PTH secretion in patients with chronic renal failure assessed by a modified CiCa clamp method: effects of 1-year calcitriol therapy.

BACKGROUND: Secondary hyperparathyroidism (secondary HPT) in patients with chronic renal failure (CRF) is characterized by parathyroid gland hyperplasia and an intrinsic defect in the recognition of parathyroid hormone (PTH) secretion. Conflicting results have been reported regarding the set point for calcium-regulated PTH release and its modification by calcitriol therapy in hemodialysis patients. Additionally, the effect of calcitriol on the calcium/PTH relationship in predialysis CRF patients with early secondary HPT has not been investigated. Our objective in this controlled study was to investigate the calcium/PTH relationship and to determine the calcium set point in patients with early stages of CRF before and after a 1-year treatment with calcitriol and in normal volunteers. METHODS: Nine patients with an early stage of CRF (GFR between 20 and 50 ml/min x 1.73 m2 b.s.) aged 35-77 years and 13 healthy volunteers (HV) aged 26-60, years were included in the study. All participants were investigated by sequential lowering and raising of serum calcium levels comprising the following phases: blood-ionized calcium (Ca2+) was lowered by about 0.2 mmol/l (3 steps), steady-state hypocalcemia of Ca2+ 0.2 mmol/l below the baseline (step 4), stop of the infusion for 5 minutes (step 5), Ca2+ was raised to about 0.2 mmol/l above baseline (steps 6 and 7), and a steady state hypercalcemia of Ca2+ 0.2 mmol/l above baseline (step 8). Ionized calcium and intact PTH (iPTH) were measured at 30 time points during 240 minutes. The calcium set point was determined using the classical 4-parameter model. The CiCa clamp test was performed before and after a 1-year treatment with 0.5 microg of calcitriol thrice weekly. RESULTS: No differences in the set point were observed between HV and CRF patients with early secondary HPT. Four of 9 patients responded to calcitriol treatment with a decrease in basal serum iPTH levels ("responders"). There was no difference between renal function (GFR 18 +/- 6 vs. 17 +/- 8 ml/min x 1.73 m2 b.s.), set point (Ca2+ 1.07 +/- 0.13 vs. 1.07 +/- 0.06 mmol/l) and suppressibility of PTH secretion (PTHmin% 7.3 +/- 1.6 vs. 8.2 +/- 2.9) in responders vs non-responders, nor did these values change after treatment with calcitriol. PTHmin% decreased significantly in the whole group after treatment (10.4 +/- 8.5 vs. 7.8 +/- 2.4). CONCLUSIONS: Although the calcium set point was not different in predialysis CRF patients with early secondary HPT compared to HV, calcitriol treatment improved the calcium-related suppression of PTH secretion (PTHmin%).

Hedgehog promotes primary osteoblast differentiation and increases PTHrP mRNA expression and iPTHrP secretion.

We used both clonal osteoblast-like cells and primary calvarial osteoblastic cells to examine the role of Hedgehog in osteoblast biology. Primary osteoblasts and several clonal osteoblast-like cell lines express Indian hedgehog (Ihh), and genes encoding both components of its receptor, patched (Ptc) and smoothened (Smo). Moreover, Ihh is relatively increased in phenotypically mature clonal cells and it increases by fivefold in primary osteoblasts as they mature in culture. Recombinant N-terminal Sonic Hedgehog (rSHH-N) upregulates Ptc and Gli-1 in osteoblasts, classical transcriptional targets. Furthermore; in response to rSHH-N, immunoreactive parathyroid hormone-related peptide (iPTHrP) secretion is transiently increased in medium conditioned by primary osteoblasts. Changes in PTHrP expression mirror those of iPTHrP, except in late cultures, when mRNA levels remain relatively elevated in response to rSHH-N. Gli-1, but not Ptc, becomes resistant to treatment with rSHH-N over a time course paralleling that of PTHrP, suggesting that mechanisms regulated by Gli-1 affect PTHrP. Last, rSHH-N increases formation of mineralized bone nodules and it accelerates expression of alkaline phosphatase, alkaline phosphatase activity, and mineralization. Taken together, these data suggest a functional role for Hedgehog protein in osteoblast recruitment and differentiation, which includes stimulation of PTHrP expression and secretion.

Molecular analysis of defect healing in rat diaphyseal bone.

