Down-Regulation of Protein Kinase C by Parathyroid Hormone and Mezerein Differentially Modulates cAMP Production and Phosphate Transport in Opossum Kidney Cells


  • Judith A. Cole

    Corresponding author
    1. The Department of Pharmacology, The University of Missouri School of Medicine, Columbia, Missouri, U.S.A.
    • Judith A. Cole, Ph.D. The Department of Pharmacology M517B Medical Sciences Building The University of Missouri School of Medicine Columbia, MO 65212 U.S.A.
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We examined the effects of prolonged exposure to parathyroid hormone (PTH) and the protein kinase C (PKC) activator mezerein (MEZ) on cyclic adenosine monophosphate (cAMP) production, PKC activity, and Na+-dependent phosphate (Na/Pi) transport in an opossum kidney cell line (OK/E). A 5 minute exposure to PTH stimulated, while a 6 h incubation reduced, cAMP production. Na/Pi transport was maximally inhibited under desensitizing conditions and was not affected by reintroduction of the hormone. MEZ pretreatment (6 h) enhanced PTH-, cholera toxin (CTX)-, and forskolin (FSK)-stimulated cAMP production, suggesting enhanced Gsα coupling and increased adenylyl cyclase activity. However, PKA- and PKC-dependent regulation of Na/Pi were blocked in MEZ-treated cells. The PTH-induced decrease in cAMP production was associated with a reduction in membrane-associated PKC activity while MEZ-induced increases in cAMP production were accompanied by decreases in membrane and cytosolic PKC activity. Enhanced cAMP production was not accompanied by significant changes in PTH/PTH related peptide (PTHrP) receptor affinity or number, nor was the loss of Na/Pi transport regulation associated with changes in PKA activity. The results indicate that down-regulation of PKC by PTH or MEZ differentially modulates cAMP production and regulation of Na/Pi transport. The distinct effects of PTH and MEZ on PKC activity suggest that agonist-specific activation and/or down-regulation of PKC isozyme(s) may be involved in the observed changes in cAMP production and Na/Pi transport.


Hormonal regulation of many cellular processes is mediated by activation of adenylyl cyclase/protein kinase A (PKA) and phospholipase C/protein kinase C (PKC). A number of studies have shown that “cross-talk” between these pathways results in further modulation of hormonal responses and cellular integration of signals. Activation of PKC produces tissue- and cell-specific changes in adenylyl cyclase activity by altering the phosphorylation state of one or more components of the receptor-G protein-effector complex. PKC induces both receptor desensitization and sensitization,1–8 PKC-induced phosphorylation of the α subunits of Gs and Gi can enhance or diminish coupling to effector,9–11 and phosphorylation of adenylyl cyclase can enhance or reduced the catalytic activity of the enzyme.5,7,12–14 The diversity of the responses to PKC activation may reflect cell-specific expression of or ligand-specific activation of PKC isozymes as well as agonist- and phorbol ester-specific effects on cross-talk between signaling pathways.

Parathyroid hormone (PTH) is an important regulator of calcium and phosphate homeostasis, acting through the G protein–coupled PTH/PTH related peptide (PTHrP) receptor in kidney and bone. In target cells, occupation of the receptor by PTH or PTHrP results in the activation of adenylyl cyclase and phospholipase C.15–22 Prolonged exposure to PTH desensitizes signaling through both the cyclic adenosine monophosphate (cAMP) and the Ca2+ pathways, an effect mimicked by phorbol ester treatment.3,6,23 These data suggest that PKC is involved in receptor desensitization but do not indicate how desensitizing conditions affect PKC activity. Since exposure to phorbol esters rapidly depletes cellular PKC,24,25 desensitization of the PTH receptor may reflect PKC down-regulation rather than activation. We and others have observed that both PKA and PKC are involved in the regulation of Na+-dependent phosphate (Na/Pi) transport in opossum kidney (OK) cells,6,15–20 but the consequences of receptor desensitization on PKC activity and transporter function have not been addressed. In this study, we have examined how long-term exposure to PTH or the PKC activator mezerein (MEZ) affects PKC activity, cAMP production, and regulation of Na/Pi transport in a PTH-responsive OK cell clone (OK/E) derived from the “wild type” OK cell line (OKwt).26 Our results indicate that PTH and MEZ both reduce PKC activity while differentially modulating PTH-stimulated cAMP production and regulation of phosphate transport.



