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*CALCIUM, ELEMENTAL
*PARATHYROID HORMONE
Endocrinology Vol. 140, No. 1 301-309
Copyright © 1999 by The Endocrine Society

ARTICLES

Cell-Specific Signaling and Structure-Activity Relations of Parathyroid Hormone Analogs in Mouse Kidney Cells1

Peter A. Friedman, Frank A. Gesek, Paul Morley, James F. Whitfield and Gordon E. Willick

Departments of Pharmacology and of Medicine, University of Pittsburgh School of Medicine (P.A.F.), Pittsburgh, Pennsylvania 15261; the Department of Pharmacology and Toxicology, Dartmouth Medical School (F.A.G.), Hanover, New Hampshire 03755; and the Institute for Biological Sciences, National Research Council of Canada (P.M., J.F.W., G.E.W.), Ottawa, Ontario, Canada K1A OR6

Address all correspondence and requests for reprints to: Peter A. Friedman, Ph.D., Department of Pharmacology, University of Pittsburgh School of Medicine, E-1347 Biomedical Science Tower, Pittsburgh, Pennsylvania 15261. E-mail: PAF10{at}pitt.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PTH is an 84-amino acid protein. Occupancy of its cognate receptor generally results in activation of adenylyl cyclase and/or phosphoinositide-specific phospholipase Cß (PLCß). In the kidney, PTH receptors are present on proximal and distal tubule cells. In proximal tubules, PTH induces calcium signaling, typified by a transient rise in intracellular calcium ([Ca2+]i) and inositol trisphosphate formation, but does not affect calcium absorption. By contrast, in distal tubules, PTH increases calcium absorption that is associated with a slow and sustained rise in [Ca2+]i, but does not stimulate phospholipase C (PLC) or cause inositol trisphosphate accumulation. Nonetheless, stimulation of distal calcium transport requires activation of protein kinase C (PKC) and protein kinase A. We now characterize the origin of the differential effects of ligand occupancy by using synthetic human PTH analogs that preferentially activate adenylyl cyclase and/or PLCß. We further tested the hypothesis that phospholipase D is responsible for PKC activation in distal tubule cells. PTH-(1–31) increased [Ca2+]i in distal tubule but not in proximal tubule cells, whereas PTH-(3–34) caused a partial increase in [Ca2+]i in proximal cells, but had no effect in distal cells. PTH-(7–34) blocked increases in [Ca2+]i in distal tubule cells stimulated by PTH-(1–34) and PTH-(1–31). The PLC inhibitor U73122 abolished the PTH-induced rise in [Ca2+]i and inositol trisphosphate formation by proximal tubule cells, but had no effect on PTH-stimulated Ca2+ uptake by distal tubule cells. These results support the view that activation of PKC by PTH in distal tubule cells does not involve PLCß. PTH did, however, activate phospholipase D with attendant formation of diacylglycerol in distal cells. As activation of PKC is required for induction of calcium transport by PTH, we conclude that PTH receptors are capable of activating multiple phospholipases and that the structural requirements for such activation differ in proximal and distal tubule cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PTH/PTH-RELATED peptide (PTHrP) receptor is a member of the structurally distinct class II subfamily of seven-transmembrane domain G protein receptors (1). Additional members of this family include the PTH2 receptor as well as receptors for calcitonin, secretin, pituitary adenylyl cyclase-activating peptide, and others (2). Class II receptors lack many of the structural signature sequences characteristic of the class I, rhodopsin/ß-adrenergic receptor family. A functional attribute of class II receptors is that they may obligatorily or facultatively activate adenylyl cyclase and phosphatidyl inositol-specific phospholipase C (PI-PLC).

Molecular cloning of the PTH receptor established three important points. First, PTH and PTHrP bind to the receptor, referred to as the type I receptor, with comparable affinity; second, as noted above, the PTH/PTHrP receptor couples, or at least is able to stimulate, adenylyl cyclase and PI-PLC (3); and third, both signaling events occur through a common receptor (4).

Nevertheless, some findings cannot be easily accommodated by the idea that all PTH effects are mediated by a canonical signaling mechanism or by a single receptor. For example, in some instances PTH activates adenylyl cyclase but not PI-PLC (5, 6, 7), whereas in others PTH activates PI-PLC but not adenylyl cyclase (8, 9, 10, 11). Second, carboxyl-terminal PTH fragments are biologically active but do not act through the type I PTH/PTHrP receptor (12, 13, 14, 15). These findings suggest that cell-specific modifications of the receptor or downstream events involved in receptor signaling are responsible for these disparities.