Spatial expression of messenger ribonucleic acid (mRNA) for osteoblastic marker in drill hole defect healing of adult male rats was analyzed by in situ hybridization. The defect was filled with hematoma 3 days after surgery, expressing Type I collagen mRNA. Hematoma was replaced with fibrous tissue on day 7, and then with new trabecular bone on day 10, originated from the intra-medullary space, respectively. mRNA for Type I collagen, parathyroid hormone 1 receptor (PTHIR), and alkaline phosphatase (ALP) were expressed in the same cell population of fibrous tissue adjacent to newly-formed trabecular bone, and in osteoblasts lining the newly-formed trabecular bone. Hematopoietic marrow with osteoclasts subsequently invaded the region, also from the intra-medullary space, replacing all the new trabecular bone by day 21, except for a thin sub-periosteal layer. mRNA for Type I collagen, PTH1R and ALP was expressed on the periosteal surface of thin layer. Although cartilage formation was not histologically visible, mRNA for Type II collagen was weakly detected in the majority of osteoblasts lining the newly-formed trabecular bone.

Fibroblast growth factor inhibits chondrocytic growth through induction of p21 and subsequent inactivation of cyclin E-Cdk2.

Fibroblast growth factor (FGF) and its receptor (FGFR) are thought to be negative regulators of chondrocytic growth, as exemplified by achondroplasia and related chondrodysplasias, which are caused by constitutively active mutations in FGFR3. To understand the growth-inhibitory mechanisms of FGF, we analyzed the effects of FGF2 on cell cycle-regulating molecules in chondrocytes. FGF2 dramatically inhibited proliferation of rat chondrosarcoma (RCS) cells and arrested their cell cycle at the G(1) phase. FGF2 increased p21 expression in RCS cells, which assembled with the cyclin E-Cdk2 complexes, although the expression of neither cyclin E nor Cdk2 increased. In addition, the kinase activity of immunoprecipitated cyclin E or Cdk2, assessed with retinoblastoma protein (pRb) as substrate, was dramatically reduced by FGF-2. Moreover, FGF2 shifted pRb to its underphosphorylated, active form in RCS cells. FGF2 not only induced p21 protein expression in proliferating chondrocytes in mouse fetal limbs cultured in vitro but also decreased their proliferation as assessed by the expression of histone H4 mRNA, a marker for cells in S phase. Furthermore, inhibitory effects of FGF2 on chondrocytic proliferation were partially reduced in p21-null limbs, compared with those in wild-type limbs in vitro. Taken together, FGF's growth inhibitory effects of chondrocytes appear to be mediated at least partially through p21 induction and the subsequent inactivation of cyclin E-Cdk2 and activation of pRb.

Roles of growth hormone and insulin-like growth factor 1 in mouse postnatal growth.

To examine the relationship between growth hormone (GH) and insulin-like growth factor 1 (IGF1) in controlling postnatal growth, we performed a comparative analysis of dwarfing phenotypes manifested in mouse mutants lacking GH receptor, IGF1, or both. This genetic study has provided conclusive evidence demonstrating that GH and IGF1 promote postnatal growth by both independent and common functions, as the growth retardation of double Ghr/Igf1 nullizygotes is more severe than that observed with either class of single mutant. In fact, the body weight of these double-mutant mice is only approximately 17% of normal and, in absolute magnitude ( approximately 5 g), only twice that of the smallest known mammal. Thus, the growth control pathway in which the components of the GH/IGF1 signaling systems participate constitutes the major determinant of body size. To complement this conclusion mainly based on extensive growth curve analyses, we also present details concerning the involvement of the GH/IGF1 axis in linear growth derived by a developmental study of long bone ossification in the mutants.

A novel putative transporter maps to the osteosclerosis (oc) mutation and is not expressed in the oc mutant mouse.

The phenotype of mice homozygous for the osteosclerosis (oc) mutation includes osteopetrosis, and a variety of studies demonstrate that osteoclasts in these mice are present but nonfunctional. We have identified a novel gene that has homology to a family of 12-transmembrane domain proteins with transport functions and maps to proximal mouse chromosome 19, in a region to which the oc mutation has been previously assigned. The putative transporter is abundant in normal kidney, but its expression is markedly reduced in kidneys from oc/oc mice when tested using Northern and Western analyses. Southern analysis of this gene, which we call Roct (reduced in oc transporter), demonstrates that it is intact and unrearranged in oc/oc mice. In situ studies show that Roct is expressed in developing bone. We propose that the absence of Roct expression results in an osteopetrosis phenotype in mice.

