA, USA); valinomycin and ionomycin (Calbiochem, San Diego, CA, USA);

Results Amiloride effects on Na and Cl influx by cultured distal convoluted tubule cells

The effects of amiloride on Na and C1 entry by distal convoluted tubule cells are shown in Figure 1 and Table 1. At an extracellular NaC1 concentration of 140 m and in the absence of CO2 and bicarbonate, 10_6 M amiloride inhibited 22Na uptake

by 39 3% (P < 0.05), similar to the inhibition reported in an earlier study [24]. Increasing the amiloride concentration to iO M caused no significant further inhibition of sodium uptake 4%; data not shown). Amiloride had no effect on 36C1 (43 uptake (Fig. 1), in contrast to chlorothiazide, which inhibits C1 uptake by distal convoluted tubule cells [7, 24]. These results are consistent with the view that amiloride selectively blocks sodium

1429

Friedman and Gesek: Amiloride-stimulated Ca2 transport

Table 1. Effect of amiloride and amiloride-analogues on 22Na and 45Ca2 uptake and on [Ca2]1

22Na uptake

nmol min' mg protein'

Final concentration Control Amiloride EIPA DMB

N

288 20

175 8

10—6 M

i0 M

5 X i0 M

45Ca2 uptake

266 38 262 41

0.24

2.75 3.83 2.82 2.53

0.16 0.25 0.16

1I3

[K1ext 1.60

5

10

>'

[Ca2] nM

101 8 332 6 102 6

99 8

3 22Na and 45Ca2 uptake or changes of intracellular Ca2 ([Ca2]) 5

Amiloride

4

were measured in the absence (control) or presence of amiloride (10—6 M), ethylisopropyl amiloride (EIPA, 10 M), or dimethylbenzamil (DMB, 5 X

i0 M). Uptake values represent means SE of triplicate determinations performed in the indicated number of independent experiments. Paired comparisons of control and drug-induced changes of [Ca2j were determined in three separate experiments. ap < 0.01 vs. Control

entry that proceeds by a chloride-independent mechanism in distal convoluted tubule cells. Hypeipolarization of membrane voltage by amiloride Amiloride depolarizes the transepithelial voltage [11, 13—15, 30,

31] and hyperpolarizes apical membrane voltage [13] of distal convoluted tubules and connecting tubules. To determine if amiloride affected membrane voltage in cultured distal convoluted tubule cells, voltage was measured with the potential-

0)

1.40

0)

C

0)

0

1.20

C

00) 0)

0)

.

1.00

0

0)

0.80

Co

0 Q a

0.60 0.40 5

0

25 Time, minutes

30

Fig. 2. Hypeipolarization of membrane voltage by amiloride. Distal convo-

luted tubule cells were grown on coverslips and loaded with the potentialsensitive dye, 3,3'-dihexyloxacarbocyanine [DiOC6 (3)]. Membrane voltage was normalized for the average fluorescence intensity during the first two minutes and reported as the ratio FI/FO. Decreases of fluorescence intensity indicate relative hyperpolarization of membrane voltage. Addi-

tion of amiloride resulted in rapid membrane hyperpolarization. The amiloride-induced hyperpolarization was completely reversed upon washout of the drug. A null-point calibration was performed at the end of each experiment using graded concentrations of external K in the presence of

10_6 M valinomycin and is shown at the right-hand portion of the recording. Similar observations were made in 7 additional independent

sensitive fluorescent dye, DiOC6 (3). A representative experiment experiments. is depicted in Figure 2. Amiloride (10—6 M) decreased fluorescence intensity, corresponding to hyperpolarization of membrane Table 2. Effects of amiloride and nifedipine on membrane voltage 5%. Upon washout of the drug, voltage, by an average of 28 fluorescence intensity returned to control levels. Since membrane Amiloride + voltage was rapidly and completely reversed when amiloride was Amiloride nifedipine Nifedipine washed out of the bath, the changes of fluorescence intensity were

not due to loss of the dye from the cells or to a toxic effect of mV SE amiloride. The right-hand portion of Figure 2 shows the calibra- N tion protocol, where alterations of DiOC6 (3) fluorescence intensity were recorded at graded concentrations of extracellular K in