In the kidneys, PTH receptors are prominently expressed on proximal and distal tubules (16, 17). However, activation of proximal and distal PTH receptors has strikingly different actions. In proximal tubules, PTH exerts a host of biochemical effects including activation of 25-hydroxyvitamin D-1{alpha}-hydroxylase, gluconeogenesis, and ammoniagenesis and inhibition of Na+-phosphate absorption, Na+/H+ exchange, and Na+/Ca2+ exchange. All of these effects may involve calcium signaling, characterized by a transient rise in intracellular calcium ([Ca2+]i) and 1,4,5-trisphosphate (Ins[1,4,5]P3) formation. The PTH-induced elevation of [Ca2+]i in proximal cells is due entirely to release from intracellular stores; neither removal of extracellular calcium nor treatment with a calcium channel blocker inhibits the transient rise in [Ca2+]i. Furthermore, PTH does not affect net cellular calcium absorption by proximal nephrons. By contrast, in distal tubules, PTH potently stimulates transcellular calcium absorption that is accompanied by a slow and sustained rise in [Ca2+]i, but does not stimulate PI-PLC hydrolysis or cause Ins[1,4,5]P3 accumulation. The rise in [Ca2+]i in distal tubule cells is dependent entirely on extracellular calcium. Addition of a calcium channel blocker (18) or removal of extracellular calcium (19) abolishes the sustained PTH-stimulated rise in [Ca2+]i. In contrast to the prompt transient rise in [Ca2+]i in proximal tubule cells, the sustained increase in [Ca2+]i in distal tubule cells begins after a latency of 8–10 min (18, 19). The origin of this delay is uncertain, but appears to involve protein translation because it is inhibited by cycloheximide (20). These features might suggest that PTH-stimulated calcium transport by distal cells does not require protein kinase C (PKC) activation. However, we established that activation of both PKC and protein kinase A (PKA) is necessary for the stimulatory effect (21).

PKC is activated by diacylglycerol (DAG), which is produced consequent to receptor-mediated hydrolysis of phospholipids, such as phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylcholine (22). It was initially assumed that DAG involved the action of PLC coupled to the breakdown of PIP2. It is now recognized that DAG may also arise from the activation of phospholipase D (PLD) and the ensuing hydrolysis of phosphatidylcholine to form phosphatidic acid, which is converted to DAG by the action of phosphatidic acid phosphohydrolase, and choline. DAG so formed may be responsible for activation of calcium-independent forms of PKC (23).

The sequence within PTH responsible for activating PKC has been suggested to be comprised of residues 29–32 (13, 14) and has been referred to as the PKC-activating domain of PTH. Amino-terminal PTH analogs shorter than 32 residues failed to activate PKC in rat osteosarcoma (ROS) cells (13, 14, 24).

Notably, although PKC activation was mandatory for the action of PTH on distal tubule cells (21), the findings suggested that stimulation of transport proceeded through a pathway that did not involve hydrolysis of phosphatidylinositol 4,5-bisphosphate, because measurable accumulation of Ins[1,4,5]P3 did not occur in distal tubule cells after challenge with PTH-(1–34). Comparable experiments on proximal tubule cells, in which PTH inhibits Na+-dependent phosphate absorption, revealed Ins[1,4,5]P3 formation (21).

In the present investigation we took advantage of the presence within the kidney of the two cell types (proximal and distal tubule cells) exhibiting distinct patterns of PTH receptor signaling to characterize the origin of the differential effects of ligand occupancy. For these studies we employed synthetic PTH analogs to test the hypothesis that stimulation of calcium transport and activation of PKC in distal tubule cells are mediated by a PLCß-independent mechanism.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Immortalized mouse proximal convoluted tubule S1 cells and distal convoluted tubule cells were established as described previously (25, 26). The proximal tubule cells exhibit functional Na+-dependent phosphate cotransport and formation of cAMP in response to PTH (26). Distal convoluted tubule cells exhibit a phenotype consisting of PTH-stimulated adenylyl cyclase, calcium transport, and thiazide diuretic-sensitive Na:Cl cotransport (21, 27). Distal and proximal cell lines were grown on 100-mm dishes (Corning Glass Works, Corning, NY) in DMEM/Ham’s F-12 medium (Sigma Chemical Co., St. Louis, MO) supplemented with 5% heat-inactivated (56 C, for 20 min) FCS (Sigma Chemical Co.) and PSN antibiotic mixture (50 µg penicillin, 50 µg streptomycin, and 100 µg neomycin/100 ml medium; Life Technologies, Gaithersburg, MD) in a humidified atmosphere of 95% O2-5% CO2 at 37 C. Proximal tubule cell passages 40–60 and distal convoluted tubule cell passages below 40 were employed.

Primary cultures of mouse proximal and distal tubule2 cells were also used. These were isolated by a double antibody immunodissection procedure (28). Primary cultures were grown under the same conditions as the cell lines, but with 10% FCS (Sigma Chemical Co.). Cells were placed in serum-free DMEM/Ham’s F-12 medium 16 h before use.

