Parathyroid Hormone Increases Sodium/Calcium Exchange Activity in Renal Cells and the Blunting of the Response in Aging*

Na+-dependent Ca" efflux was demonstrated in cells isolated from the rat renal cortex, suggestive of the presence of a Na+/Ca2+ exchange carrier in the cells. Parathyroid hormone, when incubated with the cells in vitro, increased Na+-dependent Ca2+ efflux about 60%. The effect of the hormone was specific for biolog-ically active parathyroid hormone analogs and could be mimicked by cyclic nucleotides and forskolin. The effects of parathyroid hormone concentration on Ca2+ efflux and cyclic AMP formation were similar. These findings would be consistent with the view that the cyclic nucleotide might act as the intracellular messenger to increase Na+/Ca2+ exchange activity. Cells iso- lated from parathyroidectomized rats had decreased Na+-dependent Ca2+ efflux. When these cells were treated in vitro with parathyroid hormone, Na+-de-pendent Ca" efflux was enhanced to the same rate as found with cells from sham-operated animals. Parathyroid hormone-sensitive Na+/Ca2+ exchange activity was markedly blunted in cells from senescent (24 months) rats. Basal Na+-dependent Ca2+ efflux and Na+-independent Ca2+ efflux were not altered in the aged animal. Parathyroid-stimulated adenylate cy- clase was also decreased in aging. In contrast, forsko-lin-stimulated Na+-dependent Ca" efflux and adenyl- ate cyclase did not change with senescence. These findings would be compatible with a mechanism of desen- sitization that occurred at the level of the a-methylglucoside at equilibrium (19), indicated a relationship of 3.3 pl . mg" of cell protein. The general metabolism of cells was also examined by measuring the rate of COS formation from [6-'4C]glucose. Cells from rats 2, 6, 12, and 24 months of age produced 1.38 f 0.27, 1.35 f 0.22, 1.39 f 0.28, and 1.51 f 0.53 nmol of COS- h-l. mg" of protein, respectively, values which were not significantly different. Cellular integrity was assessed by measuring the leakage of lactic dehydrogenase activity. Cells were suspended in incubation medium for 1 h at 37 "C. They were reincu-bated for 20 min at 37 "C in the absence and presence of 0.3% Triton X-100. After the reincubation, the cells were centrifuged and dehydrogenase activity measured in the supernatant. Lactic dehydrogen- ase activity in the extracellular medium after detergent treatment increased more than 6 times, indicating that the plasma membrane of the cells not exposed to Triton X-100 was highly preserved. There were no significant differences between cells from various aged ani- mals nor between cells tested prior to or after the 1-h incubation period. Measurement of Calcium Efflux-A 50-pl aliquot of the freshly cell suspension preincubated at 37 "C for 20 min. cells were preloaded with calcium by adding to the suspension 50 pl of the incubation medium containing 2 mM CaCL labeled with 0.25 pCi of '%a and incubating the mixture with constant shaking for 30 min at 37 "C. %a content the min.) efflux England Nuclear. PTH(1-84) (lot 6009 A, 1227 units/mg) was purchased from Inolex. PTH(1-34), lot 006946, 6,800 units/mg) and PTH(3-34) were from Peninsula. Hyaluronidase, Type I-S, dibutyryl CAMP, and 8-bromo-CAMP were purchased from Sigma. Forskoli was obtained from Calbiochem-Behring. Collagen- ase CLS I1 was from Cooper Biomedical.

3 To whom correspondence may be addressed. intracellular level of Ca" (4). Confirmation of the presence of the exchange system has come from direct examinations of carrier activity in basolateral membrane vesicles prepared from the plasma membrane fraction of the renal cortex (5)(6)(7)(8).