18.1

0.4

0.8 8

0.1 3

17.0 2.2 3

Values represent means SE for N independent experiments. Changes in membrane voltage (z mV) were measured with the potential-sensitive,

the presence of the K ionophore, valinomycin. Resting mem- fluorescent dye, DiOC6 (3). Cells were exposed to amiloride (10_6 M) or brane voltage, which averaged —68 5 mV, was calculated from nifedipine (10 M) along or in combination. Calibration was performed as the average null-point value for extracellular K (7.9 0.5 mM, N described in Methods using grading concentration of extracellular K in = 8) and the measured intracellular K concentration of 119 mM the presence of valinomycin (10—6 M). in cultured distal convoluted tubule cells determined in previous studies [19]. As summarized in Table 2, amiloride hyperpolarized distal convoluted tubule cells by 18 1 mV (P < 0.001). The would facilitate Ca2 movement into the cell. Since amiloride

effect of amiloride on membrane voltage provides functional evidence for the presence of Na channels in distal convoluted

reduced the rate of sodium uptake (Fig. 1) and hyperpolarized distal convoluted tubule cells (Fig. 2) its effects on calcium tubule cells and is consistent with the view that 22Na uptake that transport and on [Ca2]1 were determined. As shown in Table 1, was sensitive to inhibition by amiloride (Table 1) was mediated by amiloride significantly increased the rate of 45Ca2 entry from these plasma membrane channels. 2.75 to 3.83 nmol/min1 x mg protein' (P < 0.01). The relative Effect of amiloride and amiloride analogues on 45Ca2 uptake

and on [Ca2J

magnitude by which amiloride decreased 22Na uptake (39%) was identical to the fractional increase of calcium influx, consistent with the notion that sodium and calcium transport are functionally linked in distal convoluted tubule cells. Stimulation of calcium

Sodium and calcium absorption normally proceed in parallel [1]. However, inhibition of sodium transport in distal convoluted uptake also resulted in sustained elevation of [Ca2]1 (Fig. 3). tubules in certain circumstances accompanied by enhanced cal- Upon addition of 10—6 M amiloride, [Ca2]1 began to increase cium absorption. Costanzo [10] suggested that amiloride, by within 1.4 0.3 minutes from control levels of 101 8 nM to blocking Na entry and hyperpolarizing membrane voltage, reach plateau concentrations of 332 6 nM. [Ca2]1 remained

1430

Friedman and Gesek: Amiloride-stimulated Ca2 transport

I Amiloride

10-6 M

I

I

1OM

I

I

400

300

L il//I..

E

c 04

200

(6

0 100

0

I

0

5

10

Amiloride EIPA DMB

30 35 Time, minutes

55

60

Fig. 3. Amiloride-induced increases of free intracellular calcium ([Ca2]1) activity. [Ca2]1 was measured in single distal convoluted tubule cells loaded with Fura2 as described in Methods. After a three minute control period, 10—6 M amiloride was added to the bath and

caused a prompt increase of [Ca2} that was maintained in the presence of amiloride. Upon washout of the drug [Ca2]1 returned to control levels. Subsequent addition of io— M amiloride cause a comparable elevation of [Ca2]1.

V/////A

400

300 E

i'(6 200

0

100 Fig. 4. Specificity of amiloride stimulation of [Ca2]. Amiloride (10—6 M) reversibly increased [Ca2]1 as shown in Figure 3.

0 0

5

20

40

45

Time, minutes

elevated in the presence of amiloride and, upon washout, re-

50

Ethylisopropyl amiloride (EIPA; 10 M) and dimethylbenzamil (DMB; 5 X iO M) had no effect on [Ca2]1. Comparable results were obtained in two additional experiments.

(EIPA) is a relatively selective and potent inhibitor of Na/H

turned to control levels. As in the case of sodium uptake, exchange [34—36], whereas dimethylbenzamil (DMB) is a more increasing the concentration of amiloride to i0 M had no potent inhibitor of Na7Ca2 exchange [32, 33, 37]. As shown in further effect on [Ca2]1 (Fig. 3). In addition to its potent effect on epithelial sodium channels, amiloride also inhibits a variety of sodium-coupled transport processes including Na/H and Na/Ca2 exchange [32, 33]. To determine the selectivity of amiloride on Na and Ca2 transport

Table 1, neither EIPA nor DMB had significant effects on 22Na or on 45Ca2 uptake by distal convoluted tubule cells. Similarly, neither analogue affected [Ca2]1 (Table 1; Fig. 4). Thus, amiloride rather specifically inhibited Na uptake and increased Ca2 transport by distal convoluted tubule cells.

by distal convoluted tubule cells the effect of amiloride was Amiloride evoked concentration-dependent increases of compared with two of its analogues. Ethylisopropyl amiloride [Ca2]1 (Fig. 5). At iO M, amiloride increased [Ca2]1 by 17%

1431

Friedman and Gesek Amiloride-stimulated Ca2 transport

400

Discussion

Sodium and calcium absorption normally proceed in parallel

along the nephron [39, 40]. In humans and in experimental

300

animals changes of sodium excretion are accompanied by propor-

tionate and corresponding alterations of calcium excretion [2]. +

0

However, certain physiologic and pathophysiologic conditions, as well as pharmacologic treatment, notably with thiazide diuretics, dissociate the parallel transport of sodium and calcium [4, 6, 10, 41]. Thiazide diuretics increase sodium excretion while decreasing

200 -

calcium elimination. The reduction of calcium excretion promoted by thiazide diuretics is useful therapeutically in the man100

-

10

10.8

1 o-

10-6

1O

Amiloride, log M Fig. 5. Dose-response relations of amiloride on [Ca2],. Addition of amilo-

ride resulted in dose-dependent increases of [Ca2],. Each point denotes

the mean

sa of 4 independent experiments at each indicated

concentration.