Tissue culture medium and fetal bovine serum (FBS) were obtained from Life Technologies (Grand Island, NY, U.S.A.). Tissue culture plasticware was purchased from Corning Glass Works (Corning, NY, U.S.A.). MEZ, phorbol didecanoate (PDD), phorbol myristate acetate (PMA), 4α PMA, bPTH(1–34), cholera toxin (CTX), and 8 Bromo-cAMP (8Br-cAMP) were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Forskolin (FSK) was purchased from Calbiochem-Behring (La Jolla, CA, U.S.A.), and hPTHr(1–34) was obtained from Peninsula Laboratories, Inc. (Belmont, CA, U.S.A.). The synthetic protein kinase substrate peptides [ser25] PKC 19–31 (for PKC) and KEMPTIDE (for PKA) were purchased from Life Technologies. Carrier-free32Pi (as ortho-phosphoric acid) and [2,8-3H] adenine (specific activity 36 Ci/mmol) were purchased from ICN Radiochemicals (Irvine, CA, U.S.A.). [Na125I] (specific activity 14–17 μCi/μg) was obtained from Amersham (Arlington Hills, IL, U.S.A.) and [γ-32P]ATP (specific activity 3000 Ci/mmol) was from DuPont-NEN (Boston, MA).

Cell culture

OK/E cells were maintained in 75 cm2 flasks in a humidified atmosphere of 95% air/5% CO2. Cells were grown to confluence in Dulbecco's modified Eagle's Medium (DMEM) supplemented with 5% FBS and 100 U/ml penicillin. Confluent monolayers were subcultured weekly using Ca/Mg-free Hank's balanced salt solution and 0.025% trypsin/0.02% EDTA and were plated at a density of ∼80,000 cells/ml (24-well dishes [1 ml/well], 35 mm dishes [2.5 ml], or 60 mm dishes [5 ml]). Cultures were confluent in 3–4 days and routinely used 5–8 days after plating.

cAMP formation

cAMP formation was determined by the conversion of [3H]ATP to [3H]cAMP in cells prelabeled with [3H]adenine.18 Cells grown in 24-well multiwell plates were changed to DMEM containing 1 mg/ml bovine serum albumin (DMEM/BSA) and treated for 5 h at 37°C with MEZ or PTH. The preincubation medium was removed, replaced with 0.5 ml DMEM/BSA containing [3H]adenine (5 μCi/ml) and MEZ or PTH, and the dishes were incubated for 1 h at 37°C (neither PTH nor MEZ had an effect on [3H]adenine accumulation). Following the labeling period, medium was removed and each well was washed three times with DMEM containing 20 mM HEPES (DMEM/HEPES, pH 7.4). Adenylyl cyclase activation was initiated by adding 0.2 ml of DMEM/HEPES containing 1 mM isobutylmethylxanthine (IBMX) ± PTH or MEZ and increasing concentrations of PTH, CTX, or FSK. Dishes were incubated for 5 minutes (PTH, FSK) or 30 minutes (CTX) at 37°C, and the reaction was terminated by adding 0.4 ml of 5% trichloroacetic acid. [3H]cAMP was separated from3[H]ATP using Dowex H and neutral alumina column chromatography. [3H]cAMP and3[H]ATP were determined by liquid scintillation spectrophotometry, and data are calculated as the percent conversion of ATP to cAMP (cpm [3H]cAMP/(cpm [3H]cAMP + cpm [3H]ATP) × 100) and reported as the fold increase over basal production in untreated cells.

