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CRF is the primary neuroregulator of the function of the hypothalamic-pituitary-adrenal axis. We have recently cloned a mouse CRF receptor (mCRF-R) complementary DNA (cDNA) from an AtT-20 cell cDNA library by polymerase chain reaction. To compare the functions of mCRF-R to those of the human type I and type II CRF receptors (hCRF-RI and hCRF-RII), cDNAs were cloned into the expression vector pcDNA1 and transfected into COS-7 cells. CRF binding and CRF-stimulated cAMP accumulation as well as phosphoinositide hydrolysis were measured. Scatchard analysis of the binding of 125I-labeled [Tyr0]r/hCRF ([125I]CRF) to COS-7 cells expressing mCRF-R and hCRF-RI cDNAs revealed the same apparent Kd (9 nM). In contrast, the apparent binding Kd for hCRF-RII was 20 nM CRF. Maximal stimulatory concentrations (1 microM) of rat/human CRF-(1-41) (r/hCRF) increased cAMP accumulation in COS-7 cells transfected with mCRF-R, hCRF-RI, and hCRF-RII cDNA plasmid (10 micrograms each) from basal values of 8-19 pmol/10(5) cells.15 min to 84 +/- 10, 87 +/- 16, and 45 +/- 16 pmol/10(5) cells.15 min, respectively. The EC50 values of r/hCRF-stimulated cAMP accumulation in COS-7 cells expressing mCRF-R and hCRF-RI cDNAs were similar at 0.4 +/- 0.2 and 0.7 +/- 0.2 nM, respectively. Conversely, the EC50 of r/hCRF-stimulated cAMP accumulation in hCRF-RII-transfected COS-7 cells was 47.5 +/- 18.9 nM. As the level of expression of hCRF-RII was lower than that of hCRF-RI, we compared r/hCRF-stimulated cAMP accumulation in COS-7 cells expressing low and high levels of hCRF-RI. The EC50 for r/hCRF-stimulated cAMP accumulation in COS-7 cells transfected with hCRF-RI did not change when receptor expression was varied by a factor of 1- to 8.4-fold. In contrast, the EC50 for r/hCRF-stimulated cAMP accumulation mediated by hCRF-RII was at least 100-fold higher than that mediated by the hCRF-RI in COS-7 cells, which suggests poor coupling between hCRF-RII and adenylate cyclase. Inositol phosphate (IP) levels were also determined in mCRF-R, hCRF-RI, and hCRF-RII cDNA-transfected COS-7 cells stimulated with increasing concentrations of r/hCRF. r/hCRF-stimulated IPs accumulation was dose dependent in COS-7 cells expressing mCRF-R and hCRF-RI using 100 and 1000 nM r/hCRF. Concentrations of 10 (or less) nM r/hCRF had no effect on IP generation. hCRF-RII did not mediate stimulation of IP even at 1000 nM r/hCRF.(ABSTRACT TRUNCATED AT 400 WORDS)

Homologous down-regulation of PTH/PTH-related peptide (PTHrP) receptor expression occurs in several PTH-responsive osteoblastic cell lines, but the mechanisms responsible are not well understood. We have used wild-type SaOS-2 human osteoblastic cells, in which homologous PTH/PTHrP receptor down-regulation occurs within 4 h, and a mutant cAMP-resistant subclone (Ca4A strain), to investigate the mechanisms by which PTH/PTHrP receptor mRNA is regulated. SaOS-2 cells expressed a single 2.2- to 2.5-kilobase transcript of PTH/PTHrP receptor mRNA, as assessed by Northern blot analysis of total RNA with a cDNA probe encoding the human PTH/PTHrP receptor. Homologous down-regulation of this PTH/PTHrP receptor mRNA first became significant when SaOS-2 cells had been treated with human (h) PTH-(1-34) (10(-7) M) for 8-12 h. By 24 h, steady state levels of PTH/PTHrP receptor mRNA were reduced by about 50%. This effect was mimicked by both (Bu)2cAMP (DBcAMP; 0.5 mM) and forskolin (Fsk; 10(-5) M). In contrast, down-regulation of PTH/PTHrP receptor mRNA by hPTH-(1-34), DBcAMP or Fsk was almost completely blocked in cAMP-resistant Ca4A cells. Short term (4-6 h) treatment with hPTH-(1-34), DBcAMP, or Fsk did not reduce steady state levels of PTH/PTHrP receptor mRNA in either SaOS-2 or Ca4A cells, although down-regulation was induced by 4-6 h of treatment with active phorbol esters such as 12-O-tetradecanoyl phorbol-13-acetate (200 nM) or phorbol-12,13-didecanoate (200 nM). Neither thapsigargin (1 microM) nor ionomycin (200 nM), both of which stimulate calcium transients in these cells, altered PTH/PTHrP receptor mRNA expression. Treatment with hPTH-(39-84) and hPTH-(53-84), which do not activate either cAMP-dependent protein kinase or protein kinase-C, but do stimulate 45Ca2+ uptake in these cells, did not alter PTH/PTHrP receptor mRNA expression. In the presence of actinomycin-D (1 microgram/ml), down-regulation of PTH/PTHrP receptor mRNA by hPTH-(1-34) was not observed. Cycloheximide (10 micrograms/ml) did not block down-regulation of PTH/PTHrP receptor mRNA induced by hPTH-(1-34). We conclude that homologous down-regulation of PTH/PTHrP receptor mRNA in SaOS-2 cells occurs later than the decline in functional surface receptors via a mechanism that does not involve enhanced mRNA degradation or new protein synthesis, but is dependent upon cAMP/cAMP-dependent protein kinase.