Because the renal cytosolic free Ca2+ concentration is in the submicromolar range (9) whereas plasma and filtrate Ca" concentrations are about 2.5 mM and the membrane potential is cell interior negative, it is generally assumed that Ca2+ enters the tubular cell crossing the luminal segment of the plasma membrane by a diffusional mechanism, but that the cation has to be transported actively out of the cell crossing the basolateral segment of the plasma membrane. Two different Ca" transport systems, localized in the basolateral membrane, have been proposed to translocate the divalent cation; one is a high affinity Ca2+ ATPase which serves as a Ca2+ pump (7, [10][11][12][13], the other is the secondary active process mediated by the Na+/Ca2+ exchanger. It is not clear whether the two systems operate in parallel nor how they are regulated by intrinsic and extrinsic effectors. Parathyroid hormone (PTH') has long been known to stimulate renal Ca" reabsorption (14,15). The biochemical mechanism by which the hormone enhances Ca2+ transport has remained largely unknown. PTH receptors have been localized to the basolateral membrane of the tubular cell, and reception is coupled to increases in CAMP (15)(16)(17). Recently, we reported that Na+/Ca2+ exchange activity in basolateral membrane vesicles prepared from rats thyroparathyroidectomized 48 h previously is decreased about 40%, and this activity is restored when synthetic PTH  is infused in the thyroparathyroidectomized animal (6). These results have been confirmed in the dog (8). Moreover, it has been found that the alteration in activity is an apparent Vmax effect with no change in the apparent K,,, for Ca" (8). However, these earlier findings do not establish that when PTH is administered in uiuo the hormone acts directly on renal tubular cells to modulate Na+/Ca2+ exchange activity. In the present paper, we report experiments describing the effects of PTH and other agonists when incubated with isolated renal cells in uitro. We also report that the action of PTH is blunted in the senescent animal. A portion of this work has appeared in abstract form (18).

EXPERIMENTAL PROCEDURES
Isolation of Renal Cells-Wistar-derived male rats were obtained from the Animal Facility, Gerontology Research Center, National Institute on Aging. The animals were 2 months old, except as given in the experiments examining the effect of age. The kidneys were perfused in situ with Hanks' balanced salt solution containing 11 mM * The abbreviations used are: PTH, parathyroid hormone; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; Hepes, 4-(2-hy-droxyethy1)-1-piperazineethanesulfonic acid; CAMP, adenosine 3'5'monophosphate. glucose (isolation medium). The renal cortices from the 2 kidneys from each rat were finely minced and the tissue suspended in 30 ml of the isolation medium containing collagenase (1-1.67 mg/ml) and hyaluronidase (1 mg/ml). The suspension was incubated with constant agitation in a metabolic shaker for 30-40 min at 37 "C. The suspension was centrifuged at 130 X g for 1 min, and the supernatant was discarded. The pellet was resuspended in 10-15 ml of isolation medium and the mixture recentrifuged. The pellet was resuspended and the suspension was centrifuged at 43 X g for 10 s. The resultant supernatant, containing the cells, was centrifuged again at the higher gravitational force, and the sedimented cells were washed twice by alternating suspension and centrifugation. Then, the cells were suspended in an incubation medium containing 140 mM KCI, 10 mM Hepes-Tris buffer, pH 7.4, 10 mM mannose, 0.5 mM P-hydroxybutyrate, and 2.5 mM glutamine and washed twice. Finally, the cell preparation was suspended in the same incubation medium at a concentration of 10-15 mg of cell protein/ml.
Microscopic examinations revealed that the preparations consisted of single cells with clusters of from 2 to 6 cells. At least 85% of the cells excluded trypan blue. The general metabolism of the cells was evaluated by determining ATP content of cells when freshly isolated and after the cells were incubated for 1 h under conditions (described below) for preloading the cells with calcium and measuring calcium efflux. The ATP contents of freshly prepared cells of 6-and 24month-old rats were 7.7 f 0.7 and 7.4 f 0.5 nmol . mg-* of cell protein, respectively. After 1 h, the ATP contents were 7.1 f 0.1 and 7.6 f 1.0 nmol . mg-* of cell protein for the respective two ages. Intracellular space determinations, based on the uptake of a-methylglucoside at equilibrium (19), indicated a relationship of 3.3 pl . mg" of cell protein.