Functional analysis of the PTH/PTHrP network of ligands and receptors.

Parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) are two related proteins that activate a common PTH/PTHrP receptor, yet have quite distinct physiologic missions. PTH is the major peptide regulator of blood calcium in higher vertebrates, while PTHrP predominantly acts as a paracrine regulator of differentiation and local intercellular signaling. To analyze the physiological roles of PTHrP and the PTH/PTHrP receptor, "knockout" mice missing either the PTHrP or the PTH/PTHrP receptor gene were developed. Both the PTHrP (-/-) mice and the PTH/PTHrP receptor (-/-) mice exhibit a growth plate chondrodysplasia that reflects accelerated differentiation of proliferating chondrocytes. Growth plate chondrocytes regulate the local production of PTHrP by secreting the protein, Indian hedgehog (Ihh), as they are leaving the proliferative pool. Ihh stimulates the production of PTHrP, which then slows the differentiation of chondrocytes, thereby delaying the production of Ihh. PTHrP also stimulates transport of calcium across the placenta. PTHrP (-/-) mice lack the normal elevation of fetal blood calcium (when compared to maternal levels) and have low placental transport of calcium. Fragments of PTHrP that do not bind to the PTH/PTHrP receptor can correct the defect of placental calcium transport in these mice. Thus, this action of PTHrP is not mediated by the PTH/PTHrP receptor. The "knockout" mice thus help delineate the roles of PTH. PTHrP, and the PTH/PTHrP receptor in an interacting network of ligands and receptors.

Intermittent calcitriol therapy in secondary hyperparathyroidism: a comparison between oral and intraperitoneal administration.

BACKGROUND: Intermittent oral or intravenous doses of calcitriol given two or three times per week are commonly used to treat secondary hyperparathyroidism (secondary HPT). This study was undertaken to compare the biochemical and skeletal responses to thrice weekly intraperitoneal (i.p.) versus oral doses of calcitriol in children with secondary HPT undergoing peritoneal dialysis (CCPD). METHODS: Forty-six patients aged 12.5+/-4.8 years on CCPD for 22+/-25 months were randomly assigned to treatment with oral (p.o.) or i.p. calcitriol for 12 months; 17 subjects given p.o. calcitriol and 16 subjects given i.p. calcitriol completed the study. Bone biopsies were performed at the beginning and at the end of the study, while determinations of serum and total ionized calcium, phosphorus, alkaline phosphatase, parathyroid hormone (PTH) and calcitriol levels were done monthly. RESULTS: Serum total and ionized calcium levels were higher in subjects treated with i.p. calcitriol, P < 0.0001, whereas serum phosphorus levels were higher in those given p.o. calcitriol, P < 0.0001. For the i.p. group, serum PTH levels decreased from pre-treatment values of 648+/-125 pg/ml to a nadir of 169+/-57 pg/ml after nine months. In contrast, serum PTH levels did not change from baseline values of 670+/-97 pg/ml in subjects given p.o. calcitriol, P < 0.0001 by multiple regression analysis. Serum alkaline phosphatase levels were also lower in patients treated with i.p. calcitriol, P < 0.0001, but there was no difference between groups in the average dose of calcitriol given thrice weekly. The skeletal lesions of secondary HPT improved in both groups, 33% of patients developed adynamic bone lesion. CONCLUSION: Differences in the bioavailability of calcitriol and/or in phosphorus metabolism may account for the divergent biochemical response to p.o. and i.p. calcitriol.

Heterogeneity among cells that express osteoclast-associated genes in developing bone.

