Phosphate Transport Inhibition by KW-3902, an Adenosine A1 Receptor Antagonist, Is Mediated by Cyclic

Adenosine Monophosphate Hui Cai, MD, Diane El. Puschett, Shangbo Guan, MD, Vecihi Batuman, MD,

and Jules 6. Puschett, MD

0 We have previously demonstrated that 1,3-dipropyl-8-(3-noradamantyl) xanthine (KW-3902) has an inhibitory effect on phosphate (Pi) transport with no effect on glucose transport in the rat renal proximal tubular cell, similar to that of parathyroid hormone (PTH). In the current studies we investigated the effect of KW-3902, rat PTH (l- 34), and 1,3-dipropyl-Ecyclopentylxanthine (DPCPX), another selective adenosine A, receptor antagonist, on Pi transport and the production of cyclic adenosine monophosphate (CAMP). We then compared these effects of KW-3902 with those of rat PTH in rat renal proximal tubule cells. The results showed that both KW-3902 (30 pmol/ L) and rat PTH (l-34, 5 pmol/L) significantly inhibited Pi uptake in proximal cells from a control level of 81 2 3 to 19 ? 3 (a reduction of 89%) and 49 2 4 picomoles phosphate/mg protein/min (a reduction of W%), respectively (f < 0.01). The inhibitory effect of 30 pmol/L KW-3902 alone on Pi transport was more than twice that of 5 pmol/ L rat PTH (l-34) alone (f < 0.01). KW-3902 stimulated the production of CAMP in a dose-dependent manner (r = 0.997, P < 0.01). Rat PTH (l-34; 5 pmol/L) also stimulated CAMP production, which was greater than that induced by 30 pmol/L KW-3902 alone. A significant increase in CAMP production by 30 pmol/L DPCPX was also observed. When 30 pmol/L KW-3902 and 5 pmol/L rat PTH (l-34) were added to the medium together, their combined effect on Pi transport and CAMP production was additive and significantly greater than that of either agent alone (f < 0.01). DPCPX (30 pmol/L) also significantly inhibited Pi uptake by proximal cells. However, when 30 pmol/L KW-3902 and 30 pmol/L DPCPX were combined, no additional inhibitory effect on Pi transport was observed. In additional experiments, the addition of adenosine deaminase (10 units/ml) to the transport medium, resulting in the inhibition of adenosine binding to its receptors, also decreased phosphate uptake (by 42.8%, P < 0.01) in renal proximal cells. These data suggest that the inhibitory effect of both KW-3902 and PTH on Pi transport is mediated through the stimulation of CAMP. The effect of KW-3902 and PTH on CAMP production was additive, suggesting that their effects on Pi transport are mediated by different mechanisms. 0 1995 by the National Kidney Foundation, inc.

INDEX WORDS: KW-3902; cyclic AMP; phosphate transport; renal proximal tubular cell.

K W-3902, 1,3-dipropyl-8-(3-noradamanta- nyl)xanthine, is a newly synthesized selec-

tive adenosine A, receptor antagonist.’ This agent has been reported to have diuretic and na- triuretic actionse4 accompanied by little change in potassium excretion.5 Recent data from our laboratory have demonstrated that KW-3902 sig- nificantly inhibits phosphate (Pi) transport with no effect on glucose transport in the rat renal proximal tubular cell.6 These effects of KW-3902 are mediated largely by an inhibition of water and sodium transport at the level of the proximal nephron.‘,7 The pharmacological basis for these actions is believed to be adenosine antagonism.x The physiological effects of adenosine are thought to be mediated primarily by its interac- tion with extracellular adenosine receptors. Adenosine receptors are then coupled to adenyl- ate cyclase through the guanine nucleotide regu- latory proteins, inhibitory G-protein (Gi), and stimulatory G-protein (Gs).‘.’ ’ Adenosine can in- hibit and stimulate cyclic adenosine monophos- phate (CAMP) production by activation of A, and

A, receptors through either Gi or Gs proteins, respectively.12

Transport in the renal tubule is known to be responsive to a number of hormones that stimu- late the production of CAMP.” Parathyroid hor- mone (PTH) regulates the excretion of phospho- rus by inhibiting renal Pi reabsorption. It has been demonstrated that the PTH-induced inhibi- tion of Pi transport is preceded by a marked in- crease in the production of CAMP in the renal proximal tubular cell. Renal PTH receptors cou-

From the Section of Nephrology, Department of Medicine, Tulane University School of Medicine, and VA Medical Cen- ter, New Orleans, LA.