Binding of [125I]hPTH-related peptide to intact cells

Radiolabeled hPTHrP(1–34) was prepared using a lactoperoxidase Enzymobead Radiation Reagent according to the manufacturer's instructions (Bio-Rad Laboratories, Hercules, CA, U.S.A.) and was purified by reverse phase high performance liquid chromatography (HPLC). Radioligand binding was assessed on confluent monolayers of cells grown in 24-well dishes. Each well was washed three times with 0.5 ml of DMEM/HEPES, and binding was initiated by adding [125I]hPTHrP(1–34) (∼50,000 cpm/well) and increasing concentrations of unlabeled PTH. Dishes were incubated 1 h at room temperature, and the binding reaction was terminated by aspirating the medium. Each well was washed three times with ice-cold phosphate-buffered saline, and monolayers were dissolved with 1.0 ml of 0.2 N NaOH. Radioactivity and protein content were determined in aliquots of the solubilized cells. Data were corrected for nonspecific binding, normalized to counts per minute per milligram protein, and reported as the percent of total binding in untreated cells.

PKC assay

PKC activity was measured as described previously.27 Briefly, cells grown in 60 mm dishes were changed to DMEM/BSA containing PTH or MEZ and incubated at 37°C in a CO2 incubator for 1 minute or 6 h. The medium was removed and each dish was washed three times with 1 ml of ice-cold homogenization buffer (20 mM Tris-HCl, 0.25 M sucrose, 1 mM EDTA, 2 mM EGTA, 20 μg/ml leupeptin, and 0.1 mM phenylmethylsulfonyl fluoride, pH 7.5). The cells were scraped into 0.5 ml of the same buffer and homogenized by vortexing at high speed for 1 minute. The homogenate was spun in a microfuge for 30 minutes at 4°C, and the supernatant (cytosol) was collected. The resulting pellet was resuspended in 0.4 ml of homogenization buffer containing 0.01% Triton X-100 and incubated for 2 h on ice. The suspension was then microfuged for 30 minutes at 4°C and the supernatant (membrane fraction) was collected. The PKC assay mixture (100 μl) consisted of 60 μl of assay buffer (25 mM Tris-HCl [pH 7.5], 6.25 mM Mg acetate, 0.125 mM [γ-32P]ATP [10 μCi/ml], 10 μg/ml of the synthetic PKC peptide substrate [ser25] PKC 19–31), 10 μl of 5 mM EDTA (basal PKC activity), or 10 μl of a 10X stock solution consisting of 10 mM CaCl2, 250 μg/ml phosphatidylserine, and 5 μg/ml diolein (Ca/phospholipid-dependent PKC activity). The reaction was initiated by adding 30 μl of the cytosol or membrane preparation (10–30 μg protein) and was incubated for 10 minutes at 30°C. The assay was terminated by spotting 50 μl of the assay mixture on 2 × 2 cm pieces of Whatman P81 filter paper. The filters were washed three times in 75 mM phosphoric acid, once in 95% ethanol, dried, and counted. Kinase activity was estimated as the incorporation of32P per minute and expressed as pmol/mg protein/10 minutes.

32P transport assay

Cells grown in 24-well multiwell dishes were changed to DMEM/BSA containing MEZ or PTH 2 h prior to the addition of CTX, FSK, 8 Br-cAMP, or re-exposure to PTH or MEZ. After incubating at 37°C for an additional 4 h, the medium was removed and each well was washed three times with uptake solution (150 mM NaCl, 1.8 mM MgSO4, 1.0 mM CaCl2, and 10 mM HEPES, pH 7.4). Transport was initiated by adding 0.2 ml of uptake solution containing 0.1 mM K2H32PO4 (5 μCi/ml), and culture dishes were incubated for 5 minutes at 37°C. Uptake was terminated by adding 1 ml of ice-cold uptake solution in which 150 mM choline chloride replaced NaCl. Each well was washed three times, the monolayers were dissolved with 1.0 ml of 0.2 N NaOH, and protein and32P content were determined in aliquots of the solubilized cells. Phosphate uptake was measured in nmol/mg/5 minutes and expressed as the ratio of experimental to control uptake (E/C).