In the present study, we characterized the phenotype of cells in the osteoclast lineage by in situ hybridization, using antisense complementary RNA probes that encode three genes typically expressed by osteoclasts, tartrate-resistant acid phosphatase (TRAP), type IV collagenase (matrix metalloproteinase-9), and c-fms, the receptor for macrophage colony-stimulating factor. By using complementary RNA probes labeled with 35S, digoxygenin, or a combination of the two labeling methods (dual labeling in situ hybridization), we found that each of these genes exhibited a distinct expression pattern during early stages of endochondral bone development [embryonic day 15 (ED15) to ED17] in fetal mouse hind limbs. Type IV collagenase messenger RNA (mRNA) was first expressed in or just outside of the cellular layers that define perichondrium/periosteum, earlier than transcripts for TRAP or c-fms appeared at the same sites (ED15). Although transcripts for TRAP and c-fms colocalized within the skeleton, c-fms was also found in surrounding soft tissue, whereas TRAP mRNA was never detected outside the skeleton (ED16). Type IV collagenase mRNA was uniquely distributed at the chondro-osseous border, being distinct from the distribution of TRAP or c-fms (ED17). At later stages of skeletal development (ED18 to 15-day-old postnatal bone), however, there was more overlap among TRAP, type IV collagenase, and c-fms mRNAs in cells throughout bone, except at the chondro-osseous junction, where type IV collagenase continued to be uniquely localized to some cells at all developmental stages. Whereas the levels of type IV collagenase mRNA expression was most intense at the chondro-osseous margin, the levels of c-fms and TRAP mRNA expression appeared to be more uniform throughout the developing bone. The results indicate that there is considerable heterogeneity among cells expressing osteoclast-associated genes, particularly during early stages of endochondral bone development, but that this difference becomes less pronounced later in the more mature skeleton. Distinct expression patterns of these markers may represent different stages of osteoclastogenesis. Alternatively, type IV collagenase-positive and TRAP/c-fms-positive cells may represent distinct subpopulations of cells of the osteoclast lineage.

Mutations in the second cytoplasmic loop of the rat parathyroid hormone (PTH)/PTH-related protein receptor result in selective loss of PTH-stimulated phospholipase C activity.

To define the structural requirements of the parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor necessary for activation of phospholipase C (PLC), receptors with random mutations in their second cytoplasmic loop were synthesized, and their properties were assessed. A mutant in which the wild type (WT) rat PTH/PTHrP receptor sequence EKKY (amino acids 317-320) was replaced with DSEL had little or no PTH-stimulated PLC activity when expressed transiently in COS-7 cells, but it retained full capacity to bind ligand and to generate cAMP. This phenotype was confirmed in LLC-PK1 cells stably expressing the DSEL mutant receptor, where both PTH-stimulated PLC activity and sodium-dependent phosphate co-transport were essentially abolished. Individual mutations of these four residues point to a critical role for Lys-319 in receptor-G protein coupling. PTH-generated IPs were reduced to 27 +/- 13% when K319E, compared with the WT receptor, and PLC activation was fully recovered in a receptor revertant in which Glu-319 in the DSEL mutant cassette was restored to the WT residue, Lys. Moreover, the WT receptor and a mutant receptor in which K319R had indistinguishable properties, thus suggesting that a basic amino acid at this position may be important for PLC activation. All of these receptors had unimpaired capacity to bind ligand and to generate cAMP. To ensure adequacy of Galphaq-subunits for transducing the receptor signal, Galphaq was expressed in HEK293 and in LLC-PK1 cells together with either WT receptors or receptors with the DSEL mutant cassette. PTH generated no inositol phosphates (IPs) in either HEK293 or LLC-PK1 cells, when they expressed DSEL mutant receptors together with Galphaq. In contrast, PTH generated 2- and 2. 5-fold increases in IPs, respectively, when these cells co-expressed both the WT receptor and Galphaq. Thus, generation of IPs by the activated PTH/PTHrP receptor can be selectively abolished without affecting its capacity to generate cAMP, and Lys-319 in the second intracellular loop is critical for activating the PLC pathway. Moreover, alpha-subunits of the Gq family, rather than betagamma-subunits, transduce the signal from the activated receptor to PLC, and the PLC, rather than the adenylyl cyclase, pathway mediates sodium-dependent phosphate co-transport in LLC-PK1 cells.

Parathyroid hormone-related peptide delays terminal differentiation of chondrocytes during endochondral bone development.