The general metabolism of cells was also examined by measuring the rate of COS formation from [6-'4C]glucose. Cells from rats 2, 6, 12, and 24 months of age produced 1.38 f 0.27, 1.35 f 0.22, 1.39 f 0.28, and 1.51 f 0.53 nmol of COS-h-l. mg" of protein, respectively, values which were not significantly different. Cellular integrity was assessed by measuring the leakage of lactic dehydrogenase activity. Cells were suspended in incubation medium for 1 h at 37 "C. They were reincubated for 20 min at 37 "C in the absence and presence of 0.3% Triton X-100. After the reincubation, the cells were centrifuged and dehydrogenase activity measured in the supernatant. Lactic dehydrogenase activity in the extracellular medium after detergent treatment increased more than 6 times, indicating that the plasma membrane of the cells not exposed to Triton X-100 was highly preserved. There were no significant differences between cells from various aged animals nor between cells tested prior to or after the 1-h incubation period.
Measurement of Calcium Efflux-A 50-pl aliquot of the freshly prepared cell suspension was preincubated at 37 "C for 20 min. The cells were preloaded with calcium by adding to the suspension 50 pl of the incubation medium containing 2 mM CaCL labeled with 0.25 pCi of '%a and incubating the mixture with constant shaking for 30 min at 37 "C. (Preliminary study showed that the %a content of the cells reached a steady state in 20-25 min.) After the preloading period, efflux of calcium was initiated by the addition of 900 pl of efflux medium containing either 140 mM NaCl or choline chloride, plus 2 mM EGTA and 10 mM Hepes-Tris buffer, pH 7.4. Efflux at 5 s, unless noted otherwise, was terminated by the addition of 3 ml of ice-cold efflux medium and the mixture rapidly filtered on 5-pm Millipore filters (SMWP02500) (20). The reaction tube and filter were washed 3 times, each time with 3 ml of ice-cold stopping solution. The cells and filter were digested with 1 ml of 0.1 N NaOH for several hours, 10 ml of scintillation fluid (Beckman Ready Solv. MP) was then added, and radioactivity counted. Zero time values (0% efflux) were estimated from reactions in which the ice-cold stopping solution was added prior to the efflux medium and the contents of the reaction tube immediately filtered. All incubations were carried out at least in triplicate. Each experiment was repeated a minimum of 5 times, each with different cell preparations. Values are reported as the mean k S.E. for the different experiments.
Other Assays-The synthesis of cAMP by hormone-stimulated renal cells was determined as previously described (21). Briefly, cAMP in neutralized perchloric acid extracts of the incubation reactions was duted from AG 1-X2 columns (recovery of [3H]cAMP was 80-90%) and then estimated with a commercial radioimmunoassay kit (Immuno Nuclear). Adenylate cyclase activity in renal cell membranes was measured as reported (22). Lactic dehydrogenase activity (23), cell ATP content (24), and protein (25) were estimated by standard procedures.
Puruthyroldectomy-Rats were anesthetized with ether and sodium pentobarbital (5 mg/100 g body wt) and parathyroid glands surgically removed by electrocautery or sham-operated. Parathyroidectomized rats were used 48 h after surgery. Completeness of gland removal was verified by the decrease in serum calcium concentration, from 2.14 f 0.04 mM before to 1.55 0.04 mM 48 h after surgery, whereas the concentrations in the sham-operated animals did not change, being 2.30 1-0.08 mM and 2.28 C 0.07 mM before and after the sham procedure, respectively. Each datum represents the mean f S.E. of 10-12 rats.