PKA assay

Cells grown in 35 mm dishes were treated for 6 h with MEZ in DMEM/BSA. The medium was removed, replaced with 1 ml of DMEM/HEPES containing PTH ± MEZ, and dishes were incubated for 5 minutes at 37°C. Incubations were terminated by aspirating the PTH-containing medium and washing each dish twice with 1 ml of homogenizing buffer (150 mM NaCl, 10 mM KH2PO4, 1 mM EDTA, 5 mM IBMX, and 1 mg/ml BSA, pH 6.8). The dishes were scraped and washed with an additional 1 ml of homogenization buffer. The cell lysate was homogenized with a 20-s high-speed burst of an Ultra-Turrax homogenizer (Tekmar Co., Cincinnati, OH, U.S.A.), centrifuged 10 minutes at 10,000g, and the supernatant (cytosol) was collected. The reaction mixture (100 μl) consisted of 50 μl of a 2× assay buffer (40 mM Tris-HCl, pH 7.4, 20 mM Mg acetate, 0.2 mM [γ-32P]ATP (10 μCi/ml), 0.1 mM IBMX, and 100 μM KEMPTIDE ± 10 μl of 100 μM cAMP. The reaction was initiated by adding 20 μl of cytosol (10–25 μg of protein), incubated for 5 minutes at 30°C, and terminated by spotting 50 μl of the reaction mixture on Whatman P81 2 × 2 cm filter papers. The filters were washed three times in 75 mM phosphoric acid, once in 95% ethanol, dried, and counted. Kinase activity was estimated as32P incorporation per minute (pmol/mg protein/minute) and expressed as a ratio of enzyme activity in the absence of cAMP divided by that in the presence of cAMP (cAMP/+cAMP).

Statistical analysis

All data are expressed as means ± SE of at least three experiments performed on several passages of OK/E cells. Dose-response curves for radioligand binding, cAMP formation, and inhibition of phosphate transport were fit using nonlinear regression analysis (Prism; GraphPad Software, Inc., San Diego, CA, U.S.A.). Statistical significance was evaluated by one-way analysis of variance, and differences between means were considered significant at p ≤ 0.05.


Effect of PTH and MEZ pretreatment on PTH-stimulated cAMP production

In OKwt cells, prolonged treatment with PTH or phorbol esters reduced PTH-stimulated cAMP production.6 To determine if comparable responses were produced in OK/E cells, we assessed the effects of PTH or MEZ pretreatment on cAMP production. A 5 minute incubation with PTH increased [3H]cAMP production with an EC50 of 5 ± 1 nM in control cells and a 30-fold increase at 100 nM (Fig. 1A). A 6-h pretreatment with PTH (100 nM) elevated basal cAMP production ∼6-fold (from 127 ± 11 to 739 ± 217 cpm [3H]cAMP/well) but significantly reduced PTH potency (from 5 ± 1 to 30 ± 10 nM) and efficacy (Fig. 1A). In contrast, MEZ (100 nM) pretreatment did not affect basal cAMP production (77 ± 8 vs. 118 ± 41 cpm/well, respectively) but enhanced the cAMP response to PTH. PTH potency was increased from 13.3 ± 3.9 to 0.9 ± 0.4 nM (p ≤ 0.01), while efficacy was slightly but not significantly reduced (Fig. 1B). Although the effects of MEZ are distinctly different from those reported in OKwt cells exposed to phorbol didecanoate (PDD)6 both PDD and PMA significantly (p ≤ 0.05) increased PTH potency (from 20 ± 5 to 5 ± 3 and 3 ± 2 nM, respectively) while the inactive phorbol ester 4α-PMA had no effect on PTH-stimulated cAMP production (EC50 of 18 ± 5nM) (Fig. 1C). These data indicate that the increase in PTH potency was not unique to MEZ and that a PKC activating phorbol ester is required to produce this response.

Figure FIG. 1.

The effect of PTH or MEZ pretreatment on cAMP production. Cells were treated for 6 h with 100 nM PTH or MEZ and cAMP formation was assessed as described in Materials and Methods. (A) PTH increased cAMP production in control cells with a maximally effective concentration of the hormone increasing cAMP production ∼30-fold (from 127 ± 11 to 3791 ± 221 cpm/well). (B) MEZ had no effect on basal cAMP production but increased PTH potency. Data in (A) and (B) are means ± SE of seven experiments assayed in triplicate; *, ** p ≤ 0.05, p ≤ 0.01 versus cAMP production in control cells. (C) A 6-h exposure to 100 nM PDD or PMA increased while the inactive phorbol ester 4α PMA had no effect on PTH potency. Data are means of three experiments assayed in triplicate. Standard errors have been omitted for clarity.

