Targeted deletion of histidine decarboxylase gene in mice increases bone formation and protects against ovariectomy-induced bone loss.

Targeted disruption of the histidine decarboxylase gene (HDC(-/-)), the only histamine-synthesizing enzyme, led to a histamine-deficient mice characterized by undetectable tissue histamine levels, impaired gastric acid secretion, impaired passive cutaneous anaphylaxis, and decreased mast cell degranulation. We used this model to study the role of histamine in bone physiology. Compared with WT mice, HDC(-/-) mice receiving a histamine-free diet had increased bone mineral density, increased cortical bone thickness, higher rate of bone formation, and a marked decrease in osteoclasts. After ovariectomy, cortical and trabecular bone loss was reduced by 50% in HDC(-/-) mice compared with WT. Histamine deficiency protected the skeleton from osteoporosis directly, by inhibiting osteoclastogenesis, and indirectly, by increasing calcitriol synthesis. Quantitative RT-PCR showed elevated 25-hydroxyvitamin D-1alpha-hydroxylase and markedly decreased 25-hydroxyvitamin D-24-hydroxylase mRNA levels. Serum parameters confirming this indirect effect included elevated calcitriol, phosphorus, alkaline phosphatase, and receptor activator of NF-kappaB ligand concentrations, and suppressed parathyroid hormone concentrations in HDC(-/-) mice compared with WT mice. After ovariectomy, histamine-deficient mice were protected from bone loss by the combination of increased bone formation and reduced bone resorption.

Novel regulators of vitamin D action and metabolism: Lessons learned at the Los Angeles zoo.

We undertook an investigation of an outbreak of rachitic bone disease in the Emperor Tamarin New World primate colony at the Los Angeles Zoo in the mid-1980s. The disease phenotype resembled that observed in humans with an inactivating mutation of the vitamin D receptor (VDR), hypocalcemia, high 1,25-dihydroxyvitamin D (1,25-(OH)(2)D) levels, and rickets in rapidly growing adolescent primates. In contrast to the human disease, the New World primate VDR was functionally normal in all respects. The proximate cause of vitamin D hormone resistance in New World primates was determined to be the constitutive overexpression of a heterogeneous nuclear ribonucleoprotein in the A family which we coined the vitamin D response element binding protein (VDRE-BP). VDRE-BP competed in trans with the VDR-retinoid X receptor (RXR) for binding to the vitamin D response element. VDRE-BP-legislated resistance to 1,25-(OH)(2)D was antagonized (i.e., compensated) by another set of constitutively overexpressed proteins, the hsp-70-related intracellular vitamin D binding proteins (IDBPs). IDBPs, present but expressed at much lower levels in Old World primates including man, exhibited a high capacity for 25-hydroxylated vitamin D metabolites and functioned to traffic vitamin Ds to specific intracellular destinations to promote their action and metabolism.

Vitamin D receptor and retinoid X receptor interactions in motion.

Vitamin D receptor (VDR) and retinoid X receptor (RXR) are members of the nuclear receptor superfamily and they bind target DNA sequences as heterodimers to regulate transcription. This article surveys the latest findings regarding the roles of dimerizing RXR in VDR function and emphasizes potential areas for future developments. We first highlight the importance of dimerization with RXR for both the ligand-independent (hair growth) and ligand-dependent functions of VDR (calcium homeostasis, bone development and mineralization, control of cell growth and differentiation). Emerging information regarding the regulatory control of dimerization based on biochemical, structural, and genetic studies is then presented. Finally, the main focus of this article is a new dynamic perspective of dimerization functions, based on recent research with fluorescent protein chimeras in living cells by microscopy. These studies revealed that both VDR and RXR constantly shuttle between the cytoplasm and the nucleus and between subnuclear compartments, and showed the transient nature of receptor--DNA and receptor--coregulator interactions. Because RXR dimerizes with most of the nuclear receptors, regulation of receptor dynamics by RXR has a broad significance.