MuteTiak+"Ca (50 mCi/ml) and [3H]cAMP (30 Ci/mmol) were obtained from New England Nuclear. PTH(1-84) (lot 6009 A, 1227 units/mg) was purchased from Inolex. PTH , lot 006946, 6,800 units/mg) and PTH  were from Peninsula. Hyaluronidase, Type I-S, dibutyryl CAMP, and 8-bromo-CAMP were purchased from Sigma. Forskoli was obtained from Calbiochem-Behring. Collagenase CLS I1 was from Cooper Biomedical. Fig.  1 shows the time course of 45Ca2+ efflux from renal cells preloaded with &Ca2+ and then diluted 1:lO with a medium containing EGTA and either NaCl or choline chloride. When the extracellular medium contained choline+, efflux was relatively slow, only 14% of the "Ca2+ was released after 20 s. Similar results were obtained when KC1 was substituted for choline chloride (data not illustrated). In the presence of extracellular Na+, efflux of 45Ca2+ was rapid, being greater at all measured time points than those in the presence of choline+. At 5 s, the time interval used to roughly approximate initial rates, 16% of the 45Ca2+ effluxed, compared to 5% in the presence of extracellular choline+. When the calcium ionophore A23187 (1 PM) was added to the medium, efflux was nearly complete, 91% and 95% in the presence of EGTA and, respectively, choline+ and Na+. This finding indicated that the total exchangeable Ca2+ was not altered by the extracellular cation. Assuming isotopic equilibrium, the cell calcium content immediately prior to the initiation of efflux was 2.66 nmol-mg" of cell protein and this decreaseed to about 0.2 nmol.mg-l of cell protein after the exposure to ionophore. Previous studies on Na+/Ca2+ exchange in renal basolateral membrane vesicles demonstrated that the Na+ effect on Ca" flux was specific since Na+ could not be replaced by Li+, K+, or Rb+ (6,8). In addition, dissipation of the Na+ gradient by monensin inhibited Ca2+ transport (6). Also, the possibility that Na+ enhanced the debinding of Ca2+ from noncarrier binding sites on the membrane was largely excluded (6). Hence, the finding that extra- Cells were preloaded with 1 mM &CaCl as described in the text and %a2+ efflux initiated by the dilution of the cell suspension with a medium containing either Na+ or choline+. Each datum represents the mean f S.E. of 5 experiments, each with different cell preparations. Each experiment was carried out at least in triplicate.

Nu+-dependent Efflux of Calcium-
cellular Na+ increased the efflux of Ca2+ from the renal cell provided evidence for the presence of Na+/Ca2+ exchange in these isolated renal cells. The effect of the concentration of extracellular Na+ on the Na+-dependent rate of Ca2+ efflux is shown in Fig. 2. The Ca2+ efflux system was saturated with respect to extracellular Na+ at a concentration of about 100 mM. The relationship between Na+ concentration and rate (5 s) of efflux was sigmoidal, suggesting the interaction of more than one class of Na+ sites. A Hill transformation of the data (Fig. 2, inset), yielded a straight line (r = 0.99), with a calculated [Na+]o.5 of 10 mM. This value was similar to that obtained for the halfmaximal concentration of Na+ effecting Ca2+ efflux from basolateral membrane vesicles (6,8).