PTH fragments
Carboxyl-terminal amide fragments of human PTH or PTHrP, except where noted [PTH-(1–27)NH2, PTH-(1–28)NH2, PTH-(1–29)NH2, PTH-(1–30)NH2, PTH-(1–31)NH2, PTH-(1–34)NH2, PTHrP-(1–31)NH2, PTH-(1–34)NH2, and PTH-(3–34)], and PTHrP analogs [PTHrP-(1–31)NH2 and PTHrP-(1–34)NH2] were synthesized using the 9-fluorenylmethoxycarbonyl (F-moc) procedure (29) and a continuous flow peptide synthesizer (model 9050, PerSeptive Biosystems, Framingham, MA) as described previously (30).

Intracellular calcium
[Ca2+]i was measured in single cells or clusters of up to three cells as detailed previously (27). In brief, proximal or distal convoluted tubule cells plated on coverslips were incubated for 60 min at 37 C with 10 µM fura-2/AM (Molecular Probes, Inc., Eugene, OR) in a buffer consisting of 140 mM Na+, 148 mM Cl-, 5 mM K+, 1 mM Ca2+, 1 mM Mg2+, 28 mM HEPES, and 18 mM Tris-HCl with 10 mM glucose at pH 7.4 and adjusted to 295 mosmol. The cells were then rinsed several times, placed in a temperature-controlled incubator (Narishige Medical Systems Corp., Greenvale, NY), and mounted on the stage of an inverted microscope (Nikon Diaphot, Nikon, Melville, NY). Emitted fluorescence was measured with a Nikon Photoscan-2. Calibration was performed as previously described (27).

45Ca2+ uptake
Calcium uptake was measured by a rapid filtration technique (27). Five to 6 x 106 cells/60-mm dish were removed by brief (<=5 min) treatment with 0.125% trypsin and rinsed with a modified Krebs-Ringer solution containing 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, and 1 mM MgCl2 buffered with 18 mM Tris base and 28 mM HEPES to pH 7.40 ± 0.01 (to 295 ± 1 mosmol). Buffer, with or without PTH or other test drugs (total volume, 100 µl), was placed in 12 x 75-mm polystyrene tubes. A 50-µl aliquot of cells was added for 1 min before addition and rapid mixing of 50 µl 45Ca2+ to initiate isotope uptake. Sample tubes were maintained at 37 C in a shaking incubator. Tracer uptake was terminated after 1 min by rapid addition of ice-cold isoosmotic, Li2SO4-HEPES rinse buffer [140 mM Li2SO4 and 10 mM HEPES (pH 7.40); 295 ± 2 mosmol] and filtered onto Whatman GF/C filters (Clifton, NJ) using a Millipore Corp. 12-port manifold (Bedford, MA) followed by two additional rinses with Li2SO4-HEPES buffer. Nonspecific binding to filters and cells was determined in the presence of Li2SO4-HEPES buffer added to the cells before the addition of 45Ca2+. Calcium uptake was normalized for protein content, which was measured using the Lowry procedure on 50-µl aliquots of cells (31). Tracer uptakes are expressed as nanomoles per min/mg protein.

cAMP
Cells were trypsinized, washed, and resuspended in buffer 135 mM NaCl, 4 mM KCl, 1.0 mM KH2PO4, 1.2 mM CaCl2, 1.2 mM MgCl2, 10 mM HEPES, and 5 mM glucose) to a final concentration of 1 x 106 cells/ml. A 50-µl aliquot of the cell suspension was taken for protein determination. A second 50-µl aliquot was added to a prewarmed plastic tube of 50 µl cAMP buffer containing 0.4% BSA and 0.2 mM rolipram in the presence or absence of the indicated PTH analog. After 15 min in a 37 C shaking water bath, 1 ml ice-cold 0.132 M trichloroacetic acid was added, and samples were vortexed and stored at -70 C. For measurement of cAMP, the tubes were thawed and centrifuged at 1200 x g for 30 min, and 0.9 ml supernatant was removed and transferred to a glass tube. The sample was extracted twice with 0.9 ml H2O-saturated ether, and the final aqueous solution was dried. cAMP was measured by RIA using a kit from Diagnostic Products Corp. (Los Angeles, CA).

Inositol trisphosphate and DAG
Ins[1,4,5]P3 was measured by RIA. Primary cultures of proximal tubule cells were grown to confluence in six-well plates. Cells were serum and antibiotic starved overnight before treatment with PTH. Cells were rinsed with a buffer composed of 140 mM NaCl, 4.6 mM KCl, 1 mM CaCl2, and 1 mM MgCl2 adjusted to pH 7.40 ± 0.01 with 28 mM HEPES and 18 mM Tris base (to 295 ± 2 mosmol). A second rinse used a buffer containing 50 mM LiCl, which isoosmotically replaced NaCl. Cells were treated in the wells with a total of 500 µl buffer with or without PTH for 2 min at 37 C on a shaking incubator. The treatment was terminated by the addition of 100 µl ice-cold 20% perchloric acid. Wells were scraped, and the contents were placed in a 1.5-ml conical centrifuge tube and pelleted by centrifugation at 2000 x g for 15 min at 4 C. The supernatant was neutralized to approximately pH 7.5 by the addition of 175 µl buffer consisting of 1.5 M KOH and 120 mM HEPES. The mixture was centrifuged as described above to remove KClO4 precipitate. A 500-µl aliquot of the supernatant was removed and placed in a 1.5-ml conical centrifuge tube. Samples were evaporated to dryness at medium heat (43 C) with a Speed-Vac (Savant Instruments, Farmingdale, NY) and reconstituted in 100 µl distilled water. Samples and standards (100 µl) were analyzed with an [3H]InsP3 assay kit (Amersham, Arlington Heights, IL). Protein-bound samples and standards were placed in 5 ml scintillation fluid and counted by ß-emission spectrometry.