Received March 2, 1995; accepted in revised form May 23, 1995.

Supported in part by the Veterans Administration and by a grant-in-aid from the Kyowa Hakko Kogyo Co, Ltd, Tokyo, Japan.

Address reprint requests to Jules B. Puschett, MD, Depan- ment of Medicine, Tulane University School of Medicine, New Orleans, IA 70112.

0 1995 by the National Kidney Foundation, Inc. 0272~6386/95/2605-0020$3.00/O

American Journal of Kidney Diseases, Vol26, No 5 (November), 1995: pp 825-830 825

CAI ET AL

pled to adenylate cyclase are located in regions of the nephron where phosphorus is reabsorbed.14 The general consensus has been that PTH-regu- lated alterations in Pi transport are mediated by cAMP.‘~ Recent evidence, however, has shown that mediation of the actions of PTH may be more complex. ‘5-‘8 Signal transduction as a result of the binding of PTH to its receptors involves the stimulation of adenylate cyclase, leading to increases in the production of CAMP and activa- tion of protein kinase A.19 However, phospholi- pase C also is stimulated, leading to increases in inositol triphosphate and diacylglycerol with subsequent activation of protein kinase C.*’

We have previously demonstrated that KW- 3902 inhibits Pi uptake by the renal proximal tubular cell with little effect on glucose uptake,’ which is similar to the effects of PTH on Pi and glucose transport?’ The current studies were de- signed to examine the effect of KW-3902 on Pi transport and the production of CAMP and then to compare these effects of KW-3902 on Pi trans- port and CAMP production with those of PTH in the rat renal proximal tubular cell.

MATERIALS AND METHODS

Materials KW-3902 was a gift from Kyowa Hakko Kogyo Co. Ltd

(Japan). Synthetic rat parathyroid hormone fragment l-34 (PTH l-34), 3-isobutyl- 1 -methyl-xanthine (IBMX), bovine serum albumin (BSA), hormones, growth factors, and all other chemicals were purchased from Sigma (St Louis, MO). Radioactive “P-dipotassium phosphate (1 CiAmnol) was ob- tained from DuPont NEN Research Products (Boston, MA). All media and solutions were prepared using water purified with a milli-Q reagent grade water system (Millipore Barnstead, Dubuque, LA) The culture media (DME-F,,) con- sisted of a 50:50 mixture of Dulbecco’s modified Eagle’s (G&co, Grand Island, NY, H3-1700) and Ham’s Flz nutrient media (Gibco, 430-2100) supplemented with 15 mmol/L HEPES, pH 7.4, 14.3 mmol/L sodium bicarbonate, 5 &nL bovine insulin, 5 /~g/mL transferrin, 0.05 PmolIL hydrocorti- sone, 10 ng/mL epidermal growth factor (EGF), and 50 pmol/ L prostaglandin E, (PGE,). Hormones and growth factors were added to the medium immediately before use.

Primary Cell Culture Rat renal proximal tubules were isolated from female

Sprague-Dawley rats weighing approximately 250 g (Charles River, Wilmington, MA), under sterile conditions as de- scribed by Vinay et al*’ and as modified by Chen et al.zs The tubular suspension was filtered through a tea strainer and then centrifuged for 5 seconds at the setting of 5 in an JEC centri- fuge (Clinical Model). The supematant was transferred to a 50-mL centrifuge tube and saved. The pellet was washed

twice by resuspending it in 25 mL ice-cold Krebs-Henseleit saline (KHS) and spun again for 5 seconds at the same setting. The combined supematants were centrifuged in the IEC cen- trifuge (Needham Heights, MA) for 15 seconds. The pellet was then resuspended in 47% Percoll solution and centrifuged for 30 minutes at 19,000 rpm (43,000g) in a Sorvall centri- fuge. The F4 fraction” containing proximal tubules was washed four times with KHS containing 0.3% BSA, once with DME-F,2, and then resuspended in the hormone-supple- mented defined medium to a protein concentration of approxi- mately 0.3 mg/mL. Two-milliliter aliquots of tubule suspen- sion were then plated in 35-mm dishes. These cultures were maintained at 37°C in a humidified 95% air, 5% CO* incuba- tor. For the first 2 days the medium was changed daily to remove unattached tubules, and it was changed every 2 days thereafter.