Dimerization with retinoid X receptors promotes nuclear localization and subnuclear targeting of vitamin D receptors.

The vitamin D receptor (VDR) acts as heterodimer with the retinoid X receptor alpha (RXR) to control transcriptional activity of target genes. To explore the influence of heterodimerization on the subcellular distribution of these receptors in living cells, we developed a series of fluorescent-protein chimeras. The steady-state distribution of the yellow fluorescent protein-RXR was more nuclear than the unliganded green fluorescent protein (GFP)-VDR. Coexpression of RXR-blue fluorescent protein (BFP) promoted nuclear accumulation of GFP-VDR by influencing both nuclear import and retention. Fluorescence resonance energy transfer microscopy (FRET) demonstrated that the unliganded GFP-VDR and RXR-BFP form heterodimers. The increase in nuclear heterodimer content correlated with an increase in basal transcriptional activity. FRET also revealed that calcitriol induces formation of multiple nuclear foci of heterodimers. Mutational analysis showed a correlation between hormone-dependent nuclear VDR foci formation and DNA binding. RXR-BFP also promoted hormone-dependent nuclear accumulation and intranuclear foci formation of a nuclear localization signal mutant receptor (nlsGFP-VDR) and rescued its transcriptional activity. Heterodimerization mutant RXR failed to alter GFP-VDR and nlsGFP-VDR distribution or activity. These experiments suggest that RXR has a profound effect on VDR distribution. This effect of RXR to promote nuclear accumulation and intranuclear targeting contributes to the regulation of VDR activity and probably the activity of other heterodimerization partners.

1,25-Dihydroxyvitamin D3 hypersensitivity of osteoclast precursors from patients with Paget's disease.

Our previous studies suggested that increased osteoclast formation and activity in Paget's disease may be related in part to increased responsiveness of highly purified osteoclast precursors to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. However, the basis for this enhanced sensitivity to 1,25-(OH)2D3 is unclear. To address this question, we examined 24-hydroxylase and 1,25-(OH)2D3 receptor (VDR) messenger RNA (mRNA) expression during human osteoclast differentiation from normal subjects and patients with Paget's disease in response to 1,25-(OH)2D3 as well as VDR content and affinity. Reverse-transcription polymerase chain reaction (RT-PCR) analysis of granulocyte-macrophage colony-forming unit (GM-CFU), the earliest identifiable osteoclast precursor, derived from patients with Paget's disease demonstrated 24-hydroxylase mRNA expression in response to 1,25-(OH)2D3 was induced at concentrations of 1,25-(OH)2D3 that were at least one log less than that required for normal GM-CFU. VDR mRNA and VDR protein were detected in both immature and more differentiated osteoclast precursors, as well as in osteoclast-like multinucleated cells (MNCs). However, VDR expression was lower in MNCs than the mononuclear precursor cells. Osteoclast precursors and MNCs from patients with Paget's disease had levels of VDR expression similar to those of normal subjects but showed increased VDR affinity for 1,25-(OH)2D3. Because the effects of 1,25-(OH)2D3 are in part mediated by induction of expression of RANK ligand on marrow stromal cells, which in turn stimulates osteoclast formation, we examined expression of RANK ligand mRNA by marrow stromal cell lines derived from patients with Paget's disease and normal subjects in response to 1,25-(OH)2D3. RT-PCR analysis showed no difference in sensitivity of marrow stromal cells to 1,25-(OH)2D3 from normal subjects or patients with Paget's disease although the Paget's stromal cells expressed increased basal levels of RANK ligand mRNA. These results show that VDR protein is expressed in early and more differentiated osteoclast precursors, that expression levels of VDR decline with osteoclast differentiation, and that 1,25-(OH)2D3 has direct effects on osteoclast precursors. The enhanced sensitivity to 1,25-(OH)2D3 is an intrinsic property of osteoclast precursors from patients with Paget's disease that distinguishes them from normal osteoclast precursors. Furthermore, our results suggest that an increased affinity of VDR for 1,25-(OH)2D3 may be responsible for the enhanced 1,25-(OH)2D3 sensitivity of osteoclast precursors in patients with Paget's disease compared with normal subjects.