To test the hypothesis that PTH-related peptide (PTHrP) is a paracrine regulator of endochondral bone development, we localized PTHrP and its cognate receptor during normal skeletal development at both messenger RNA (mRNA) and protein levels and compared the growth plate phenotypes of PTHrP-deficient [(PTHrP(-/-)] mice to those of normal littermates [PTHrP(+/+]. PTHrP mRNA was expressed adjacent to uncavitated joints, in the perichondrium of long bones and to a lower level in proliferating chondrocytes. In contrast, PTHrP protein was most evident at the interface of proliferating and hypertrophic zones, where it colocalized with PTH/PTHrP receptor mRNA and protein. Most strikingly, the proliferating zone was dramatically shorter in PTHrP(-/-) cartilage, although the percentage of cells in S-phase of the cell cycle in the proliferating zone was indistinguishable between PTHrP(+/+) and PTHrP(-/-) mice. Terminal differentiation of chondrocytes, which was characterized by cell hypertrophy, apoptosis (DNA fragmentation and decreased bcl-2 mRNA expression), and matrix mineralization, was more advanced in growth cartilage of PTHrP(-/-), compared with PTHrP(+/+) animals. These data demonstrate that PTHrP acts principally as a paracrine factor, which promotes elongation of endochondral bone by restraining or delaying the pace of chondrocytic development and terminal differentiation of growth-plate chondrocytes.

Localization of the extracellular Ca(2+)-sensing receptor and PTH/PTHrP receptor in rat kidney.

Using a strategy based on homology to the bovine parathyroid Ca(2+)-sensing receptor previously identified by us (5), we have recently isolated an extracellular, G protein-coupled Ca2+/ polyvalent cation-sensing receptor, RaKCaR (22), from rat kidney. The localization and physiological role(s) of this receptor in the kidney are not well understood. In the present study, we assessed the distribution of mRNAs for RaKCaR and the parathyroid hormone/parathyroid hormone-related protein (PTH/PTHrP) receptor along the rat nephron by in situ hybridization and reverse transcriptase-polymerase chain reaction of microdissected nephron segments. Our results show that transcripts for both receptors coexpress at glomeruli, proximal convoluted tubule, proximal straight tubule, cortical thick ascending limb, distal convoluted tubule, and cortical collecting duct. In addition, RaKCaR (but not PTH/PTHrP receptor) transcripts were found in the medullary thick ascending limb and outer medullary and inner medullary collecting ducts. These findings raise the possibility of roles for RaKCaR not only in the regulation of divalent mineral reabsorption but also in water reabsorption and urinary concentration. Taken together, our results provide new insights in understanding the effects of hypercalcemia on hormone-stimulated salt and water transport.

Down-regulation of the receptor for parathyroid hormone (PTH) and PTH-related peptide by PTH in primary fetal rat osteoblasts.

We studied the effects of parathyroid hormone (PTH) on PTH parathyroid hormone related peptide (PTHrP) receptor mRNA level, PTHrP binding and PTH-stimulated cyclic adenosine monophosphate (cAMP) accumulation in osteoblasts, derived from fetal rat calvariae (ROB). Cells isolated during 10-70 minutes of collagenase treatment were seeded at a density of 25,000 cells/cm2 and cultured for 4 days. These cells show a fast increase in cAMP production after stimulation for 5 minutes with 20 nM bovine parathyroid hormone(1-34) (bPTH(1-34)). When ROB are incubated with bPTH(1-34) (0.04-40nM) for 24 h, a dose-dependent decrease of the PTH/PTHrP receptor mRNA level, PTHrP binding, and PTH-stimulated cAMP accumulation can be observed. Pretreatment of ROB with a high concentration of bPTH(1-34) (40 nM) leads within 15 minutes to a decrease in PTH-stimulated cAMP accumulation. However, it takes > or = 3 h before a significant decrease in PTH/PTHrP receptor mRNA level can be observed. Also a significant decrease in PTHrP binding is observed after only 4 h of incubation with bPTH(1-34). Compared with bPTH(1-34), pretreatment of ROB with bPTH(3-34) (40 and 100 nM) for 24 h causes smaller decreases in PTH-stimulated cAMP accumulation, PTHrP binding, and in the PTH/PTHrP receptor mRNA level. We investigated the possible involvement of the protein kinase A signaling pathway in the regulation of the PTH/PTHrP receptor mRNA expression. Both forskolin and (Bu)2cAMP decreased PTHrP binding and PTH/PTHrP mRNA levels. These observations suggest that chronic activation of the PKA signaling pathway may down-regulate PTH/PTHrP receptor expression and thus hormone responsiveness in "normal" osteoblasts. In short, we found that the decrease of the PTH-stimulated cAMP accumulation after long-term pretreatment with bPTH(1-34) is correlated with both PTH/PTHrP receptor mRNA level and PTHrP binding. These data also suggest that the initial desensitization (< 30 minutes) of PTH-stimulated cAMP responsiveness by pretreatment with a high concentration of bPTH(1-34) (40 nM) is not dependent on the number of available PTH/PTHrP receptors. The protein kinase A signaling pathway is involved in the regulation of the PTH/PTHrP receptor, but, regarding the effect of bPTH(3-34), other signaling systems are also involved.

Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein.

Proper regulation of chondrocyte differentiation is necessary for the morphogenesis of skeletal elements, yet little is known about the molecular regulation of this process. A chicken homolog of Indian hedgehog (Ihh), a member of the conserved Hedgehog family of secreted proteins that is expressed during bone formation, has now been isolated. Ihh has biological properties similar to those of Sonic hedgehog (Shh), including the ability to regulate the conserved targets Patched (Ptc) and Gli. Ihh is expressed in the prehypertrophic chondrocytes of cartilage elements, where it regulates the rate of hypertrophic differentiation. Misexpression of Ihh prevents proliferating chondrocytes from initiating the hypertrophic differentiation process. The direct target of Ihh signaling is the perichondrium, where Gli and Ptc flank the expression domain of Ihh. Ihh induces the expression of a second signal, parathyroid hormone-related protein (PTHrP), in the periarticular perichondrium. Analysis of PTHrP (-/-) mutant mice indicated that the PTHrP protein signals to its receptor in the prehypertrophic chondrocytes, thereby blocking hypertrophic differentiation. In vitro application of Hedgehog or PTHrP protein to normal or PTHrP (-/-) limb explants demonstrated that PTHrP mediates the effects of Ihh through the formation of a negative feedback loop that modulates the rate of chondrocyte differentiation.

PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth.

The PTH/PTHrP receptor binds to two ligands with distinct functions: the calcium-regulating hormone, parathyroid hormone (PTH), and the paracrine factor, PTH-related protein (PTHrP). Each ligand, in turn, is likely to activate more than one receptor. The functions of the PTH/PTHrP receptor were investigated by deletion of the murine gene by homologous recombination. Most PTH/PTHrP receptor (-/-) mutant mice died in mid-gestation, a phenotype not observed in PTHrP (-/-) mice, perhaps because of the effects of maternal PTHrP. Mice that survived exhibited accelerated differentiation of chondrocytes in bone, and their bones, grown in explant culture, were resistant to the effects of PTHrP and Sonic hedgehog. These results suggest that the PTH/PTHrP receptor mediates the effects of Indian Hedgehog and PTHrP on chondrocyte differentiation.

G alpha q family members couple parathyroid hormone (PTH)/PTH-related peptide and calcitonin receptors to phospholipase C in COS-7 cells.

The PTH/PTH-related peptide (PTHrP) receptor and the calcitonin receptor mediate the action of their physiological ligands by activating two different effectors, adenylyl cyclase and phospholipase C. Whereas regulation of adenylyl cyclase via both receptors is thought to involve the G protein G(s), it is not known whether activation of phospholipase C results from coupling of the receptors to G(q) family members or whether beta gamma-subunit released from receptor-activated G(s) lead to phospholipase C activation. To elucidate the mechanism of this type of dual signaling, we reconstituted the signal transduction of the PTH/PTHrP and the calcitonin receptor in COS-7 and HEK293 cells. In COS-7 cells expressing the receptor alone, addition of the respective ligands resulted in the accumulation of cAMP and inositol phosphates. When cells were cotransfected with the cDNAs of receptor and different alpha-subunits of the Gq family (G alpha q, G alpha 11, G alpha 14, G alpha 15, and G alpha 16, a severalfold increase in the ligand-dependent inositol phosphate production could be observed, indicating that the receptors functionally interacted with all alpha-subunits of the G alpha q family. Additionally, whereas PTH treatment of HEK293 cells coexpressing both the PTH/PTHrP receptor and G alpha q increased both second messengers, the same treatment in cells expressing the PTH/PTHrP receptor alone increased only cAMP. Under all conditions tested, activation of phospholipase C via the PTH/PTHrP and calcitonin receptor required higher ligand concentrations than receptor-mediated adenylyl cyclase activation. Our data strongly support the idea that dual signaling of the PTH/PTHrP and calcitonin receptors is due to the a activation of different G proteins belonging to the G(s) and G(q) families.