DAG accumulation was determined using the method developed by Griendling et al. (32) and modified as described previously (33). Distal convoluted tubule cells were grown to 90% confluence on 100-mm dishes as described above and labeled with [3H]arachidonic acid (New England Nuclear Corp., Boston, MA) for 4 h. PTH was added for the times shown in Results. The experiment was terminated, and DAG was extracted and analyzed by TLC as detailed previously (33).

PLD
PLD was assayed by exploiting its unique ability to catalyze the transphosphatidylation of a primary electrophile, butanol in this case. Distal convoluted tubule cells were grown to confluence on 35-mm dishes. At the time of the experiment, 20 µCi [4-N-3H]butanol (ARC, St. Louis, MO) were added to each dish, with or without 100 nM PTH-(1–84), for 30 min at 37 C. The reaction was stopped by adding 0.9 ml ice-cold 0.6 N perchloric acid. The cells were scraped in to a 15-ml centrifuge tube, resuspended in 1 ml buffer A (CHCl3-CH3CHCOOH, 100:0.5) and applied to 500-mg, 6-cc silica Sep-Pak columns (Waters, Milford, MA) that had been preequilibrated with 10 ml CHCl3. The column was washed with 6 ml buffer A, and the eluate was discarded. Phospholipids were eluted with 5 ml buffer C (CHCl3-MeOH-H2O, 2:1:0.8), evaporated to dryness in a Speed-Vac, and resuspended in 100 µl CHCl3-MeOH (4:1) for analysis by TLC. Phosphatidylbutanol was resolved by short bed, continuous development TLC, using silica gel G-precoated, channeled plates (Analtech, Newark, DE) and a mobile phase consisting of CHCl3-MeOH-CH3COOH (65:15:2) as described by Sarri et al. (34). A phosphatidylbutanol standard (1,2-dimyristoyl-sn-glycero-3-phosphobutanol; Avanti Polar Lipids, Alabaster, AL) was run in parallel. Identification was performed with iodine. Samples were cut from the poly-backed plate and counted by ß-scintillation spectrometry.

Materials
U73122 and U73343 were purchased from Research Biochemicals International (Natick, MA). Rolipram was purchased from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA). Human PTH-(19–84) was provided by Dr. Harald Jüppner, Massachusetts General Hospital (Boston, MA). 45Ca2+ (1 µCi/ml; carrier-free) was obtained from New England Nuclear (Boston, MA). Chemicals and other reagents were of the highest grade commercially available. Solutions containing drugs were prepared fresh daily.

Statistical analysis
The effects of experimental treatments were assessed by paired comparisons within experiments and are reported as the mean ± SE of n independent experiments. Comparisons between control and experimental treatment groups were evaluated by Student’s t test (Instat, GraphPad Software, Inc., San Diego, CA). P <= 0.05 was assumed to be significant. Kinetic parameters and curve fitting were performed with Prism software (GraphPad).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PTH effects on intracellular calcium in distal tubule cells
PTH increases calcium transport by renal distal convoluted tubules, and this effect involves activation of PKA and PKC (21). To determine the structural requirements of PTH necessary for stimulation of calcium entry, we analyzed the effects of synthetic amino-terminal fragments of human PTH. [Ca2+]i was measured in single mouse distal convoluted tubule cells. As shown in the example in Fig. 1aGo, PTH-(1–34) increased [Ca2+]i by 200 nM, similar to the effects reported previously with the comparable bovine analog (18). PTH-(1–31) also increased [Ca2+]i, although to a somewhat lesser extent. Figure 1bGo shows a complete dose-response curve for PTH-(1–31). The maximal sustained increase in [Ca2+]i elicited by PTH-(1–34) (330 ± 5 nM; n = 4) was comparable to that caused by bovine PTH-(1–34) (18), but was greater than that caused by PTH-(1–31) (275 ± 4; n = 6). The smaller maximal effect, an index of intrinsic activity, is consistent with the view that in the distal tubule, PTH-(1–31) is a partial receptor agonist.