The human vitamin D receptor gene (VDR) is localized to region 12cen-q12 by fluorescent in situ hybridization and radiation hybrid mapping: genetic and physical VDR map.

The vitamin D receptor (VDR) is a member of the steroid hormone receptor superfamily of ligand-activated transcription factors. The VDR gene was previously mapped to human chromosome 12q13-12q14, but its precise physical and genetic localization are unknown. The present study reports the mapping of the human VDR gene by radiation hybrid (RH) analysis, the isolation of a bacterial artificial chromosome (BAC) containing this gene, and physical mapping of the VDR gene by fluorescent in situ hybridization (FISH). RH analysis placed the VDR gene locus at chromosome 12cen-q12, flanked by Stanford Human Genome Center (SHGC) 30216 and SHGC 9798 (D12S1892) markers. FISH analysis of a BAC containing the VDR gene confirmed its centromeric location. Thus, we have identified a BAC and genetic markers which can be used in the genetic analysis of the VDR gene and investigation of its involvement in osteoporosis and related disorders. We conclude that the VDR gene is centromeric to its previously reported locus on chromosome 12.

Hormone-dependent translocation of vitamin D receptors is linked to transactivation.

Vitamin D receptor (VDR) acts as a transcription factor mediating genomic actions of calcitriol. Our earlier studies suggested that calcitriol induces translocation of cytoplasmic VDR, but the physiologic relevance of this finding remained uncertain. Previous studies demonstrated that the activation function 2 domain (AF-2) plays an essential role in VDR transactivation. To elucidate hormone-dependent VDR translocation and its role, we constructed green fluorescent protein (GFP) chimeras with full-length VDR (VDR-GFP), AF-2-truncated VDR (AF-2del-VDR-GFP), and ligand-binding domain (LBD)-truncated VDR (LBDdel-VDR-GFP). COS-7 cells were transiently transfected with these constructs. Western blot analysis, fluorescent microscopy, and transactivation assays showed that the generated chimeras are expressed and fluoresce and that VDR-GFP is transcriptionally active. After hormone treatment, cytoplasmic VDR-GFP translocated to the nucleus in a concentration-, time-, temperature-, and analog-specific manner. Hormone dose-response relationships for translocation and for transactivation were similar. Truncation of LBD and truncation of AF-2 each abolished hormone-dependent translocation and transactivation. Our data confirm a hormone-dependent VDR translocation, demonstrate that an intact AF-2 domain is required for this translocation, and indicate that translocation is part of the receptor activation process.

Intranuclear trafficking and gene targeting by members of the steroid/nuclear receptor superfamily.

Upon binding to regulatory elements in mammalian chromosomes, steroid receptors induce specific transitions in the nucleoprotein structure of the template. These transitions reflect, in part, the reorganization of chromatin structure to permit interaction of secondary factors with target sequences in promoter regulatory regions. Steroid receptors represent a class of transcriptional activators that are able to interact with repressed nucleoprotein templates and recruit necessary activities for chromatin remodeling. The ligand-induced movement of nuclear receptors from inactive states, either in the cytoplasm or in the nucleus, to productive interactions with chromatin is complex and likely reflects the interaction with multiple protein complexes and subcellular structures. Regulation of gene expression by nuclear receptors is thus mediated through the subcellular distribution of inactive receptors, the redistribution of activated receptor complexes to appropriate nuclear domains, the reorganization of chromatin structures for interaction with soluble components of the nucleoplasm, and direct protein-protein contacts between receptors and the basal transcription apparatus.

Subcellular distribution of normal and mutant vitamin D receptors in living cells. Studies with a novel fluorescent ligand.