(P < 0.01). Maximal increases of [Ca2j1 to 332 6 n were observed with 10—6 M amiloride. Concentrations of amiloride above 10—6 M that were tested (10 M to i0 M) elicited no

of hypercalciuria and nephrocalcinosis [5, 42—44]. Side effects of thiazide diuretics, including hypokalemia, glucose intolerance, and altered serum lipids, limit their utility [45].

Although less extensively studied, amiloride has also been reported to reduce renal calcium excretion, while causing a modest increase of sodium excretion [4, 5, 15, 46, 47]. To characterize the mechanism by which amiloride stimulates calcium absorption, the effect of amiloride on sodium transport and membrane voltage were first assessed. The data in Table 1 and Figure 1 demonstrate that amiloride inhibits Na uptake, but not C1 uptake, by distal convoluted tubule cells. These findings

suggest that amiloride blocks a Na entry mechanism that is

chloride-independent. In contrast, chlorothiazide inhibits sodium and chloride to an equivalent extent [7]. The inhibitory effect of further elevation of [Ca2]1. Half-maximal stimulation occurred at amiloride on sodium uptake by distal convoluted tubule cells is approximately 3.5 X 108 M. The magnitude of the maximal additive to the action of maximally inhibitory concentrations of increase of [Ca2]1 induced by amiloride is comparable to that chlorothiazide [24], consistent with the view that amiloride does reported with chlorothiazide (50 LM), parathyroid hormone, or not block NaC1 cotransport. The absence of an effect of EIPA or calcitonin [7, 18, 19, 38].

Cellular ently mechanism for amiloride-stimulated Ca2 influx

DMB on 22Na accumulation (Table 1) further supports the specificity of amiloride under the present experimental conditions. It should be noted that EIPA, but not amiloride, blocks Na47H exchange in distal convoluted tubule cells [24]. Likewise,

Parathyroid hormone stimulates Ca2 entry through dihydro- DMB inhibits Na/Ca2 exchange in these cells [24, 48]. Amiloride hyperpolarized membrane voltage of distal convopyridine-sensitive Ca2 channels in primary cultures of cortical thick ascending limb plus distal convoluted tubule cells [18] and in luted tubule cells (Fig. 2), similar to its reported effect on apical immortalized distal convoluted tubule cells [20]. Thiazide diuret- membrane voltage of rabbit distal convoluted tubules [14]. Taken ics also stimulate Ca2 entry into distal convoluted tubule cells by together, the inhibition of 22Na uptake by amiloride and its a dihydropyridine-sensitive mechanism [7]. To determine if Ca2 action on membrane voltage are consistent with the presence of entry stimulated by amiloride was mediated through a similar amiloride-sensitive Na channels in mouse distal convoluted dihydropyridine-sensitive mechanism the effects of the nifedipine tubule cells. Microperfusion studies in the rat have generally pointed to a on 45Ca2 uptake and [Ca2]1 were determined. Figure 6 shows that nifedipine alone did not alter [Ca2]1. In the presence of spatial separation of thiazide-sensitive NaCl cotransport to "early nifedipine, amiloride failed to increase [Ca2]1. Challenge with distal tubule," presumably distal convoluted tubules, and amiloamiloride alone caused a rapid and sustained elevation of [Ca2]1. ride-sensitive sodium transport to "late distal tubules," likely to Following washout of amiloride, the level of intracellular Ca2 represent predominantly connecting tubules [6, 49]. Other studies returned to control levels. The effects of nifedipine on amiloride- report amiloride-sensitive Na channels in distal convoluted stimulated 45Ca2 influx and [Ca2]1 are summarized in Table 3. tubules [14, 30, 50, 51] suggesting co-localization of the two In this series of experiments amiloride increased 45Ca2 influx by transport mechanisms. The distal convoluted tubule of the mouse an average of 33 5%. Nifedipine alone had no significant effect and the rat exhibits a gradual transition to the connecting tubule, on the rate of 45Ca2 uptake by distal convoluted tubule cells but whereas sharp demarcations characterize the transition in the abolished the stimulation of 45Ca2 uptake by amiloride. Nifedip- rabbit [6, 52, 53]. The divergent findings with respect to localizame also inhibited the increase of intracellular Ca2 due to tion of diuretic action may be partially ascribed to anatomical amiloride. There was no effect of nifedipine alone on intracellular differences of the distal convoluted tubule between species [54]. Ca2. Taken together, the finding that nifedipine abolished the Recent evidence clearly localizes mRNA and protein for the rise of [Ca2] and inhibited 45Ca2 uptake elicited by amiloride pore-forming, alpha subunit of the amiloride sensitive Na chanis consistent with the conclusion that Ca2 entry was mediated by nel (rENaC) to the distal convoluted tubule of the rat [55]. The dihydropyridine-sensitive Ca2 channels. present studies establish that clonal mouse distal convoluted