Esfects of KW-3902 With and Without PTH and Adenosine Deaminase on Phosphate Transport

Phosphate (Pi) uptake by renal proximal tubular cells was assayed on day 5 or 6 of cell culture after seeding as pre- viously described.6,“,34 The culture medium was removed, and the cells were washed with a sodium-free solution con- taining 152 mmoliL choline chloride, 2.5 mmol/L KHCO,, 3.5 mmol/L KCl, 0.5 mmol/L MgSO,, 1 mmol/L CaC12, 2.5 mmol/L glutamine, and 10 mmoliL mannose, adjusted to pH 7.5.” These cells were preincubated in 1 mL sodium-free solution for 30 minutes at 37°C 95% air, 5% CO2 in the incubator and then replaced with working solution containing 152 mmol/L NaCl, 2.5 mmol/L KHCO,, 3.5 mmol/L KCl, 0.5 mmol/L MgSO,, 1 mmol/L CaC&, 2.5 mmol/L gluta- mine, and 10 mmoJ/L mannose, pH 7.5. When the effects of KW-3902 with and without rat PTH (l-34) and with DPCPX, another A, receptor antagonist, were investigated, the cells were exposed to vehicle or to the maximal inhibitory concen- tration (30 pmol/L) of KW-39026 with and without 5 pmol/ L of rat PTH(l-34) and with DPCPX (30 pmol/L) and incu- bated at 37°C for 2 hours. In an additional experiment, 10 U/mL adenosine deaminase was added and incubated with the cells at 37°C for 2 hours. Pi uptake was initiated by adding 10 PL 0.1 mmol/L KzH9*PO~. After 10 minutes at room temperature, transport was terminated by removing the working solution and by adding 1 mL ice-cold stopping solu- tion containing 1 mmol/L Tris-HEPES, 100 mmol/L NaCl, and 1 mmol/L sodium arsenate, pH 7.5. At this time, Pi transport is known to be linear.” Cells were washed twice and solubilized in 1 mL 0.2 N NaOH for 30 minutes at 30°C. The cell solution was aliquotted for scintillation counting and protein determination. Pi transport was expressed as pmol/ mg protein/min. Protein measurement was done by a micro- method using the principle of protein-dye binding as de- scribed by Bradford.” We determined phosphate transport in the presence of 152 mmol/L NaCl and did not correct total transport for its sodium-independent component in sodium- free medium. This sodium-independent transport has usually been less than 1% to 2% of total transport in our previous experiments and in the experience of other workers. Thus, the phosphate transport data presented here represent predom- inantly sodium-dependent uptake.

PHOSPHATE TRANSPORT INHIBITION BY KW-3902 827

Table 1. Effects of Rat PTH (l-34) and KW-3902, Alone and in Combination, on Phosphate (Pi) Uptake and CAMP Production in the Rat Renal Proximal Tubular Cell (RRPTC)

Additions Pi Uptake by RRPTC Picomoles CAMP Production in RRPTC

(Pi/mg protein/min) n (pmol/mg protein) n

Vehicle 61 2 3 9 18.2 t 1.3 14 Rat PTH(l-34) (5 pmol/L) 46 2 4 9 95.1 2 9.5 6 KW-3902 (30 pmol/L) 19 2 3 9 56.3 ? 4.6 15 KW-3902 (30 pmol/L) +Rat PTH(i -34) (5 pmol/L) 11 2 1=t 9 113.1 + 10.4*t 6

* P < 0.01 Y vehicle. t P < 0.01 v either KW-3902 or rat PTH (l-34) alone.