The effect of PTH or MEZ pretreatment on PKC activity

The preceding data are consistent with PKC involvement in the regulation of PTH-stimulated cAMP production. However long-term exposure to PKC activators can down-regulate PKC. To determine the effects of prolonged exposure to PTH or MEZ on PKC activity, we assayed Ca2+-dependent PKC activity in cytosol and membranes following short- and long-term treatment with PTH or MEZ. A 1 minute exposure to PTH (100 nM) increased while MEZ (100 nM) decreased membrane-associated PKC activity (Fig. 2). Neither PTH nor MEZ affected cytosolic PKC under these conditions. Membrane-associated PKC activity was significantly reduced in PTH or MEZ pretreated cells while MEZ pretreatment also reduced cytosolic activity. In addition, PTH activation of PKC was blunted in MEZ-treated cells. These data indicate that prolonged incubations with either PTH or MEZ down-regulate PKC but the qualitative differences in the responses suggest each agent regulates PKC in different fashions.

Figure FIG. 2.

Effect of PTH or MEZ pretreatment on PKC activation. PKC activity in OK/E cells was determined in cells treated for 1 minute or 6 h with 100 nM PTH or MEZ. Cytosol and membrane fractions were prepared and PKC activity was assayed as described in Materials and Methods. Values are means ± SE, n = 6; *, ** p ≤ 0.05, p ≤ 0.01 versus controls, respectively.

MEZ pretreatment does not affect PTH receptor binding

The MEZ-induced increase in PTH-stimulated cAMP production and the associated increase in PTH potency may result from alteration in any one of the three components of the receptor-Gs-adenylyl cyclase complex. To determine if the increased potency was associated with a change in receptor number and/or affinity, cells were incubated for 6 h with MEZ, and inhibition of [125I] human PTHrP ([125I] hPTHrP(1–34)) binding to the PTH receptor was evaluated (Fig. 3). The IC50 for inhibition of [125I] hPTHrP(1–34) binding was not significantly (p ≥ 0.06) affected by MEZ pretreatment (1.9 ± 0.2 vs. 0.8 ± 0.4 nM in control and MEZ-treated cells, respectively). Although total binding of [125I] hPTHrP(1–34) was consistently higher in MEZ treated cells (128 ± 30% of control; 8338 ± 1415 vs. 12,110 ± 3549 cpm/mg; control and MEZ-treated, respectively), large standard errors precluded the differences from being statistically significant (p ≥ 0.3). The lack of statistically significant changes in either receptor affinity or number suggests that increases in PTH potency cannot be attributed to PTH/PTHRrP binding characteristics.

Figure FIG. 3.

MEZ pretreatment does not affect PTH/PTHrP receptor number or affinity. OK/E cells were treated for 6 h with 100 nM MEZ and PTH/PTHrP receptor binding was assessed as described in Materials and Methods. MEZ pretreatment did not affect total binding of [125I] hPTHrP(1–34) or receptor affinity. Values are means ± SE of four experiments assayed in triplicate.

The effect of MEZ pretreatment on cholera toxin- and forskolin-stimulated cAMP production

The fact that MEZ pretreatment did not alter PTH/PTHrP binding suggests that cAMP production was affected at a site distal to the receptor. Therefore, we assessed cholera toxin (CTX)- and forskolin (FSK)-stimulated cAMP production in MEZ-treated cells. CTX produces a persistent activation of adenylyl cyclase by ADP-ribosylating the α subunit of Gs,28 while FSK directly activates the catalytic subunit of the enzyme.29 MEZ significantly enhanced CTX-stimulated cAMP production, nearly doubling the cAMP produced at each concentration of CTX (Fig. 4A). FSK-stimulated cAMP formation was also enhanced by MEZ with significant increases occurring at most concentrations of the cyclase activator (Fig. 4B). These data indicate that enhanced PTH-stimulated cAMP production was accompanied by increased receptor-effector coupling and catalytic activity of adenylyl cyclase.