Effect of PTH on Na+-dependent Calcium Efflm-Highly purified bovine PTH(1-84) or vehicle control was incubated with the renal cells for 1.5 min prior to initiation of Ca2+ efflux, and then efflux was measured in the Na+-or choline+containing medium (Fig. 3). Earlier experiments revealed that the maximal effect of PTH on efflux was obtained with a 1.0-1.5-min exposure of the cells to the hormone (data not shown). As illustrated, 10 units/ml PTH stimulated the Na+-dependent efflux (5 s) of 45Ca2+ 55%, from 17.6 f 4.6% in the control The specificity of the action of PTH is indicated by the experiments shown in Fig. 4. Equivalent units (10 units/ml) of the synthetic tetratriacentapeptide PTH(1-34) resulted in an increase in Na+-dependent 45Ca2+ efflux comparable to that found with PTH(1-84). In contrast, the equivalent weight of the inactive form of the hormone, PTH(3-34), did not significantly affect Na+-stimulated Ca2+ efflux. The biological activity of the different PTH analogs was confirmed in the renal cell system used in the present study by measuring the cAMP formed. After the 1.5-min preincubation of the cells with the analogs, the CAMP found in the cells and medium was 8 f 2 pmol-mg" of cell protein in control cells and 12 f 3 (p < 0.05) and 13 & 3 (p < 0.02) for cells exposed to PTH(1-84) and PTH(1-34), respectively. The cAMP found when the cells were treated with PTH(3-34) was 9 f 2 pmol. mg" of cell protein, a value not significantly different from the value measured with control cells. The data in Fig. 4 show additionally that forskolin, an activator of renal cell adenylate cyclase (26), and the cAMP analogs, dibutyryl cAMP and 8bromo-CAMP, also stimulated Na+-dependent Y!a2+ efflux.
The effects of the concentration of PTH(1-84) on 45Ca2+ efflux and on the production of cAMP are shown in Fig. 5. Efflux was enhanced with increased concentration of hormone with a maximal effect obtained with about 10 units/ml. Elevating the hormone concentration to 50 units/ml did not stimulate efflux additionally. Na+-independent Ca2+ efflux was not affected at any of the tested concentrations of hormone (data not shown). The formation of cAMP also increased with increasing concentrations of PTH (Fig. 5).
Effect of Parathyroidectomy on Na+-dependent Calcium Efflux-Parathyroid glands were removed from rats, and 48 h later renal cells were prepared. At the time of death, the endogenous levels of PTH in the parathyroidectomized rats would be negligible in view of the short (24-min) half-life of the hormone in the parathyroidectomized rat (27), and this would be supported by the decrease in serum calcium concentration, noted under "Experimental Procedures." As shown CONTROL . .  (PTH 1-84) on Na+-dependent '5Ca2+ efflux and generation of CAMP. Na+-dependent efflux was measured as the difference (A) between the efflux in the presence of extracellular Na' , without added PTH, shown as zero A efflux, and the efflux in the presence of Na+, with the indicated concentrations of PTH. The A cAMP formed was calculated in a similar manner. Cells were exposed to PTH for 1.5 min prior to measurements of efflux and CAMP. The cAMP in cells as well as in the medium was determined. Each datum represents the mean of 4-6 experiments, each replicated. in Fig. 6, Na+-dependent 45Ca2+ efflux with cells from parathyroidectomized animals was decreased 25% from the value found with cells from sham-operated rats, 10.6 f 0.6% to 8.0 f 0.6% (p < 0.01). 45Caz+ efflux measured in the presence of extracellular choline+ instead of Na' was not altered by the removal of the parathyroids (data not illustrated). These findings suggested that Na+-dependent Ca2+ flux in renal cells was modulated by endogenous levels of PTH.
Cells from control and parathyroidectomized rats were incubated in vitro for 1.5 min with 10 units/ml PTH(1-84).
With cells from sham-operated rats, Na+-dependent 45Ca2' efflux was enhanced 61%, from 10.6 +-0.6 to 17.1 & 0.9% (p < 0.01). With cells from parathyroidectomized animals, PTH increased efflux 106%, from 8.0 k 0.6 to 16.5 t-1.5% (p < 0.01). The difference between 17.1 t 0.9%, the efflux found when control cells were incubated with PTH in vitro, and 16.5 & 1.5%, the efflux found when cells from parathyroidectomized animals were incubated with PTH in vitro, was not significantly different. These results indicated that 45Ca2+ efflux from control and parathyroidectomized rats treated with PTH in vitro could be increased to the same level.