The recombinant human PTH/PTH-related peptide (PTHrP) receptor, when transiently expressed in COS-7 cells, binds [Nle8,18,Tyr34] bovine PTH-(7-34)amide [PTH-(7-34)], human PTH-(10-34)amide [PTH-(10-34)], and bovine PTH-(15-34)amide [PTH-(15-34)] with at least 50-fold higher affinity than does the rat receptor homolog. In contrast, PTH-(1-34) binding affinities are similar for both receptor homologs. To map those areas of the PTH/PTHrP receptors that determine the binding specificity for carboxyl-terminal fragments of PTH-(1-34), we constructed chimeric rat/human PTH/PTHrP receptors. These bound PTH-(1-34) with normal affinity and, therefore, must have an overall conformation that resembles that of native receptors. Chimeras with the amino-terminal extracellular domain of the human PTH/PTHrP receptor have a considerably higher binding affinity for PTH-(7-34), PTH-(10-34), and PTH-(15-34) than do the reciprocal receptor constructs in which the amino-terminal region is from the rat PTH/PTHrP receptor. The opossum PTH/PTHrP receptor homolog also binds PTH-(7-34) with higher affinity than the rat receptor, and studies of rat/opossum chimeras confirm the importance of the amino-terminal extracellular domain in determining the PTH-(7-34) binding specificity. Mutant rat and human PTH/PTHrP receptors in which either residues 61-105 of the extracellular region or most of the intracellular tail were deleted have PTH-(7-34) binding characteristics indistinguishable from those of either wild-type receptor. These findings indicate that the amino-terminal extracellular region of the PTH/PTHrP receptor contains a domain(s) that largely determines the binding affinity of amino-terminally truncated PTH analogs. This region, therefore, is likely to constitute a site for ligand-receptor interaction.

PTH elicits multiple second messenger signals in target cells. This signaling diversity may reflect coupling of a single species of PTH receptors to multiple effectors, the action of different subtypes of PTH receptors, or both. We recently reported the expression cloning, from rat and opossum cells, of closely related cDNAs encoding receptors for PTH [and PTH-related peptide (PTHRP)]. To determine if these cloned PTH/PTHRP receptors can activate multiple intracellular effectors when present at near-physiological levels in intact target cells, we have stably expressed the rat and opossum PTH/PTHRP receptor cDNAs in LLC-PK1 porcine renal epithelial cells. These cells lack endogenous PTH/PTHRP receptors, but do express abundant calcitonin receptors and many features of a proximal tubular phenotype. Subclones of transfected LLC-PK1 cells exhibited high affinity binding (Kd, 1-5 nM) of [Nle8.18,Tyr34]bovine PTH-(1-34)amide (PTH) and dose-dependent activation by PTH of both cAMP accumulation (EC50, 1 nM) and increased release of cytosolic free calcium from intracellular stores (EC50,or = 20-50 nM) across a wide range of receptor expression. Expressed rat and opossum receptors exhibited similar properties, except for a 5-fold lower binding affinity of the rat receptor for PTH-(7-34). Stimulation by PTH of both cAMP accumulation and elevated cytosolic free calcium was augmented in cells expressing higher numbers of PTH/PTHRP receptors. Like calcitonin, PTH (1-100 nM) reduced the rate of cell proliferation and augmented the rate of inorganic phosphate transport after 24 and 5 h of preincubation, respectively. The growth effect was mimicked by cAMP analogs, forskolin, phorbol esters, and calcium ionophores. Regulation of phosphate transport, however, was mimicked by phorbols, but not by cAMP analogs or forskolin. We conclude that LLC-PK1 cells provide a useful model in which to study the function of cloned PTH/PTHRP receptors. In these cells, a single species of cloned PTH/PTHRP receptors, stably expressed at near-physiological numbers, activates multiple second messenger responses and regulates subsequent biological responses, including at least one (phosphate transport) that is mediated by mechanisms independent of cAMP.