Figure FIG. 4.

MEZ pretreatment enhances CTX- and FSK-stimulated cAMP formation. OK/E cells were treated for 6 h with 100 nM MEZ, then stimulated with CTX (30 minutes) or FSK (5 minutes). The reaction was terminated and cAMP formation was assessed as described in Materials and Methods. MEZ pretreatment enhanced CTX- (A) and FSK-stimulated (B) cAMP production over that of control cells. Values are means ± SE of three experiments assayed in triplicate for CTX and four experiments determined in triplicate for FSK. Significant increases in cAMP production are indicated by * (p ≤ 0.05) and ** (p ≤ 0.01).

Regulation of phosphate transport in MEZ pretreated OK/E cells

To determine if the changes in cAMP production altered phosphate transport regulation, we measured PTH-dependent phosphate uptake in PTH- and MEZ-pretreated cells. In control cells, PTH was a potent inhibitor of phosphate transport (EC50 of ∼50 pM) with maximally effective concentrations of the hormone reducing transport by ∼40% (from 3.0 ± 0.02 to 1.9 ± 0.1 nmol/mg/5 minutes) (Fig. 5A). A 6-h treatment with PTH (100 nM) maximally inhibited transport, and there was no further reduction in uptake following re-exposure to the hormone (Fig. 5A). Prolonged incubations with MEZ did not affect basal Na/Pi transport but prevented PTH-dependent inhibition of the transport process (Fig. 5B). Shorter exposures to MEZ (2 h) inhibit Na/Pi transport (see below and Ref. 16). However, after 3 h, MEZ effects on Na/Pi transport are lost and PTH-dependent inhibition of transport is markedly attenuated (data not shown).

Figure FIG. 5.

Effect of PTH or MEZ pretreatment on PTH-dependent inhibition of phosphate transport. Cells were treated for 6 h with 10 nM PTH or 100 nM MEZ, and hormone was added 2 h into the pretreatment period. (A) In control cells, a 4 h exposure to PTH produced dose-dependent decreases in phosphate transport. Transport was maximally inhibited in PTH-pretreated cells and re-exposure to the hormone produced no further reduction. (B) MEZ pretreatment had no effect on basal transport but blocked inhibition of phosphate transport by PTH. Values are means ± SE of four experiments assayed in triplicate. *, ** p ≤ 0.05, 0.01, respectively, compared with the control E/C ratio of 1.

Although PKC down-regulation enhances cAMP production by CTX and FSK, PTH-dependent regulation of Na/Pi transport was blocked in MEZ-treated cells. In fact, MEZ treatment also blocked CTX- and FSK-dependent regulation of Na/Pi transport even in the face of enhanced cAMP production (Fig. 6). In addition, a 4 h incubation with 8 Br-cAMP (100 μM) or a 2 h incubation with MEZ (100 nM) significantly (p ≤ 0.05) reduced transport from 3.2 ± 0.3 in control cells to 1.8 ± 0.3 or 2.4 ± 0.2 nmol/mg/5 minutes in 8Br-cAMP and MEZ-treated cells, respectively. However, MEZ pretreatment blocked inhibition by both agents (control 3.2 ± 0.3 vs. 2.5 ± 0.4 and 3.1 ± 0.4, 8Br-cAMP and MEZ, respectively), suggesting that MEZ affects transport regulation at a site distal to the generation of cAMP as well as causing the loss of both PKA- and PKC-dependent regulation of phosphate transport.

Figure FIG. 6.

MEZ pretreatment blocks inhibition of phosphate transport by CTX and FSK. Cells were treated 6 h with 100 nM MEZ. After 2 h, CTX or FSK was added to the incubation medium. (A) In control cells, CTX inhibited transport with maximal responses appearing at 0.3 μg/ml. MEZ treatment increased basal transport but attenuated CTX inhibition at each toxin concentration. Data are means ± SE of three experiments, determined in triplicate. (B) Forskolin produced a dose-dependent inhibition of phosphate transport in control cells. MEZ had no significant effect on basal phosphate transport and blocked the forskolin response. Data are means ± SE of four experiments assayed in triplicate. Significant differences from the control E/C ratio of 1 are indicated by *, ** p ≤ 0.05, 0.01, respectively.