Friedman and Gesek: Amiloride-stimulated Ca2 transport

1432 AMIL NIF

350

250 +

0

50//i,

150

//_________________

0

5

25

10

30

Fig. 6. Inhibition of amiloride-induced increases of [Ca2']1 by nifedipine. Addition of the dihydropyridine calcium-channel blocker

nifedipine (10 NI) alone did not alter [Ca2],. Subsequent addition of amiloride (10—6 M)

45

Time, minutes

Table 3. Effect of nifedipine on amiloride-stimulated 45Ca2 uptake

50

failed to increase [Ca2],. Following washout of the drugs for 12 minutes, a second application of amiloride in the absence of nifedipine increased [Ca2]1. Equivalent results were obtained in two additional experiments.

Three theories have been proposed to account for the dissoci-

ation of Na and Ca2 transport in distal convoluted tubules

and [Ca2]1

induced by amiloride [2, 10]. Walser suggested [2] that reductions

45Ca2 uptake

of transepithelial voltage are responsible for enhanced calcium transport. The decrease of the transepithelial voltage, in turn, 98 13 would result in diminished calcium backflux from the peritubular 2.81 0.38 Control 340 12 space into the lumen, thereby increasing net calcium absorption. 3.74 0.15 Amiloride 99 15 However, backfiux of calcium in distal convoluted tubules is 2.81 0.50 Amiloride + nifedipine 98 14 negligibly small [41, 62] and can't explain the effect of amiloride 2.68 0.32 Nifedipine One minute rates of 45Ca2 uptake or alterations of intracellular Ca2 on net calcium absorption. A second explanation implicates ([Ca2]) were measured in the absence (Control) and presence of enhanced basolateral membrane Na/Ca2 exchange as mediatamiloride (106 M) alone or in combination with nifedipine (10 M). SE for triplicate measurements per- ing the increase of calcium absorption [9, 10]. According to this Uptake values represent means formed in 6 separate experiments; [Ca21 was determined in 3 indepen- view, by blocking Na entry amiloride decreases intracellular Na dent experiments for each treatment. activity, which enhances the driving force for basolateral memP < 0.01 vs. control brane Na']Ca2 exchange. The results presented in Table 1 and Figures 3 and 5 show that amiloride increased [Ca2]1, opposite to nmol min1 mg

protein'

[Ca2]1 nM

the expected effect if enhanced Na'iCa2 exchange were the tubule cells express amiloride-sensitive Na channels, as well as thiazide-sensitive NaCl cotransport [23, 56].

mechanism responsible for the amiloride-induced stimulation of calcium absorption. Such an inhibitory, but minor, action of high concentrations of amiloride was shown by Yamasaki et al [611. A

inhibitory actions of amiloride on cellular metabolism or Na,KATPase activity [57, 58], intracellular buffering [59], or blockade of L-type Ca2 channels at millimolar concentrations of amiloride [60]. High concentrations of amiloride (10 p.M) have been reported to decrease [Ca2]1 in rabbit connecting tubules [61]. The effect of amiloride on 45Ca2 uptake and on [Ca2] was specific since the amiloride analogues EIPA and DMB at 10 to 50 times greater concentrations, respectively, had no detectable effects (Table 1, Fig. 4). In the present investigation amiloride stimulated calcium transport without treatment with parathyroid hormone, as has generally been reported by others [4]. However, in rabbit connecting tubules prior treatment with parathyroid hormone was apparently required to observe the stimulatory effect of amiloride or trichlormethiazide on calcium absorption [11, 61].

luminal fluid in to the cell. The results reported herein provide direct support this mechanism. According to this scheme, amiloride-induced stimulation of calcium absorption by distal convoluted tubules involves the following events: (1) amiloride blocks apical membrane Na channels; (2) the reduced Na conductance results in hyperpolarization of apical plasma membranes; (3) hyperpolarization activates and increases the driving force for Ca2 entry through dihydropyridine-sensitive Ca2 channels. Evidence that amiloride blocks Na channels in distal convoluted tubule cells is supported by the data presented in Table 1 and in Figures 1 and 2. Amiloride inhibited Na uptake by 39% in distal convoluted tubule cells with no detectable effect on C1 uptake. These findings are similar to the reported magnitude of