Identical complementary DNAs (cDNAs) that encode a 593-amino acid human PTH (PTH)/PTH-related peptide (PTHrP) receptor were isolated by hybridization techniques from two cDNA libraries which had been constructed from human kidney and human osteoblast-like osteosarcoma cells (SaOS-2). Northern blot analysis of total RNA from human bone- and kidney-derived tissue revealed one single major messenger RNA species of about 2.5 kilobases in both tissues. The human PTH/PTHrP receptor has 91% and 81% identity, respectively, with the previously cloned rat and opossum receptors, indicating a high degree of conservation among mammals. Despite this striking degree of amino-acid conservation, the human PTH/PTHrP receptor has several unique biological properties when transiently expressed in COS-7 cells. The apparent dissociation constants for [Nle8,18,Tyr34] bovine PTH(1-34) amide [bPTH(1-34)] are similar for the human and the rat receptor (approximately 8 vs. approximately 15 nM) whereas [Tyr36]PTHrP(1-36) amide has a slightly lower affinity for the human (15-40 nM) than for the rat receptor (approximately 15 nM). Both ligands stimulate efficiently and with similar efficacy the accumulation of intracellular cAMP. The affinities for the antagonists [Nle8,18,Tyr34] bPTH(3.34) amide [bPTH(3-34)] and in particular for [Nle8,18,Tyr34] bPTH(7-34) amide [bPTH(7-34)] are considerably higher for the human receptor, e.g. approximately 8 nM vs. 30 nM for bPTH(3-34) and approximately 100 nM vs. 5000 nM for bPTH(7-34), respectively. Similar biological findings were previously attributed to differences in species- and/or organ-specific PTH/PTHrP receptors. The expression of the recombinant, highly homologous rat and human receptors in a uniform environment indicate that the moderate differences in the primary receptor structure have profound consequences for the receptor binding affinity of amino-terminally truncated PTH analogs. Furthermore, the molecular cloning of identical cDNAs encoding a human PTH/PTHrP receptor from the two major target organs for PTH, bone and kidney, provides strong evidence for one single PTH/PTHrP receptor in both organs, although additional and/or alternatively spliced receptors cannot be excluded.

To study mechanisms controlling the expression of PTH/PTH-related peptide (PTHrP) receptors in ROS 17/2.8 and OK cells, we investigated the regulation of PTH/PTHrP receptor availability and receptor mRNA levels by glucocorticoids and PTH. Treatment of ROS 17/2.8 cells with dexamethasone (1 microM) for 2, 4, and 6 days increased specific binding of PTH to 148 +/- 12%, 203 +/- 10%, and 344 +/- 9% (mean +/- SD), respectively, compared to that in untreated control cells. PTH-stimulated cAMP accumulation also increased with dexamethasone treatment (1 microM) from 230 +/- 15%, 382 +/- 9%, and 820 +/- 9% after 2, 4 and 6 days, respectively, compared to that in untreated cells. Treatment of ROS 17/2.8 cells with [Nle8,Nle18,Tyr34]bovine PTH-(1-34) amide (NlePTH; 100 nM) alone or together with dexamethasone (1 microM), however, markedly decreased PTH binding and PTH-stimulated cAMP accumulation. Northern blot analysis showed that dexamethasone dramatically increased steady state levels of PTH/PTHrP receptor mRNA in a time- and dose-dependent manner, which did not occur when NlePTH (100 nM) was added concomitantly to the cultures. As previously reported, daily NlePTH treatment of ROS 17/2.8 cells reduced PTH/PTHrP receptor availability and PTH-stimulated cAMP accumulation markedly within 2 days, which remained at these low levels during continued PTH treatment. In contrast, the identical treatment reduced steady state levels of PTH/PTHrP receptor mRNA in ROS 17/2.8 transiently and to only a slight extent, which then returned to pretreatment levels. Treatment of OK cells with NlePTH (100 nM) for 1, 2, and 4 days decreased PTH binding to 56 +/- 6%, 44 +/- 4%, and 64 +/- 4% (mean +/- SD) and PTH-stimulated cAMP accumulation to 42 +/- 6%, 19 +/- 4%, and 21 +/- 3% (mean +/- SD), respectively, compared to values in untreated control cells. The same treatment, however, had no significant effect on steady state levels of PTH/PTHrP receptor transcripts. In contrast to its effects in ROS 17/2.8 cells, dexamethasone (1 microM) treatment of OK cells for 1-4 days did not affect PTH binding, nor did it significantly affect steady state levels of PTH/PTHrP receptor mRNA, although the latter was slightly lowered by dexamethasone treatment. PTH-stimulated cAMP accumulation was unchanged after 1-day treatment with dexamethasone and modestly rose to 142 +/- 4% of the control value by day 4 of glucocorticoid exposure.(ABSTRACT TRUNCATED AT 400 WORDS)