The effect of MEZ pretreatment on PKA activity

The loss of cAMP-dependent inhibition of phosphate transport suggested that MEZ affects PKA as well as PKC activity. We examined this possibility by assaying PTH-dependent activation of PKA in control and MEZ-treated cells. MEZ pretreatment had no effect on basal PKA activity, and PTH produced comparable increases in activity in control and MEZ-treated cells (Fig. 7). These data suggest that the loss of cAMP-dependent regulation of phosphate transport was not due to a reduction in PKA activity but may reside at a step distal to the activation of this enzyme.

Figure FIG. 7.

MEZ pretreatment does not affect PTH-stimulated protein kinase A activity in OK/E cells. OK/E cells were treated with 100 nM MEZ for 6 h followed by 100 nM PTH for 5 minutes. PKA activity was estimated as described in Materials and Methods and reported as an activity ratio (activity in the absence of cAMP divided by that in the presence of cAMP). MEZ pretreatment had no effect on basal or PTH-stimulated PKA activity. Values are means ± SE of three experiments assayed in triplicate. ** p ≤ 0.01 control versus PTH and * p ≤ 0.05 MEZ control versus MEZ + PTH.


In this study, we examined the effects of prolonged exposure to PTH and MEZ on PKC activity, cAMP production, and the regulation of phosphate transport in clonal OK/E cells. As reported in a number of studies, PTH pretreatment blunts subsequent PTH-stimulated cAMP production by reducing the potency and efficacy of the hormone.2,3,30,31 In OKwt and OK/P cells, homologous desensitization was mimicked by phorbol esters and blocked by PKC inhibitors, suggesting PKC involvement in this response.6,23 However, PKC activity was not assessed in these studies. Our data indicate that PTH pretreatment decreases membrane-associated PKC activity, suggesting that PKC down-regulation rather than activation is involved in agonist-induced desensitization. Like PTH, MEZ pretreatment reduced membrane-associated PKC while also markedly reducing cytosolic activity. However, PKC down-regulation was associated with an increase in PTH potency rather than receptor desensitization. Phorbol ester-induced sensitization in not unique to OK/E cells because phorbol esters also enhance PTH-stimulated cAMP production in osteoblast-like osteosarcoma cells. However, PTH efficacy rather than potency was affected by phorbol ester treatment.1,10,32,33 Although the reasons for such differences are unclear, cross-talk between second messenger pathways often reflects ligand- and cell-specific responses and phorbol esters are likely to produce different responses in bone and kidney cells. However, MEZ-induced sensitization of cAMP production in OK/E cells is also distinct from the responses produced by PDD in OKwt cells.6 Although PKC activators can differentially activate or down-regulate PKC,8,34–37 MEZ, PDD, and PMA all sensitize cAMP production in OK/E cells, indicating this response is not unique to MEZ. Since these three PKC activators desensitize PTH-stimulated cAMP production in OKwt cells maintained in our laboratory (unpublished observations), it seems likely that cell-specific expression of PKC isozymes explains the differences between OK/E and OKwt cells. Since the OKwt cell line is composed of several distinct cell types,30 the changes in cAMP production associated with PKC down-regulation would reflect the responses produced in all cell types. In addition, these data illustrate the differences in activation of PKC by phorbol esters and receptor agonists (see Ref. 25 for review). Phorbol esters can activate and induce the translocation of Ca-dependent PKC isoforms in the absence of calcium. Furthermore, agonists usually cause the redistribution of one PKC isoform, while phorbol esters often cause the redistribution of several. Finally, prolonged exposure to phorbol esters often results in the loss of total cellular PKC, a condition not produced by agonist treatment. The latter appears to occur in MEZ-treated OK/E cells where both membrane and cytosolic activities are down-regulated. The differences in PKC activation suggest that OK/E cells express a PKC isozyme down-regulated by MEZ but not by PTH, resulting in a MEZ-specific change in PTH-stimulated cAMP production. Resolution of the differences between PTH- and MEZ-induced down-regulation of PKC will require identification of the PKC isozymes present in the OK/Ecell line as well as their sensitivities to PTH and MEZ regulation.