Effect of Age of the Rat on the Sensitivity of Nu+-dependent Calcium Efflux to PTH-Because calcium homeostasis is a critical problem in the aging animal and the senescent rat was reported to have increased levels of immunoreactive PTH (28,29) and the accumulation of cAMP in response to PTH in renal cortical slices from 12-month-old rats was found to be decreased in comparison to the accumulation in slices from 2-month-old animals (30), we examined the effect of age of the rat on renal cell 45Ca2+ efflux and the responsiveness of the system to PTH. 45Ca2+ efflux into a choline+-containing medium was not altered by the age of the animal, from 2 to 24 months (data not shown). Fig. 7 shows that Na+-dependent 45Ca2+ efflux also did not significantly differ in the cells from the different aged rats, varying from a low of 8.3 f 1.4% in cells from 12-month-old rats to a high of 10.3 f 1.0% in the cells from 24-month-old animals. In contrast, the PTH responsiveness of the transport system did depend on age. When the cells were preincubated for 1.5 min with 10 units/ml PTH(1-34), Na+-dependent 45Ca2+ efflux from cells from 2month-old animals increased from 10.0 k 1.0% to 17 2.2%, a stimulation of 69% (p < 0.01). The PTH stimulation was 62% (p < 0.01) and 59% (p < 0.01) with cells from 6-and 12month-old rats, respectively. However, stimulation with cells from senescent animals (24 months) was markedly decreased, being 10.3 f 1.0% without PTH and 12.8 & 1.3% with PTH, a change of 24% which was not statistically significant. These findings indicated that the PTH sensitivity of the renal cell Na+-dependent 45Ca2+ efflux system was blunted in the senescent rat. The decrease in PTH responsiveness with aging could not be attributed to an age-dependent difference in the cellular uptake of labeled Ca" during preloading of the cells. The calcium contents of cells from the different aged rats at steady state immediately prior to initiation of efflux varied from 2.45 to 2.66 nmol.mg-' of protein, values which were not significantly different.
This loss in the responsiveness of the transport system to PTH with age was in accord with the finding of a decrease in PTH-sensitive adenylate cyclase in membranes prepared from the rend cells from senescent rats. Table I shows FIG. 8. Effect of age of the rat on the sensitivity Na+-dependent 46Ca2+ to forskolin. Cells were treated with 10 p~ forskolin for 1.5 min prior to initiation of efflux. Na+-dependent *Ca2+ efflux was calculated as described in Fig. 6. Each datum represents the mean f S.E. for 5-8 experiments, each replicated. adenylate cyclase did not change significantly with age.
The decreases with age in PTH-sensitive Na+-dependent 45Ca2+ efflux and PTH-sensitive adenylate cyclase, but the lack of a significant effect of aging on forskolin-stimulated adenylate cyclase, prompted us to examine whether the Na+dependent 45Ca2+ efflux enhanced by forskolin showed an agedependent decline. The results of this experiment are illustrated in Fig. 8. In agreement with the results of the experiment shown in Fig. 7, the basal rate of Na+-dependent 45Ca2' efflux as well as the efflux in the presence of extracellular choline+ (not illustrated) did not change significantly with age. In contrast to the decrease found with senescence in PTH-sensitive Na+-dependent 45Ca2+ efflux, forskolin-sensitive Na+-dependent 45CaZ+ efflux remained unchanged by the aging process. As shown (Fig. 8), forskolin (10 p~) increased Na+-dependent 45Ca2+ efflux 115, 104, 103, and 111% in 2-, 6-, 12-, and 24-month-old animals respectively.

DISCUSSION
The present study showed that the efflux of Ca2+ from renal cortical cells was increased specifically by extracellular Na+. The efflux system was saturable with respect to Na+, with a calculated [Na+]O.s of 10 mM. Na+ did not alter the total exchangeable Ca", measured in cells treated with A23187. Similar properties of the Na+/Ca2+ exchange system were found in previous studies on the exchange mechanism in renal cortical basolateral membrane vesicles (5)(6)(7)(8). Thus, the present findings provide evidence consistent with the presence of a Na+/Ca2+ carrier in the isolated rat renal cortical cells.