PTH/PTH-related peptide (PTHrP) receptor mRNAs are widely distributed in rat tissues. PTH and PTHrP, a peptide responsible for hypercalcemia associated with cancers, bind equivalently to common receptors that initially were cloned from rat bone and opossum renal cell cDNA libraries. In this study we used rat PTH/PTHrP receptor cDNA to probe for receptor expression in different rat tissues by Northern blot analysis. PTH/PTHrP receptor transcripts are highly expressed in PTH target tissues, kidney and bone. Receptor transcripts, however, also are expressed in many other tissues, including aorta, adrenal gland, bladder, brain, cerebellum, breast, heart, ileum, liver, lung, skeletal muscle, ovary, placenta, skin, spleen, stomach, uterus, and testes. The major transcript in most tissues is 2.3-2.5 kilobases in size. At least two larger mRNAs are observed in kidney and liver, and smaller transcripts are found in kidney, skin, and testes. The most abundant testicular transcript is 1.4-1.5 kilobases in size, and it hybridizes with two different cDNA probes that encode portions of the receptor sequence from the putative fourth transmembrane domain to its C-terminal end. It does not hybridize, however, with a probe encoding the first 107 residues of the receptor sequence. Although, PTH/PTHrP receptor mRNAs are highly expressed in kidney and bone, classic PTH targets that are associated with calcium homeostasis, their wide tissue distribution suggests that PTH and/or PTHrP have other physiological roles, particularly in these other tissues. The mechanisms leading to tissue-specific expression of PTH/PTHrP receptor transcripts of different sizes and the functions of these mRNAs remain to be determined.

The involvement of protein kinase C in normal corticotroph function was studied by analysis of the effects of the phorbol ester derivative phorbol 12-myristate-13-acetate (PMA) and the synthetic diacylglycerol dioctanoylglycerol (DOG) on basal and stimulated ACTH release in cultured rat anterior pituitary cells. Incubation of rat pituitary cells with increasing concentrations of PMA or DOG caused dose-related increases in ACTH release up to 13.4 +/- 2.1- and 10.1 +/- 0.9-fold, respectively, similar to that caused by CRF (9.8 +/- 1.6-fold). Also, stimulation of endogenous diglyceride formation by phospholipase C (100 mU/ml) stimulated ACTH release by 2.5 +/- 0.1-fold. In cells incubated with maximum stimulatory concentrations of CRF (10 nM) or 8-bromo-cAMP (8-Br-cAMP; 5 mM), addition of either 100 microM DOG or 100 nM PMA caused significantly higher ACTH responses than those obtained with CRF, 8-Br-cAMP, DOG, or PMA alone. 8-Br-cAMP (5 mM) and 10 nM CRF significantly increased the effect of 100 nM PMA by 1.4 +/- 0.2- and 1.5 +/- 0.1-fold, respectively. Combinations of 10 nM CRF with either vasopressin (VP) or angiotensin II (AII) increased ACTH secretion to values higher than those produced by CRF, VP, or AII alone. However, addition of maximal stimulatory concentrations of VP or AII (10 nM) did not further increase the effects of either PMA alone or PMA/CRF combinations, indicating that their mechanisms of action may be similar to that of PMA. These results indicate that in addition to the established cAMP-dependent mechanism, stimulation of ACTH release in normal pituitary cells may be elicited by activation of protein kinase C. The evidence also suggests that protein kinase C is involved during stimulation of ACTH release by the cAMP-independent regulators VP and AII and in the synergistic effects of VP and AII with CRF on the corticotroph.