At 10_6 M, amiloride maximally inhibited Na entry and stimulated Ca2 transport. Although not shown in Figure 5, third mechanism proposed by Costanzo [10] envisioned that concentrations of amiloride greater than iO M elicited smaller inhibition of apical membrane Na channels hyperpolarizes memrises of [Ca2]. This attenuated effect may be due to non-specific brane voltage resulting in enhanced calcium entry from the

1433

Friedman and Gesek: Amilonde-stimulated Ca2 transport

inhibition (46%) of sodium transport by amiloride in microperfused rabbit distal convoluted tubules [14]. The second step in the proposed mechanism of amiloride action is hyperpolarization of

membrane voltage. The data Table 2 and Figure 2 show that amiloride-hyperpolarized membrane voltage by 18 mV. Yoshitomi et al [13] reported that, in rabbit distal convoluted tubules, amiloride hyperpolarized apical membrane voltage by 7 mV. Resting membrane voltage in the distal convoluted tubule cells

dissociates Na and Ca2 transport in the distal convoluted tubule and augments Ca2 transport in this segment. Acknowledgments These studies were supported by National Institutes of Health grants GM 34399 (PAF) and DK 46064 (FAG). Dimethylbenzamil (DMB) was generously provide by Merck, Sharp & Dohme (West Point, PA, USA). Portions of this study have been presented in preliminary form at the Calcium Antagonists Investigators' Meeting, Santa Fe, New Mexico,

(—72 mV [7, 19]) measured with fluorescent dyes is comparable to April, 1992 [65]. The technical assistance of Ms. B. Coutermarsh is greatly that measured with glass microelectrodes [63]. The reason for the appreciated. References were prepared with Reference Manager (RIS,

difference in the magnitude of the induced hyperpolarization by Inc., Carlsbad, CA, USA). amiloride is not obvious. Several possibilities that may account for Reprint requests to Peter A. Friedman, Ph.D., Department of Pharmacolthe difference include the techniques employed, and differences in ogy & Toxicology, Dartmouth Medical School, 7650 Remsen, Hanover, New the membrane resistance of intact tubules compared with cells Hampshire 03 755-3835, USA.

grown in tissue culture. Irrespective of the difference in the relative magnitude of the observed effect, amiloride unquestionably hyperpolarizes membrane voltage.

The last part of the proposed mechanism for stimulation of calcium transport by amiloride involves enhanced Ca2 entry through dihydropyridine-sensitive Ca2 channels. Recent patchclamp experiments demonstrate the presence of dihydropyridinesensitive calcium channels in plasma membranes of distal convo-

luted tubule cells [20, 64] that are activated upon membrane hyperpolarization, which also increases the electrochemical gradient for calcium entry [20]. The data in Table 3 and Figure 6 demonstrate that the dihydropyridine calcium channel blocker, nifedipine, inhibited amiloride-stimulated increases of 45Ca2

References 1. COSTANZO LS, WINDHAGER EE:

Transport functions of the distal

convoluted tubule, in Physiology of Membrane Disorders, edited by TE ANDREOLI, JF HOFFMAN, DD FANESTIL, SG SCHULTZ, New York, Plenum Medical Book Company, 1986, pp 727—750 2. WALSER M: Calcium-sodium interdependence in renal transport, in

Renal Pharmacology, edited by JW FISHER, New York, AppletonCentury-Crofts, 1971, pp 21—41 3. FRIED TA, KUNAU RT: Thiazide diuretics, in Diuretics: Physiology, Pharmacology & Clinical Use, edited by JH DIRK5, RAL SUTFON, Philadelphia, WB Saunders, 1986, pp 66—85 4. COSTANZO LS, WEINER TM: Relationship between clearances of Ca and Na: Effect of distal diuretics and PTH. Am J Physiol 230:67—73, 1976

uptake and [Ca2J1, consistent with the view that amiloridestimulated calcium entry is mediated by these channels. The

5. MASCHIO G, TESSITORE N, D'ANGELO A, FABRIS A, PAGANO F, TASCA

calcium channel blocker nimodipine also caused concentrationdependent reductions of amiloride-induced increases of [Ca2]1 [65]. The observation that amiloride and hydrochlorothiazide produce additive effects on renal calcium transport is consistent with their acting through distinct mechanisms. Thus, although parathyroid hormone [19], chlorothiazide [7], and amiloride hyperpolarize membrane voltage by different mechanisms, they activate a common calcium entry mechanism that is inhibitable by dihydropyridine-type calcium channel blockers. Taken together, the results described herein and those of clearance or microperfusion experiments [4, 6, 101 provide unequivocal evidence that amiloride dissociates calcium and sodium transport. The present work establishes that the dissociation results from inhibition of Na entry leading to hyperpolarization of membrane voltage and stimulation of Ca2 entry through dihydropyridine-sensitive channels. Only limited information is available regarding amiloride effects on renal calcium excretion in humans [5, 46, 47, 66]. The present study (Fig. 1, Table 1) underscores the functional relation between sodium and calcium transport by distal convoluted tubule cells. By extrapolation, the present findings would imply that adequate delivery of sodium to the site of amiloride action in distal convoluted or connecting tubules is required for a significant pharmacologic action of amiloride on calcium transport. In summary, the present results show that amiloride inhibits