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  		Figure 1. Effects of selected PTH analogs on [Ca2+]i in distal convoluted tubule cells. A, Representative sample traces of effects of PTH-(1–34), PTH-(1–31), and PTH-(1–30) on [Ca2+]i in single cells. After a 2-min control period, the indicated PTH analog was added to the bath at 10-9 M. B, Dose-response curve for PTH-(1–31) on [Ca2+]i. Each point represents the mean ± SE of three independent measurements. Maximal stimulation was at 275 ± 3 nM; half-maximal stimulation occurred at 0.22 nM. C, Summary of the effects of the indicated PTH analogs on the change in [Ca2+]i ([/{delta}[Ca2+]i) in distal convoluted tubule cells.

 
In contrast to the stimulatory actions of PTH-(1–34) and PTH-(1–31) in distal convoluted tubule cells, shorter carboxy-truncated analogs (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) were devoid of activity as was PTH-(3–34). The results for all analogs are compiled in Fig. 1cGo. We also examined the action of the carboxyl-terminal peptide, PTH-(19–84). It had no detectable effect on [Ca2+]i in distal convoluted tubule cells (data not shown).

If a common PTH/PTHrP receptor mediates PTH actions in distal tubules, then the receptor antagonist PTH-(7–34) should block the rise in [Ca2+]i. We tested this postulate by analyzing the effects of PTH-(7–34) on changes in [Ca2+]i induced by concentrations of PTH-(1–34) and PTH-(1–31) that elicited half-maximal increases in [Ca2+]i (10-10 M). The results, shown in Fig. 2Go, revealed that intermediate concentrations of PTH-(7–34) (10-9 M) caused partial suppression of PTH-(1–34)-stimulated increases in [Ca2+]i; higher concentrations (10-8 M) abolished the effects of PTH-(1–34) or PTH-(1–31).



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  		Figure 2. Competitive inhibition by PTH-(7–34) of PTH-(1–34)- and PTH-(1–31)-induced rises in [Ca2+]i in distal convoluted tubule cells. The indicated concentration of PTH-(7–34) was added to the bath concurrently with 10-10 M of either PTH-(1–34) or PTH-(1–31).

 
PTH effects on intracellular calcium in proximal tubule cells
The actions of these PTH analogs were examined under identical conditions in proximal tubule cells. PTH-(1–34) caused a transient, spiking elevation of [Ca2+]i, as described previously by several groups (3, 35, 36, 37). As depicted in Fig. 3aGo, PTH-(1–31) had no measurable on [Ca2+]i in a single proximal tubule S1 cell. Equivalent results were obtained in four paired independent experiments (Fig. 3bGo). The absence of an effect of PTH-(1–31) on [Ca2+]i in proximal tubule cells contrasts with its prominent action on distal convoluted tubule cells (Fig. 1Go, a–c). The spiking rise in [Ca2+]i that attends PTH action in proximal tubule cells is primarily due to the release of calcium from intracellular stores, which is secondary to activation of PLCß. Hence, the failure of PTH-(1–31) to elevate [Ca2+]i in proximal tubule cells is consistent with the view that in these cells PTH-(1–31) does not activate PLCß, as originally proposed by Whitfield et al. (13, 14). PTH-(19–84) had no detectable effect on [Ca2+]i in proximal tubule cells (data not shown). This latter observation suggests that the 29–32 region is not sufficient to activate PLCß and that amino-terminal sequence is required.



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  		Figure 3. Effects of PTH-(1–31), PTH-(3–34), or PTH-(1–34) on [Ca2+]i in proximal convoluted tubule cells. A, Representative sample traces of effects of PTH-(1–31), PTH-(3–34), and PTH-(1–34) on [Ca2+]i. The designated analog of PTH was added to the bathing solution at the indicated times. A 5-min washout of the peptide was followed by a control recording of [Ca2+]i before adding the next analog. B, Summary of four experiments analyzing the effects of PTH-(1–31), PTH-(3–34), and PTH-(1–34) on [Ca2+]i. Lines connect paired observations from the same cell.

 
Dependence of PTH actions on PLC
In previous studies we established that PTH causes Ins[1,4,5]P3 formation by mouse proximal tubule cells (21). To test the theory that the PTH-induced rise of [Ca2+]i in proximal tubule cells is attributable to PLCß, we used the PLC inhibitor U73122. Figure 4aGo shows that U73122 virtually abolished the PTH-(1–34)-stimulated rise in [Ca2+]i in a proximal tubule cell, but has no detectable effect of its own. The inactive analog, U73343, had no effect on resting or PTH-stimulated changes in [Ca2+]i. Likewise, U73122 inhibited PTH-stimulated Ins[1,4,5]P3 formation in proximal tubule cells (Fig. 4bGo). These findings fortify the conclusion that the effects of PTH on [Ca2+]i in proximal tubule cells are mediated by PLCß. We also determined the effects of U73122 on PTH-dependent calcium uptake by distal tubule cells. In prior work we established the ability of distal cells to mount an appropriate PLC-ß response, characterized by a transient rise in calcium and inositol trisphosphate (IP3) formation (38). Moreover, U73122 abolished the rise in both calcium and IP3 formation. In the present studies we used the same concentration of U73122 (10 µM). As shown in Table 1Go, inhibition of PLCß with U73122 under conditions identical to those that blocked hormone-stimulated increases in [Ca2+]i and Ins[1,4,5]P3 formation in proximal tubule cells had no effect on 45Ca2+ uptake by distal tubule cells. Predictably, U73122 had no detectable effect on PTH-(1–34)- or PTH-(1–31)-induced increases in [Ca2+]i in distal tubule cells (data not shown).