The increase in PTH potency was not associated with significant changes in PTH/PTHrP receptor density or affinity but was accompanied by enhanced CTX- and FSK-stimulated cAMP formation. Comparable changes in the response to CTX and FSK have been observed in several studies and suggest that the MEZ-induced increase in potency is due to enhanced Gs coupling to and increased catalytic activity of adenylyl cyclase.1,7,10,33 However, it should be pointed out that the IC50 for inhibition of [125I]hPTHrP binding as well as total binding were always greater in MEZ-treated cells. Although these differences were not statistically significant, small increases in receptor affinity and number may be magnified by a greater interaction with Gs and enhanced activation of adenylyl cyclase. More extensive analysis of receptor binding characteristics will be required to assess this possibility. Even if receptor number and affinity are not affected by MEZ treatment, it does not preclude an effect on the receptor. While G protein receptor kinases seem to regulate agonist-induced phosphorylation and desensitization of the PTH/PTHrP receptor, PKC appears to be involved in its constitutive phosphorylation.38 If PKC-dependent receptor phosphorylation dampens receptor-Gs coupling, then PKC down-regulation could reduce receptor phosphorylation and enhance receptor interaction with Gs. This possibility seems unlikely since prolonged exposure to PMA increased basal phosphorylation of PTH/PTHrP receptors expressed in human embryonic kidney cells.38 Alternatively, changes in the phosphorylation state of Gs and adenylyl cyclase may contribute to the increases in CTX- and FSK-stimulated cAMP production. Gsα and adenylyl cyclase serve as PKC substrates and, depending upon the system, PKC-induced phosphorylation can either enhance or reduce the activities of each protein.11–15 If PKC-dependent phosphorylation limits Gs and/or adenylyl cyclase activity in OK/E cells, down-regulation of PKC would release them from inhibitory control. Assessment of the phosphorylation state of the receptor, Gsα, Giα, and adenylyl cyclase following PKC down-regulation will be required to determine which of these possibilities is correct.

Prolonged exposure to PTH and MEZ also produced distinct differences in the regulation of Na/Pi transport. PTH pretreatment persistently inhibited phosphate transport, and a second challenge with the hormone produced no further response. Increases in intracellular cAMP inhibited phosphate transport in control cells, yet the MEZ-induced increase in PTH potency as well as the enhanced cAMP production produced by CTX and FSK were not accompanied by inhibition of Na/Pi transport. Total PKA activity was not affected by MEZ treatment suggesting the loss of cAMP-dependent regulation of Na/Pi transport was not the result of altered PKA activity. Although these data do not rule out changes in the EC50 for PTH activation of PKA, the affinity of cAMP for the regulatory subunit, nor in the signaling pathway distal to activation of PKA, they suggest that PKA is not directly affected by MEZ-induced down-regulation of PKC. However, it is also clear that down-regulation of PKC induced by PTH and MEZ alters Na/Pi transport regulation in a fashion distinct to each agent. As with cAMP production and PKC activity, this may reflect differences in PKC isozyme activation and down-regulation.

In summary, PTH-induced homologous desensitization is associated with a reduction in membrane-associated PKC activity and persistent inhibition of phosphate transport. In contrast, MEZ-induced down-regulation of PKC enhances PTH-stimulated cAMP production as a result of greater receptor-effector coupling. However, sensitization is associated with the loss of PKA- and PKC-dependent inhibition of phosphate transport. These data indicate that prolonged exposure to PTH and MEZ produce qualitatively different effects on the function of the PTH/PTHrP receptor-Gs-adenylyl cyclase complex, PKC activity, and the regulation of phosphate transport. They also underscore the importance of measuring PKC activity in studies where phorbol esters are used to assess the role of PKC in agonist responses. Further studies will be required to identify the common and unique site(s) where PTH and MEZ exert their regulatory influences on signal transduction and Na/Pi transport regulation.