Previous investigations demonstrated that PTH stimulated renal Ca2+ reabsorption and decreased urinary Ca2+ excretion (14,15). The hormone increased Ca2+ transport in isolated tubules, indicating direct action on the kidney (31)(32)(33)(34)(35). More recently, it was reported that Na+/Ca2+ exchange activity in basolateral membrane vesicles prepared from thyroparathyroidectomized animals was decreased and that activity could be restored when PTH was infused in vivo into the animals (6,8). However, these earlier studies did not establish whether the hormone administered in vivo acted directly on the renal tubular cells to regulate Na+/Ca2+ exchange activity or whether the modulation was effected indirectly, such as by changing renal hemodynamics, altering serum calcium levels, or influencing the titers of other hormones. The present study now demonstrated that PTH incubated in vitro with isolated renal cortical cells increased the cellular efflux of Ca2+. The response to the hormone was dependent on the presence of Na' in the extracellular medium. Thus, these findings provided evidence supporting the postulate of a direct effect of PTH on renal cells which resulted in regulation of Na+/Ca2+ exchange activity. The present observations that PTH enhanced Ca" efflux would be consistent with the recent report that PTH lowered the intracellular free Ca2+ concentration in tubular cells (36).
The specificity of the action of PTH was indicated by the findings that the biological active bovine PTH(1-84) and the synthetic tetratriacentapeptide PTH(1-34) increased Na+dependent 45Ca2+ efflux, whereas the biological inactive analog PTH(3-34) did not. In addition, the effect on PTH(1-84) was concentration-dependent. Although the concentration of hormone needed for maximal response in our in vitro system was relatively high, approximately 10 units/ml, the dose was in accord with that used in previous investigations on the in vitro effect of bovine PTH on adenylate cyclase activity in renal tubular segments (37) and cultured renal cells (38). One possible explanation for the required high dosage was the discordance between the animal species from which the cell was obtained and the animal from which the PTH molecule was derived. Recently, it was reported that rat PTH  was significantly more potent than bovine or human PTH  when tested by in vitro activation of rat renal adenylate cyclase (39). Another possible explanation for the relatively high concentration of PTH needed to demonstrate response in these isolated cells could be an effect of collagenase-hyaluronidase, used in obtaining the preparations, on the hormone receptor-adenylate cyclase complex.
The present findings that 1) PTH when incubated with renal cells for 1.5 min elicited both Ca2+ transport and cAMP formation responses in a comparable concentration-dependent manner; 2) PTH analogs which increased Na+-dependent Ca2+ efflux also increased cellular CAMP, whereas the analog that did not affect transport did not generate cAMP and 3) dibutyryl CAMP and forskolin, the activator of adenylate cyclase, mimicked the action of PTH on Ca" efflux supported the view that cAMP might act as an intracellular messenger to increase Na+/Ca2+ exchange activity. The precise region of the nephron from which the isolated cortical cells were derived has not been established. This question gains importance because of the functional heterogeneity of the nephron with respect of Ca2+ transport and of the multiple loci along the nephron of PTH-stimulated adenylate cyclase. More than half of the Ca2+ reabsorbed by the nephron was found t o take place in the proximal tubule; the remainder occurred distally, in the cortical thick ascending limb of Henle and the cortical distal convoluted and collecting tubule (31)(32)(33)(34)(40)(41)(42). Substantial PTH-stimulated adenylate cyclase was demonstrated in the rat nephron in the proximal tubule, cortical thick ascending limb, and distal convoluted tubule, with only marginal activity in the cortical connecting tubule and no measurable PTH-stimulated cyclase in medullary regions (37,43). Furthermore, it had been long argued that Ca2+ flux in the proximal tubule was mostly passive or occurred paracellularly, whereas the remaining component of the filtered Ca", which was reabsorbed distally, was regulated (14, 15). However, recent evidence would suggest that