The role of calcium in the stimulation of ACTH secretion by CRF and other regulators was studied in rat anterior pituitary cells. Incubation of cultured pituitary cells in normal calcium with CRF, vasopressin, angiotensin II, or norepinephrine increased the rate of ACTH release for up to 45 min and then became constant for up to 3 h. In the absence of extracellular calcium, the initial rate of stimulated secretion was unaffected, but after 45 min the secretion rate decreased by 40% for CRF and to a greater extent for the other stimuli. Addition of calcium after 90 min in calcium-free medium restored the CRF-stimulated ACTH release rate to the control value. The absence of extracellular calcium had no effect on CRF-stimulated cAMP accumulation, but intracellular calcium depletion by preincubation of the cells with EGTA completely inhibited CRF-stimulated cAMP production and ACTH release. The voltage-dependent calcium channel antagonist nitrendipine and the calcium channel agonist BK 8644 had little effect on the CRF-stimulated ACTH release rate, while they, respectively, inhibited and enhanced the stimulation by vasopressin and high potassium. In calcium-depleted cells incubated with the calcium ionophore A23187, CRF stimulation of cAMP production and ACTH release were dependent upon extracellular calcium concentrations from 0.1-100 microM. These findings have defined two phases in the stimulation of ACTH release by CRF and cAMP-independent stimuli in cultured pituitary cells: an early phase with a rapid increase in the ACTH release rate which is independent of extracellular calcium, and a late phase of constant secretion rate, with partial extracellular calcium dependence for the stimulation by CRF and complete calcium dependence for the stimulation by non-cAMP-mediated stimuli.

The effects of atrial natriuretic factors (ANFs) on anterior pituitary hormone secretion and cyclic nucleotide production were investigated in cultured rat pituitary cells. ANF had no effect on ACTH, GH, PRL, and TSH release or on cAMP production either on basal hormone levels or during stimulation of their secretion by the appropriate releasing factor. However, ANF markedly stimulated cGMP production in both mixed anterior pituitary cells and enriched anterior pituitary cell populations fractionated by centrifugal elutriation. Unexpectedly, certain ANF preparations, Bachem rat ANF-(5-28) and rat ANF-(5-25), markedly stimulated LH release from cultured anterior pituitary cells and gonadotroph-enriched elutriated pituitary cells. The same ANFs also displaced [125I-D-Lys6]GnRH ethylamide from binding to anterior pituitary membranes with potencies similar to their LH-releasing activities. Immunoprecipitation of ANF with a specific antiserum abolished the effect of ANF on cGMP production, but did not change the effect of ANF on LH release. In conclusion, ANF did not affect anterior pituitary hormone secretion or cAMP production, but stimulated cGMP formation. The effect of certain ANF preparations on LH release appears to be attributable to peptide contamination with a potent GnRH agonist.

We examined mechanisms of down-regulation of PTH receptors and desensitization of the PTH-stimulated increase in intracellular cAMP in clonal rat osteosarcoma cells, ROS 17/2.8. ROS cells treated with 10 nM [Nle8,Nle18,Tyr34] bovine (b) PTH-(1-34) amide (NlePTH) for 3 days showed loss of specific PTH binding and PTH-stimulated cAMP accumulation to 10% of that in vehicle-treated control cells. Treatment of these cells with both 0.5 mM 8-bromo-cAMP (8-Br-cAMP) and 1 mM methylisobutylxanthine or 100 ng/ml cholera toxin for 3 days elicited no change in either of these responses. Treatment with 10 nM NlePTH for 3 days did not modify the cAMP accumulation stimulated by 30 microM forskolin or 1 micrograms/ml cholera toxin, indicating that agonist-specific desensitization of PTH-stimulated cAMP accumulation is not due to diminished activity of either the stimulatory guanyl nucleotide regulatory subunit (Gs) or the catalytic subunit of the adenylate cyclase. Treatment of ROS cells with pertussis toxin (PT; 10 ng/ml) for 12, 24, 48, and 72 h increased specific PTH binding by 21%, 28%, 35%, and 39%. The increase in PTH binding was associated with a parallel increase in PTH-stimulated cAMP accumulation and was due to an increase in the number of PTH receptors. PTH receptor affinity remained constant (apparent Kd = 0.3 nM). PT treatment of the cells partially blocked agonist-specific PTH receptor down-regulation. PT catalyzed ADP ribosylation of 41K and 39K membrane proteins, consistent with the alpha-subunits of Gi and Go, respectively. In conclusion, agonist-induced PTH receptor down-regulation in ROS 17/2.8 cells is cAMP independent and can be reversed by PT treatment. PTH receptor expression in these cells appears to be under tonic inhibitory control by mechanisms involving a PT-sensitive G protein(s).