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  		Figure 4. Effect of PLCß inhibition in proximal convoluted tubule cells. A, The PLCß inhibitor U73122 abolished PTH-stimulated elevations in [Ca2+]i. PTH (10 nM) was added to the bath before or after treatment with the PLCß inhibitor, U73122, or its inactive control, U73343. Inhibitors were used at a final concentration of 10 µM. B, Application of U73122 eliminated the PTH-stimulated accumulation of InsP3. Data are the mean ± SE of four paired observations. P < 0.01 vs. control.

 

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  		Table 1. Effects of U73122 on calcium uptake by distal convoluted tubule cells

 
The aforementioned results with U73122 affirm the conclusion reached with PTH-(1–31) that PTH-induced calcium entry in distal convoluted tubule cells requires activation of PKC, but is independent of PLCß. The 29–32 region differs between human PTH, where the sequence is Gln-Asp-Val-His, and PTHrP, which has the sequence Ala-Glu-Ile-His. This suggested that PTHrP-(1–34) and PTHrP-(1–31) would also increase calcium entry in distal convoluted tubule cells if PKC activation proceeded through a PLCß-independent mechanism. Therefore, we compared the actions of the 1–34 and 1–31 analogs of PTH and PTHrP. As shown in Fig. 5Go, although both analogs raised [Ca2+]i, PTH-(1–34) was more efficacious that its 1–31 counterpart, whereas the two PTHrP fragments exhibited comparable activities. These findings support the view that the 29–32 sequence of PTH is not obligatorily or uniquely associated with activation of PLCß, but may activate PKC through other pathways.



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  		Figure 5. Analysis of PTH and PTHrP effects on [Ca2+]i in distal convoluted tubule cells. Data are the mean ± SE of three or more paired observations, where the effects of 10-9 M of the respective 1–34 and 1–31 analogs of PTH and PTHrP were compared on the same cell or group of cells. **, P < 0.01 vs. PTH-(1–34).

 
The fact that stimulation of calcium transport in distal convoluted tubule cells by PTH requires activation of PKC but evidently does not proceed through PLCß suggested that in these cells PKC activation may be secondary to stimulation of PLD. Figure 6Go shows that in six paired independent comparisons, PTH-(1–84) increased PLD activity (P < 0.01). Hydrolysis of phosphatidylcholine by PLD releases phosphatidic acid that, in turn, is converted to DAG by the action of phosphatidic acid phosphohydrolase. Thus, if PTH acts through PLD in distal convoluted tubule cells, it should stimulate DAG formation despite the absence of Ins[1,4,5]P3 accumulation. As illustrated in Fig. 7Go, PTH caused a time-dependent stimulation of DAG by distal convoluted tubule cells. Maximal activation occurred at 2 min, a time appreciably before PTH increases calcium entry by these cells (Fig. 1aGo). We further tested the postulate that stimulation of PKC by PTH proceeds through a PLCß-independent mechanism in distal convoluted tubule cells by analyzing the effects of U73122. The results, shown in Table 2Go, confirm that doses of U73122 that inhibit PTH-stimulated Ins[1,4,5]P3 accumulation by proximal cells (Fig. 4bGo) (21) or {alpha}-adrenergically induced Ins[1,4,5]P3 formation in distal cells (38) did not suppress activation of PKC.



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  		Figure 6. Stimulation of phospholipase D in distal tubule cells by PTH. PLD activity was measured by transphosphatidylation of [3H]butanol. Cells were stimulated with 100 nM PTH-(1–84) for 30 min.

 


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  		Figure 7. Time course of DAG formation stimulated by PTH-(1–84) in distal tubule cells. Each point represents the average of three independent determinations.

 

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  		Table 2. Effects of U73122 on PTH-stimulated PKC activity in distal convoluted tubule cells

 
Adenylyl cyclase activation by amino-terminal PTH analogs
As described above, we noted a small stimulatory action of PTH-(3–34) on [Ca2+]i in proximal convoluted tubule cells but not in distal convoluted tubule cells (Fig. 3Go, a and b). Such an effect implies that in proximal tubule cells the rise in [Ca2+]i does not require activation of adenylyl cyclase, which depends upon the presence of Ser and Ala in positions 1 and 2 (40). To test explicitly the hypothesis that the rise in [Ca2+]i in proximal tubule cells is mediated through a cAMP-independent mechanism, we examined the effects of PTH-(3–34). In seven paired studies, resting [Ca2+]i averaged 99 ± 4 nM and increased by 33% to 132 ± 5 nM (P < 0.01). Nonetheless, the magnitude of this change was less than that elicited by PTH-(1–34) (change in [Ca2+]i, 116 ± 4) or PTH-(1–84).