Evidence that implicates the parathyroid hormone-related peptide in vascular stenosis. Increased gene expression in the intima of injured carotid arteries and human restenotic coronary lesions.

Proliferation of vascular smooth muscle cells (VSMCs) is considered to be one key event underlying the pathophysiology of restenosis after angioplasty. The parathyroid hormone-related peptide (PTHrP) and its receptor, a local autocrine and paracrine regulator of cellular growth in a variety of normal cell types, have been reported in the vicinity of VSMCs. To investigate how PTHrP might be involved in the process of neointimal formation after balloon angioplasty, we examined PTHrP expression in balloon-denuded rat carotid arteries and human coronary arteries that had been retrieved by directional atherectomy. In rat carotid arteries, the RNase protection assay and in situ hybridization demonstrated that PTHrP mRNA expression increased fourfold to sixfold 1 to 7 days after denudation and continued for 28 days, coincident with downregulation of PTH/PTHrP receptor mRNA expression. In situ hybridization and immunohistochemistry revealed that PTHrP expression in balloon-denuded carotid arteries was mainly localized to the neointima. To confirm the involvement of the PTHrP in human coronary artery restenotic lesions, immunohistochemical analysis of human coronary atherectomy specimens (23 primary and 10 restenotic lesions) was then performed. The number of intimal cells that expressed PTHrP protein was significantly higher in restenotic (407 +/- 53 cells/mm2; range, 143 to 739) than in stable angina (50 +/- 12 cells/mm2; range, 18 to 132; P<.05) or unstable angina (129 +/- 16 cells/mm2; range, 21 to 232; P<.05) specimens. These data demonstrate that PTHrP gene expression in VSMCs markedly increases during neointimal formation, supporting the hypothesis that PTHrP may play an important role in vascular stenosis as a regulator of VSMC proliferation.

Downregulation of the PTH/PTHrP receptor by vitamin D3 in the osteoblast-like ROS 17/2.8 cells.

Effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the expression of the parathyroid hormone (PTH)/PTH-related peptide (rP) receptor protein and mRNA in ROS 17/2.8 cells were studied. Treatment of ROS 17/2.8 cells with 1,25(OH)2D3 caused time- and dose-dependent suppression of PTH/PTHrP receptor number and immunoreactivity. The effects required more than 24 h incubation with 1,25(OH)2D3 and were maximal by 72 h. The cells did not recover their PTH/PTHrP receptors even after 4 days of treatment with control medium. Treatment with low concentrations of 1,25(OH)2D3 (0.1 M) dramatically decreased the PTH/PTHrP receptor mRNA levels, which were maximal after 24 h of incubation. The half-life of the PTH/PTHrP receptor transcript, 6-8 h, was similar in control and 1,25(OH)2D3-treated cells, suggesting that 1,25(OH)2D3 acts in controlling transcription of the PTH/PTHrP receptor gene but does not change the degradation rate of the PTH/PTHrP receptor transcripts. These data indicate that 1,25(OH)2D3 has a potent inhibitory effect on the expression of the PTH/PTHrP receptor protein and mRNA in ROS 17/2.8 cells.

Localization of parathyroid hormone/parathyroid hormone-related peptide receptor mRNA in kidney.

In kidney, parathyroid hormone (PTH) exerts differential distal effects along the nephron. To define cells expressing receptors for PTH in kidney, we localized PTH/ PTH-related peptide (PTH/PTHrP) receptor mRNA in rat kidney by in situ hybridization. PTH/PTHrP receptor mRNA is localized to glomerular podocytes, convoluted and straight proximal tubules, the cortical portion of thick ascending limbs, and distal convoluted tubules, but was not detected in the thin limb of Henle's loop or in collecting ducts. Northern blot analysis showed that cultured human glomerular podocytes express a unique 4.0-kb PTH/PTHrP receptor transcript but do not express detectable levels of the common approximately 2.4-kb transcript found in whole kidney and in many other tissues. Whereas the tubular localization of PTH/PTHrP receptor mRNA coincides well with previously known sites of PTH action, the intense expression and the unique size of the PTH/PTHrP receptor transcript in glomerular podocytes suggest that PTH and/or PTHrP may play a role(s) in glomerular function.