calcium nephrolithiasis with low-dose thiazide, amiloride and allopurinol. Am J Med 71:623—626, 1981 6. COSTANZO LS: Localization of diuretic action in microperfused rat distal tubules: Ca and Na transport. AmJPhysiol 248:F527—F535, 1985 7. GESEK FA, FRIEDMAN PA: Mechanism of calcium transport stimulated by chlorothiazide in mouse distal convoluted tubule cells. J Clin Invest 90:429—438, 1992

A, GRAZIANI G, AROLDI A, SURIAN M, COLUSSI G, MANDRESSI A, TRINCHIERI A, ROCCO R, PONTICELLI C, MINETrI L: Prevention of

8. COSTANZO LS: Mechanism of action of thiazide diuretics. Semin Nephrol 8:234—241, 1988 9. Mom Y, MACHIDA T, MIYAKAWA S, BOMSZTYK K: Effects of amilo-

ride on distal renal tubule sodium and calcium absorption: dependence on luminal pH. Pharmacol Toxicol 70:201—204, 1992 10. COSTANZO LS: Comparison of calcium and sodium transport in early and late rat distal tubules: Effect of amiloride.Am JPhysiol 246:F937— F945, 1984 11. SHIMIZU T, NAKAMURA M, YOSHITOMI K, IMAT M: Interaction of

trichlormethiazide or amiloride with PTH in stimulating calcium absorption in the rabbit connecting tubule. Am J Physiol 261:F36—F43, 1991 12. FRIEDMAN PA, GESEK FA: Calcium transport in renal epithelial cells. Am J Physiol 264:F181—F198, 1993 13. YOSI-IITOMI K, SHIMIZU T, TANIGUCHI J, IMAI M: Electrophysiological

characterization of rabbit distal convoluted tubule cell. Pflugers Arch 414:457—463, 1989 14. SI-JIMIZU T, YOSHITOMI K, NAKAMURA M, IMAI M: Site and mecha-

nism of action of trichiormethiazide in rabbit distal nephron segments perfused in vitro. J Clin Invest 82:721—730, 1988 15. DUARTE CG, CHOMETY F, GIEBISCH G: Effect of amiloride, ouabain,

and furosemide on distal tubular function in the rat. Am J Physiol 221:632—640, 1971

Na entry and increases Ca2 uptake and intracellular Ca2

16. O'NEIL RG, BOULPAEP EL: Effect of amiloride on the apical cell membrane cation channels of a sodium absorbing potassium secreting

activity in distal convoluted tubule cells. Inhibition of Na entry leads to a membrane hyperpolarization that, in turn, stimulates Ca2 entry into distal convoluted tubule cells. Amiloride-stimulated Ca2 entry was mediated by dihydropyridine-sensitive Ca2

renal epithelia. J Membr Biol 50:365—387, 1979 17. FRIEDMAN PA, GESEK FA: Hormone-responsive Ca2 entry in distal convoluted tubules. JASN 4:1396—1404, 1994 18. BACSKAI BJ, FRIEDMAN PA: Activation of latent Ca2 channels in renal epithelial cells by parathyroid hormone. Nature 347:388—391,

channels. These results explain the mechanism by which amiloride

1990

1434

Friedman and Gesek: Amiloride-stimulated Ca2 transport

19. GESEK FA, FRIEDMAN PA: On the mechanism of parathyroid hor-

mone stimulation of calcium uptake by mouse distal convoluted tubule cells. J Clin Invest 90:749—758, 1992 20. MATSUNAGA H, STANTON BA, GESEK FA, FRIEDMAN PA: Epithelial

Ca2 channels sensitive to dihydropyridines and activated by hyperpolarizing voltages. Am J Physiol 267:C157—C165, 1994 21. PIzz0NIA JH, GESEK FA, KENNEDY SM, COUTERMARSH BA, BACSKAI