To learn whether the origin of the dissimilar actions of the PTH analogs, particularly with respect to PTH-(1–31), which increased [Ca2+]i in distal tubule cells but not in proximal tubule cells, was attributable to differences in the generation of second messengers, we analyzed the effects of selected amino-terminal PTH analogs on cAMP formation. The results, shown in Fig. 8Go, revealed distinct differences among the various analogs, although in all circumstances their effects on cAMP formation in distal convoluted tubule and primary cultures of proximal tubule cells were comparable. PTH-(1–30) failed to stimulate cAMP accumulation in either distal tubule or proximal tubule cells, whereas PTH-(1–31) caused nearly a 20-fold increase in cAMP formation in both distal tubule and proximal tubule cells. Thus, although PTH-(1–31) induced comparable cAMP formation in proximal tubule cells, it failed to increase [Ca2+]i. These findings suggest that the rise in [Ca2+]i in proximal tubule cells cannot be triggered solely by a cAMP- or PKC-dependent mechanism.



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  		Figure 8. Stimulation of cAMP formation by selected PTH analogs in proximal and distal tubule cells. cAMP formation was measured in primary cultures of proximal and distal tubule cells as described in Materials and Methods. The results show the mean ± SE of sic independent observations. Only PTH-(1–31) and PTH-(1–34) caused significant cAMP formation. The magnitude of cAMP accumulation was statistically indistinguishable for PTH-(1–31) and PTH-(1–34) and between proximal and distal tubule cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present studies were designed to take advantage of the presence within the nephron of PTH receptors that exhibit different signaling behaviors and physiological effects to characterize the signaling pathways stimulated by PTH in proximal and distal renal tubule cells. For these experiments we used synthetic PTH fragments that selectively or preferentially activate adenylyl cyclase or PLCß. The most striking finding of the present investigation is that PTH-(1–31) dissociates calcium transport in proximal and distal tubule cells; calcium entry and a rise in [Ca2+]i were stimulated in distal tubule cells but not in proximal tubule cells despite full activation of adenylyl cyclase. The results strongly demonstrate that the type I PTH/PTHrP receptor uses different effectors to trigger the transient rise in [Ca2+]i in proximal tubule cells and the delayed, but prolonged, elevation of [Ca2+]i in distal tubule cells.

Both PTH-(1–31) and PTH-(1–34) caused comparable accumulation of cAMP by proximal and distal convoluted tubule cells. Thus, it is unlikely that preferential activation of adenylyl cyclase accounts for the observed differences in calcium entry. PTH-(1–27), PTH-(1–28), PTH-(1–29), and PTH-(1–30) failed to stimulate detectable cAMP formation in proximal and distal convoluted tubule cells. The findings with distal tubule cells differ somewhat from the pattern of adenylyl cyclase stimulation by these analogs in rat osteosarcoma 17/2 cells, where PTH-(1–30)NH2 was nearly equally effective as PTH-(1–31) and PTH-(1–30) (30). The divergence in the results may be due to species differences as well as to differences in PTH/PTHrP receptor number. For instance, when the human type I PTH/PTHrP receptor was expressed in LLC-PK1 cells, PTH-(1–34), PTH-(1–31), and PTH-(1–30) caused equivalent stimulation of adenylyl cyclase, with identical half-maximal concentrations. Likewise, all three PTH analogs elicited comparable InsP3 formation and rise in [Ca2+]i. However, when equivalent numbers of the rat type I PTH/PTHrP receptors were expressed in LLC-PK1 cells, PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) was an order of magnitude less potent in activating adenylyl cyclase and failed to stimulate PLCß, as evidenced by the absence of InsP3 formation (41).