The inhibition of ACTH secretion by glucocorticoids in vivo is biphasic, with rapid early suppression followed by transient recovery and a late inhibitory phase. To evaluate whether this biphasic effect of glucocorticoids occurs at the pituitary level, the effects of corticosterone (B) on stimulated ACTH release were analyzed in rat anterior pituitary cell cultures. Preincubation with 1 microM B inhibited the ACTH response to 10 nM CRF in a biphasic manner, with rapid inhibition after 10-40 min of preincubation, followed by partial recovery between 40-80 min, and a later phase of inhibition after 80-140 min. Preincubation with B for 40 or 120 min caused a dose-dependent suppression of CRF-stimulated ACTH release, with ED50 values of 416 +/- 21 and 45 +/- 12 nM B, respectively. Pretreatment with B also caused a biphasic inhibitory effect on the stimulatory action of vasopressin, angiotensin II, and norepinephrine on ACTH release. However, addition of these stimuli in combination with CRF surmounted B inhibition of CRF-stimulated ACTH release. B also inhibited the ACTH-releasing effects of postreceptor stimuli, including 8-bromo-cAMP, phorbol 12-myristate 13-acetate, and 1,2-dioctanoylglycerol. In the presence of cycloheximide (10 microM), the early inhibitory effect of B was unchanged, but the delayed effect was decreased. Whereas preincubation with B for 40 min inhibited ACTH release, but not total intracellular plus released ACTH, preincubation for 120 min decreased both released and total ACTH. These findings demonstrate that the two inhibitory effects of B on ACTH release differ in their kinetics, steroid sensitivity, and dependence on protein synthesis. The inhibitory effect of B on ACTH responses to stimuli with different mechanisms of action suggests that the suppressive effects of B are mainly exerted at a site distal to the formation of the second messengers involved in hormonal activation of ACTH release.

PTH binds to specific receptors that are coupled to adenylate cyclase and activate cAMP-dependent protein kinase. Since it has been shown that PTH activates phospholipid inositol metabolism, we investigated whether PTH influences protein kinase-C (PKC) activity in rat osteosarcoma (ROS) cells 17/2.8 that contain a large number of PTH receptor. Incubation of ROS cells with PTH or phorbol 12-myristate 13-acetate (PMA) for 1-30 min caused a rapid and transient decrease in PKC activity in the cytosol, which was associated with a transient increase in PKC activity in the membrane fraction. After 1, 5, 15, and 30 min of incubation with PTH, cytosolic PKC activity decreased to 57%, 74%, 84%, and 93% of the control value, whereas membrane PKC activity increased to 156%, 122%, 111%, and 106% of the control value, respectively. After PMA treatment for 1, 5, 15, and 30 min, cytosolic PKC activity decreased by 81%, 74%, 63%, and 44%, whereas membrane-bound PKC activity increased by 83%, 44%, 28%, and 17%, respectively. The effects of PTH and PMA on PKC were dose dependent, with ED50 values of 0.3 nM PTH and 4 nM PMA. Chronic treatment of ROS cells for 3 days with PMA caused depletion of total PKC activity in cytosolic and membrane fractions to less than 10% of that in control cells. Conversely, chronic treatment of ROS cells with PTH did not deplete PKC. In addition, chronic treatment of ROS cells with PTH inhibited the responsiveness of PKC activity to subsequent acute PTH challenge, but not to acute PMA challenge, suggesting specific desensitization of this response by PTH. Activation of cytosolic PKC by diolein, phosphatidylserine, and calcium caused phosphorylation of many cytosolic proteins, including those having apparent mol wt of 39K, 35K, 33K, 25K, 19K, and 16K. Pretreatment of ROS cells with PTH resulted in a transient decrease in the phosphorylation of these cytosolic proteins by PKC. This decrease in cytosolic protein phosphorylation by treatment with PTH is temporally associated with PTH-stimulated translocation of PKC activity from the cytosol to the membranes. These data suggest a potential role for PKC in the mechanism of action of PTH in ROS cells.