Effects of KW-3902 With and Without PTH on CAMP Production

Cyclic AMP production in response to various concentra- tions (3, 10, 30 pmol/L) of KW-3902 with and without 5 pmol/L rat PTH (l-34) and with DPCPX (30 PmoVL) was studied in the renal proximal tubular cell. On day 5 or 6 of cell culture, KW-3902 with and without rat PTH (l-34), with DPCPX or vehicle were added in Hank’s balanced salt solu- tion containing 0.1% BSA and 1 mmoUL phosphodiesterase inhibitor, IBMX. Cells were then incubated for 10 minutes at 37”C, and the reaction was stopped by aspiration of the media. The monolayer was then extracted with 5% perchloric acid for 60 minutes at -20°C. After neutralization with 4 N KOH and centrifugation, the supematant of the extract was analyzed for CAMP by a competitive protein-binding assay using a commercial kit (Diagnostic Product Carp, Los Angeles, CA). The values were then corrected for protein by a micromethod using the principles of protein dye binding as described by Bradford.25

Statistical Analysis All results are expressed as mean 2 SEM. Significance of

differences was calculated by the unpaired Student’s t-test and the analysis of variance (ANOVA) test.

RESULTS

The effect of KW-3902 (30 pmol/L) on Pi uptake by the renal proximal tubular cell in the presence and absence of 5 pmol/L rat PTH (l- 34) was evaluated. Both rat PTH (l-34; 5 pmol/ L) and KW-3902 (30 pmol/L) significantly de- creased Pi uptake by renal proximal tubular cells (P < 0.01; Table 1). At these concentrations, determined to be maximally inhibitory in our pre- vious studies,6 the effect of KW-3902 on Pi trans- port was more than twice as great as that of rat PTH (l-34) (P < 0.05). The inhibitory effect of 30 pmol/L KW-3902 in the presence of 5 pmol/ L rat PTH (l-34) on Pi uptake by the proximal tubular cell was significantly enhanced compared

with that of either agent alone (P < 0.01, Table 1). These data indicate that the inhibitory effects on Pi transport by KW-3902 and rat PTH (l-34) are additive. Pi uptake by proximal tubular cells was also significantly decreased by 30 pmol/L DPCPX (Fig 1; P < 0.01). However, the inhibi- tory effect of KW-3902 on Pi transport was not influenced by DPCPX (Fig 1). In addition, Pi transport was decreased by 42.6% in the presence of adenosine deaminase (10 U/mL) (Fig 2).

The effects of various concentrations (3, 10, 30 PmolIL) of KW-3902 on CAMP production were investigated. KW-3902 caused a dose-de- pendent enhancement of CAMP production in the rat proximal tubular cell (r = 0.997, P -=z 0.01)

80 1

%‘aM 40-i I 1 T

Vehicle

(n=Q)

Fig 1. Inhibitory effects of DPCPX and KW-3902, singly and in combination, on phosphate transport in the rat renal proximal tubular cell (RRPTC). l : P < 0.01 v vehicle. n = 8.

828 CAI ET AL

” Vehicle ADA( 10 unit/ml)

(n=9) (n=6)

Fig 2. Effect of adenosine deaminase (ADA) on phosphate transport in the rat renal proximal tubular cell (FtRPTC). l : P < 0.01 v vehicle. n = 6.

(Fig 3). The production of CAMP was also sig- nificantly increased by DPCPX (30 pmol/L) (Fig 3). Both 30 pmol/L KW-3902 and 5 pmol/L rat PTH (l-34) significantly stimulated CAMP pro- duction in the proximal tubular cell (P < 0.01, in each case) (Table 1). The stimulation of CAMP production by KW-3902 (30 pmol/L) was less than that by 5 pmol/L rat PTH (l-34) alone (P < 0.05). When 30 pmol/L KW-3902 and 5 pmol/ L rat PTH (l-34) were added to the medium together, their stimulatory effects on CAMP pro- duction in the proximal tubular cell were also additive and significantly greater than that of either KW-3902 or rat PTH (l-34) alone (P < 0.05, Table 1).

DISCUSSION

These studies demonstrate that the inhibition of Pi transport by KW-3902 and rat PTH (l-34) is associated with increased production of CAMP, suggesting that the inhibitory effects of both KW-3902 and PTH on Pi transport are mediated through stimulation of CAMP production. We have also observed that both the inhibitory effect on Pi transport and tbe stimulatory effects on CAMP production in the rat renal proximal tubule cells by KW-3902 and rat PTH (l-34) are addi- tive, suggesting that KW-3902 and PTH stimu- late CAMP through different mechanisms. Al- though the effect of KW-3902 on CAMP

production was less than that of PTH, its inhibi- tory effect on Pi transport was significantly greater. This suggests that the inhibitory effect of KW-3902 on Pi transport is not exclusively mediated by the stimulation of CAMP.