BJ, FRIEDMAN PA: Immunomagnetic separation, primary culture and characterization of cortical thick ascending limb plus distal convoluted tubule cells from mouse kidney. In Vitro 27A:409—416, 1991 22. FRIEDMAN PA, COUTERMARSH BA, RHIM JS, GESEK FA: Character-

ization of immortalized mouse distal convoluted tubule cells. (abstract) JASN 2:737, 1991 23. GESEK FA, COUTERMARSI-I BA, FRIEDMAN PA: Mechanism of thia-

zide-stimulated calcium transport in distal convoluted tubule cells. (abstract) .JASN 2:620, 1991 24. GESEK FA, FRIEDMAN PA: Sodium entry mechanisms in distal convoluted tubule cells. Am J Physiol 268:F89—F98, 1995 25. SNEDDON WB, GESEK FA, FRIEDMAN PA: 1,25(OH)2 vitamin D3

up-regulates the expression of the parathyroid hormone receptor in distal convoluted tubule cells. (abstract) JASN 4:729, 1993 26. YANG W, FRIEDMAN P, SIU-CALDERA M-L, KUMAR R, HEBERT SC,

CHRISTAKOS S: Evidence that the distal convoluted tubule of mouse kidney is a site of production of 24,25dihydroxyvitamin D3. J Bone Miner Res (in press) 27. LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ: Protein measurement with the Folin phenol reagent.JBiol Chem 193:265—275, 1951

28. GRYNKIEWICZ G, POENIE M, TSIEN RY: A new generation of Ca2 indicators with greatly improved fluorescence properties. J Biol Chem 260:3440—3450, 1985 29. ZEIDEL ML, KIKERI D, SILVA P, BURROWES M, BRENNER BM: Atrial

natriuretic peptides inhibit conductive sodium uptake by rabbit inner medullary collecting duct cells. J Gun Invest 82:1067—1074, 1988 30. BARRA'VIT U: The effect of amiloride on the transepithelial potential

difference of the distal tubule of the rat kidney. Pflugers Arch 361:251—254, 1976 31. GROSS JB, IMAT M, KOKKO JP: A functional comparison of the cortical

collecting tubule and the distal convoluted tubule. J Clin Invest 55:1284—1294, 1975 32. FRELIN C, VIGNE P, BARBRY P, LAZDUNSKI M: Molecular properties

of amiloride action and of its Na transporting targets. Kidney mt 32:785—793, 1987 33. FRELIN C, BARBRY P, VIGNE P, CHASSANDE 0, CRAGOE EJJ, LAZ-

DUNSEI M: Amiloride and its analogs as tools to inhibit Na transport

via the Na channel, the Na/H antiport and the Na/Ca2 exchanger. Biochimie 70:1285—1290, 1988 34. VIGNE P, FRELIN C, CRAGOE EJ JR, LUDZINSKI M: Structure-activity

relationships of amiloride and certain of its analogues in relation to the blockade of the Na/11 exchange system. Mol Pharmacol 25:13 1— 136, 1984 35. ZHUANG YX, CRAGOE EJ JR, SIIAIKEWJTZ T, GLASER L, CASSEL D:

Characterization of potent Na/H exchange inhibitors from the amiloride series in A431 cells. Biochemistry 23:4481—4488, 1984

36. Rocco VK, CRAGOE EJ JR, WARNOCK DG: N-ethoxycarbonyl-2ethoxy-1,2-dihydroquinoline, amiloride analogues, and renal NaIH antiporter. Am J Physiol 252:F517—F524, 1987 37. KACZOROWSKI GJ, SLAUGHTER RS, KING VF, GARCIA ML: Inhibitors

44. YENDT ER, CowIM M: Prevention of calcium stones with thiazides. Kidney mt 13:397—409, 1978

45. KNAUF H: The role of low-dose diuretics in essential hypertension. J Cardiovasc Pharmacol 22(Suppl 6):S1—S7, 1993 46. LEPI'LA D, BROWNE R, HILL K, P CYC: Effect of amiloride with or

without hydrochlorothiazide on urinary calcium and saturation of calcium salts. J Clin Endocrinol Metab 57:920—924, 1983 47. BENTUR L, ALON U, BERANT M: Hypercalciuria in chronically insti-

tutionalized bedridden children: frequency, predictive factors and response to treatment witch thiazides. mt j Pediatr Nephrol 8:29—34, 1987 48. WHITE KE, GESEK FA, FRIEDMAN PA: Structure-function relations of

Na/Ca exchange in distal convoluted tubule cells. (abstract) FASEB J 9:A878, 1995 49. ELLISON DH, VELAZQUEZ H, WRIGHT FS: Thiazide-sensitive sodium

chloride cotransport in early distal tubule. Am J Physiol 253:F546— F554, 1987 50. ALLEN GG, BARRATr U: Origin of positive transepithelial potential difference in early distal segments of rat kidney. Kidney mt 27:622— 629, 1985 51. MEROT J, BIDET M, GACHOT B, LE MAOUT S, KOECHLIN N, TAUC M,