a significant fraction of the filtered Ca2* conserved by the proximal tubule was reabsorbed actively (3,44). Moreover, the presence of the Na+/Ca2+ exchange system in the proximal tubule was inferred from physiological experiments (3,45), and Na+/Ca2+ exchange activity was demonstrated in basolateral membranes derived largely, but not exclusively, from the proximal tubule (5-8). Because the cells used in this study were prepared from the rat renal cortex, it is not unreasonable to expect elements from all cortical nephron segments to be present in the preparation. The pattern of hormone-stimulated cyclase in the cell preparation, Le. responsive to PTH but only moderately (2-fold over basal) responsive to vasopressin and not at all responsive to calcitonin: might suggest that the cells were derived predominantly from the proximal tubule. This finding would be consistent with the fact that the bulk of the nephrogenous elements in the cortex would be proximal tubules. In addition, the proximal tubule would be the locus of PTH-sensitive phosphate transport (46) and 25hydroxyvitamin D, hydroxylation (47). On the other hand, the cortical thick ascending limb of Henle in the rat should contain calcitonin-and PTH-stimulated adenylate cyclase as well as some enzyme responsive to vasopressin. Thus, the failure to find activation of the cyclase by calcitonin in the isolated cells would not be concordant with their derivation from the thick ascending limb. The possibility that during the preparation of the cells calcitonin-stimulated adenylate cyclase was lost was not precluded, however. Therefore, based on the present evidence, we would propose the hypothesis that PTH modulated Na+-dependent Ca2+ efflux in proximal tubular cells. This view would be consistent with a preliminary communication that PTH increased the active transport rate for Ca2+ in the microperfused proximal tubule (48) and with the recent report that the hormone and dibutyryl CAMP decreased the concentration of intracellular Ca2+ in the proximal tubule (36). Since the effects of PTH and cyclic nucleotides in increasing Ca2+ transport in distal regions of the nephron were well documented (49), it could be postulated that the hormone also acted on Na+/Ca2+ exchange in these nephron segments. Thus, although it is clear that PTH enhanced Na'/Ca2+ exchange in renal cells, the precise loci of the carrier in the heterogenous nephron and its sensitivity to the hormone remain to be determined.
The present study demonstrated that PTH-sensitive Na+/ Ca2+ exchange activity was markedly decreased in the senescent rat. Basal Na+/Ca2+ exchange and Na+-independent efflux of Ca2+ were not altered in the aging animal. In contrast, forskolin-stimulated Na+/Ca2+ exchange activity did not decrease in the aged rat. In accord with these results, it was found that PTH-stimulated adenylate cyclase activity was decreased in cells from senescent rats, whereas forskolinstimulated adenylate cyclase activity did not change with age. These decrements with age appeared not to be due to an agedependent decrease in the general metabolism and integrity of the cells, for there were no differences with age in the ATP content of the cells, the rate of glucose catabolism, the leakage of the cytosolic enzyme lactic dehydrogenase, and the exclusion of trypan blue. However, a possible age-dependent increase in the susceptibility of the PTH receptor or hormonereceptor coupling to adenylate cyclase to damage by the cell isolation procedure has not been definitively excluded.
These results would be consistent with reports that the aged rat had increased levels of immunoreactive PTH (28,29), and cultured renal cells developed refractoriness in their CAMP response to PTH (38, 50, 51). Our findings would be compatible with a mechanism of desensitization that occurred at the level of the receptor or hormone-receptor coupling to adenylate cyclase. Additional studies on PTH receptor number and sensitivity as well as the activities of the stimulatory and inhibitory GTP-binding proteins with respect to age are indicated. In summary, the present biochemical results demonstrating PTH-sensitive Na+-dependent Ca2+ efflux in renal cells and the blunting of the response with age may be of physiological significance in understanding calcium homeostasis and the imbalances in mineral metabolism associated with old age.