The inhibition of Pi transport by PTH is associ- ated with the activation of both protein kinase A and C.‘7*‘9 Because the current studies demon- strated that the inhibitory effects of KW-3902 and PTH on Pi transport are additive, KW-3902 may have additional inhibitory effects on Pi transport.

Two main types of adenosine receptors are related to the adenylate cyclase system, Al and AZ. These receptors have been localized or char- acterized in several anatomic sites within the kid- ney and are involved in the regulation of renal hemodynamic26 and tubular functions2’ In the current studies, the inhibition of Pi transport by both selective adenosine A, receptor antagonists, KW-3902 and DPCPX, was associated with in- creased production of CAMP, indicating that A, receptor antagonism interferes with Pi transport. We also observed that removal of endogenous adenosine by adenosine deaminase significantly decreased Pi transport by proximal cells. This indicates that the inhibitory effect of KW-3902 on Pi transport is caused by preventing the bind- ing of endogenous adenosine to its A, receptor.

80 1

Fig 3. Effects of various concentrations of KW- 3902 (3,10,30 pmol/L) and DPCPX (30 pmol/L) on the production of CAMP in ths rat renal proximal tubular cell. *: P < 0.05 v vehicle; l *: P < 0.01) v vehicle.

PHOSPHATE TRANSPORT INHIBITION BY KW-3902 829

Because KW-3902 is highly specific for the A, receptor, we cannot comment on any possible role of A2 receptors on Pi transport in these stud- ies. However, it appears that the prevention of binding of adenosine to A, receptors by KW- 3902 leads to increased production of CAMP through the inhibitory G-protein, Gi,28 resulting in inhibition of Pi transport. Because it is a xan- thine derivative, KW-3902 may also have an in- hibitory effect on phosphodiesterase and may cause an increase in CAMP, at least in part, through this mechanism. The data from Coulson et alz9 showing that adenosine stimulates Pi trans- port through an A, receptor-mediated mecha- nism, are consistent with our observations. Thus, our studies confirm that the inhibitory effect of KW-3902 on Pi transport is attributable to block- ade of the interaction of endogenous adenosine with the A, receptor, which is coupled to adenyl- ate cyclase through the guanine nucleotide regu- latory protein-the inhibitory G-protein (Gi).“,28

In our previous studies, Dixon plot analysis showed that the effect of KW-3902 is associated with an alteration in the velocity (V) of the Pi uptake process, whereas Michaelis constant (Km) is unchanged.6 This pattern is consistent with pure noncompetitive inhibition, implying ei- ther interference with the carrier function through carrier-inhibitor-substrate complexing or through impairment of energy utilization by the trans- porter.“.” ’ The interference of the interaction by adenosine with the A, receptor ultimately results in inhibition of Pi transport through an inhibitory G-protein. This would most probably reduce the velocity of the transport. Although our data sug- gest a role for Gi, we do not have direct proof in these studies attesting to its role. The involve- ment of Gi in decreased Pi uptake must be con- sidered speculative until studies directly examin- ing the role of Gi are conducted.

The inhibition of Pi transport by both KW- 3902 and PTH was associated with stimulation of CAMP. However, the magnitude of transport inhibition did not correlate with the magnitude of CAMP production. Furthermore, the inhibitory effects of these two agents were additive, in con- trast to the combination of two A, receptor antag- onists, KW3902 and DPCPX, which did not yield additive effect. These observations suggest that mechanisms other than CAMP may also contrib- ute to the inhibition of Pi transport. However,

stimulation of CAMP and coupling of the A, re- ceptor to adenylate cyclase through the inhibitory G-protein appear to be the major mechanisms by which KW3902 inhibits Pi transport.

ACKNOWLEDGMENT

We thank Kyowa Hakko Kogyo Co, Ltd, Japan, for the gift of KW-3902 and Karen Williamson for the preparation of this manuscript.

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