POUJEOL P: Electrical properties of rabbit early distal convoluted tubule in primary culture. Am J Physiol 257:F288—F299, 1989 52. KAISSLING B: Structural aspects of adaptive changes in renal electrolyte excretion. Am J Physiol 243:F211—F226, 1982 53. MADSEN KM, TISHER CC: Structure-functional relationships along the distal nephron. Am J Physiol 250:F1—F15, 1986 54. KRIZ W, KAISSLJNG B: Structural organization of the mammalian kidney, in The Kidney: Physiology and Pathophysiology, edited by DW SELDIN, U GIEBISCH, New York, Raven Press, Ltd. 1992, pp 707—777 55. DUc C, FARMAN N, CANESSA CM, BONVALET JP, ROSSIER BC: Cell-specific expression of epithelial sodium channel alpha, beta, and

gamma subunits in aldosterone-responsive epithelia from the rat: Localization by in situ hybridization and immunocytochemistry. J Cell

Biol 127:1907—1921, 1994 56. FRIEDMAN PA, GESEK FA: Mechanism of action of thiazide diuretics

on sodium and calcium transport by distal convoluted tubules, in Diuretics IV. Chemistry, Pharmacology, and Clinical Applications, edited by JB PUSCHETr, A GREENBERG, Amsterdam, Excerpta Medica, 1993, pp 319—326

57. SOLTOFF SP, MANDEL U: Amiloride directly inhibits the Na,KATPase activity of rabbit kidney proximal tubules. Science 220:957— 959, 1983 58. SOLTOFF SP, CRAGOE EJJ, MANDEL U: Amiloride analogues inhibit proximal tubule metabolism. Am J Physiol 250:C744—C747, 1986 59. DUBINSKY WP JR, FRIZZELL RA: A novel effect of amiloride on

Htdependent Na transport. Am J Physiol 245:C157—C159, 1983 60. GARCIA ML, KING VF, SHEVELL JL, SLAUGHTER RS, SUAREZ-KURTZ U, WINQUIST RJ, KACZOROWSKI GJ: Amiloride analogs inhibit L-type

calcium channels and display calcium entry blocker activity. J Biol Chem 265:3763—3771, 1990 61. YAMASAKI F, Y05HIT0MI K, SHINKAWA T, IMAI M: Effects of amiloride

and a novel diuretic, 7-chloro-2,3-dihydro-1-(2-methylbenzoyl)-4(IH)quinolinone-4-oxim e-o-sulfonic acid, potassium salt (M17055), on

calcium transport in the rabbit connecting tubule. J Pharmacol Exp Ther 266:1589—1593, 1993 62. BINDELS RJM, HARTOG A, TIMMERMANS J, VAN OS CH: Active Ca2

of sodium-calcium exchange: Identification and development of

transport in primary cultures of rabbit kidney CCD: stimulation by

probes of transport activity. Biochim Biophys Acta 988:287—302, 1989 38. GESEK FA, FRIEDMAN PA: Calcitonin stimulates calcium transport in distal convoluted tubule cells. Am J Physiol 264:F744—F751, 1993 39. EDWARDS BR, BAKE PU, SUTrON RAL, DIRKS JH: Micropuncture

1,25-dihydroxyvitamin D3 and PTH. Am J Physiol 261:F799—F807,

study of diuretic effects on sodium and calcium reabsorption in the dog nephron. J Clin Invest 52:2418—2427, 1973 40. AGUS ZS, CHIU PJS, GOLDBERG M: Regulation of urinary calcium excretion in the rat. Am J Physiol 232:F545—F549, 1977 41. CosrANzo LS, WINDHAGER EE: Calcium and sodium transport by the distal convoluted tubule of the rat. Am J Physiol 235:F492—F506, 1978

42. Sw-roN RAL: Diuretics and calcium metabolism. Am J Kidney Dis 5:4—9, 1985

43. STIER CT JR, ITSKOVITZ HD: Renal calcium metabolism and diuretics. Anna Rev Pharmacol Toxicol 26:101—116, 1986

1991

63. KOEPPEN BM, GIEBISCH G, BIAGI BA: Electrophysiology of mamma-

lian renal tubules: Inferences from intracellular microelectrode studies. Annu Rev Physiol 45:497—517, 1983 64. PONCET V, MEROT J, POUJEOL P: A calcium-permeable channel in the

apical membrane of primary cultures of the rabbit distal bright convoluted tubule. Pflugers Arch 422:112—119, 1992 65. FRIEDMAN PA, GESEK FA, MATSUNAGA H, STANTON BA: Physiolog-

ical and pharmacological regulation of calcium entry in epithelial cells. Drugs Dev 2:89—99, 1993 66. ALON U, COSTANZO LS, CIIAI't JC: Additive hypocalciuric effects of

amiloride and hydrochiorothiazide in patients treated with calcitriol. Miner Electrol Metab 10:379—386, 1984