Although it could be argued that proximal and distal convoluted tubule cells express different PTH receptor isoforms, we think this is unlikely for the following reasons. First, at present there is scant evidence that alternatively spliced isoforms of the type I PTH/PTHrP receptor are functionally expressed (42, 43). Second, a PTH/PTHrP receptor cloned from mouse kidney is indistinguishable from that in the distal convoluted tubule (44, 45). Third, the EC50 values for increasing [Ca2+]i by PTH-(1–34) and PTH-(1–31) in distal tubule cells were indistinguishable: 2.3 vs. 2.9 x 10-10 M, respectively. Likewise, the EC50 values for stimulation of adenylyl cyclase by PTH-(1–84) in proximal and distal convoluted tubule cells were comparable (41 and 24 nM, respectively; calculated from data in Ref. 21). The Hill coefficient of 1 derived from the dose-response curves for PTH-(1–34) and PTH-(1–31) is also compatible with a single class of receptor. Fourth, PTH-(7–34) blocked PTH-(1–34)- and PTH-(1–31)-induced increases in [Ca2+]i, suggesting that both peptides activate the classical type I PTH/PTHrP receptor. The finding that PTH-(3–34) (13, 46) and PTH-(7–34) (Jouishomme, H., unpublished observations) activated PKC but evoked only a limited rise in [Ca2+]i in proximal tubule cells and failed to increase [Ca2+]i in distal tubule cells suggests that cAMP is needed to trigger the rise in [Ca2+]i or that the first two amino acids independently are important for maximizing the intrinsic activity of PLCß. Alternatively, the partial increase in [Ca2+]i induced by PTH-(3–34) and PTH-(7–34) may result from a failure of these analogs to stimulate InsP3 formation maximally.

The fact that the 1–34 and 1–31 analogs of PTH and PTHrP elicited similar effects in distal convoluted tubule cells is consistent with the idea that the PTH receptor mediating these actions recognized both PTH and PTHrP, as demonstrated for the type I PTH/PTHrP receptor (47). Clearly, certain PTH analogs working through a common type I PTH/PTHrP receptor activate adenylyl cyclase in some target cells whereas others do not. It is possible that this may be a consequence of the facility with which these analogs are able to induce conformational changes in the receptor.

The involvement of PLCß in transducing the action of PTH in proximal and distal tubule cells was assessed with the PLCß inhibitor, U73122. This compound abolished PTH-induced transient rises in [Ca2+]i in proximal convoluted tubule cells and InsP3 formation, but did not suppress PTH-stimulated 45Ca2+ uptake by distal convoluted tubule cells. In earlier studies (21) it was established that PTH-stimulated calcium entry in distal tubule cells required activation of both PKA and PKC. However, although PKC participation was necessary, its activation apparently proceeds through a PLCß-independent pathway, as PTH did not cause measurable InsP3 formation. It is possible, however, that large doses of PTH-(1–30) may activate PLCß in cells expressing high numbers of the human PTH/PTHrP receptor, where the Km for adenylyl cyclase is similar for PTH-(1–34), PTH-(1–31), and PTH-(1–30) (40). Nonetheless, stimulation of calcium uptake and the rise in [Ca2+]i in distal convoluted tubule cells require activation of PKC (21). The results with U73122 support the view that stimulation of PKC by PTH in distal convoluted tubule cells does not involve PLCß. Therefore, we surmise that type I PTH/PTHrP receptors are capable of activating phospholipases other than PLCß and that the structural requirements for such activation differ from the 29–32 sequence that is necessary for activation of PLCß in ROS 17/2 osteosarcoma cells (13, 14).

An attractive candidate for such a non-PLCß mechanism is PLD, which hydrolyzes membrane phosphatidylcholine and indirectly generates PKC-activating diacylglycerols without the attendant formation of InsP3 and associated mobilization of intracellular Ca2+ (48). The results depicted in Fig. 6Go show that PTH caused a moderate, but consistent, activation of PLD. The observation that PTH also induced DAG formation serves as an independent confirmation of PLD activation and the fact that DAG accumulation proceeds in the absence of InsP3. The increase in PLD activity was relatively modest, especially in the face of a comparable increase in DAG formation. These findings suggest that a relatively large pool of the DAG precursor, phosphatidic acid, or the presence of additional routes of DAG formation may exist. In this regard, it is known that DAG formation can arise from phospholipase A2 hydrolysis of phosphatidylcholine and that PTH activates PLA2 in some renal cells (49, 50, 51). A recent study likewise reported that PTH activates PLD in osteoblast-like UMR-106 cells (52). At present, the mechanism by which PTH receptors activate PLD is unclear. It is currently thought that PLD is activated by monomeric (small) G proteins or PKC (47, 53). The identities of these mediators remain to be identified. It is uncertain how or why the type I PTH/PTHrP receptor preferentially activates PLD, because distal cells express PLCß that can be activated through a Gq/11 mechanism (38).


   Acknowledgments
 
We thank Ms. B. Coutermarsh for superb technical assistance, and Dr. Thomas L. Ciardelli for helpful discussions on receptor kinetics.


   Footnotes
 
1 This work was supported by NIH Grant R01-DK-54171 and an American Society of Nephrology Career Enhancement Award. Back

2 The terms distal tubule and proximal tubule cells denote the mixed populations of cortical ascending limb and distal convoluted tubule cells, or S1 and S2 proximal tubule cells, respectively, that are isolated by immunoselection (28 ) and grown in primary cell culture. In contrast, distal convoluted tubule cells and proximal convoluted tubule cells refer to immortalized lines of clonal distal and proximal convoluted tubule cells. Back

Received May 18, 1998.


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