To understand the subcellular localization of the vitamin D receptor (VDR) and to measure VDR content in single cells, we recently developed a fluorescent labeled ligand, 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene (BODIPY)-calcitriol. This tagged hormone has intact biological activity, high affinity and specific binding to the receptor, and enhanced fluorescent emission upon receptor binding. Using BODIPY-calcitriol, here we monitored the subcellular distribution of VDR in living cultured cells by microscopy. Time course studies showed that an equilibrium between the cytoplasmic and nuclear hormone binding developed within 5 min and was maintained thereafter. We found a substantial proportion of VDR residing in the cytoplasm, colocalized with endoplasmic reticulum, the Golgi complex, and microtubules. Confocal microscopy clarified the presence of VDR within discrete regions of the nucleus and along the nuclear envelope. There was no VDR in the plasma membrane. Low affinity BODIPY-calcitriol binding sites were in the mitochondria. Mutations in the VDR gene selectively and specifically altered BODIPY-calcitriol distribution. Defects in the hormone binding region of VDR prevented both nuclear and cytoplasmic hormone binding. Defects in the DNA binding region decreased the nuclear retention of VDR and prevented localization to nuclear foci. These results with BODIPY-calcitriol reveal cytoplasmic VDR localization in living cells and open the possibility of studying the three-dimensional architecture of intranuclear target sites.

Visualization of glucocorticoid receptor translocation and intranuclear organization in living cells with a green fluorescent protein chimera.

A highly fluorescent mutant form of the green fluorescent protein (GFP) has been fused to the rat glucocorticoid receptor (GR). When GFP-GR is expressed in living mouse cells, it is competent for normal transactivation of the GR-responsive mouse mammary tumor virus promoter. The unliganded GFP-GR resides in the cytoplasm and translocates to the nucleus in a hormone-dependent manner with ligand specificity similar to that of the native GR receptor. Due to the resistance of the mutant GFP to photobleaching, the translocation process can be studied by time-lapse video microscopy. Confocal laser scanning microscopy showed nuclear accumulation in a discrete series of foci, excluding nucleoli. Complete receptor translocation is induced with RU486 (a ligand with little agonist activity), although concentration into nuclear foci is not observed. This reproducible pattern of transactivation-competent GR reveals a previously undescribed intranuclear architecture of GR target sites.

Development of a biologically active fluorescent-labeled calcitriol and its use to study hormone binding to the vitamin D receptor.

To gain better insight into the mechanism of steroid receptor activation and calcitriol action, we have developed the first pharmacologically relevant fluorescent-labeled ligand for the vitamin D receptor (VDR). Purity and structure of three BODIPY-labeled calcitriol derivatives were characterized by TLC, HPLC, and 1H-NMR spectroscopy. 3 beta-BODIPY-calcitriol was the most potent derivative to induce 25-hydroxyvitamin D3 24-hydroxylase activity and to inhibit cell proliferation. It was taken up rapidly and specifically and was not cleaved by endogenous esterases. 3 beta-BODIPY-calcitriol also retained high-affinity binding to the VDR. Hormone binding to the receptor was measured by spectrofluorometry in high-salt extracts from cultured cells with wild-type VDR, from cells virally over-expressing the human VDR, and in intact cells with and without VDR. Results from fluorescent binding studies agreed with results from radioligand assays. The most useful feature of this reagent is that its fluorescence emission increases severalfold upon binding to VDR. This allows direct monitoring by microscopy of ligand receptor interactions in living cells. Fluorescent-labeled calcitriol can be a valuable diagnostic tool for cancer research and is essential for exploring the subcellular localization of VDRs.

Molybdate increases intracellular 3',5'-guanosine cyclic monophosphate and stabilizes vitamin D receptor association with tubulin-containing filaments.

With a recently developed method we detected rapid and sequential reorganization of vitamin D receptors (VDR), including their temporary association with fibers, and we showed that calcitriol induces cGMP accumulation around reorganizing VDRs. In this report we first identified the VDR-associated fibers as microtubules: they show immunoreactivity with tubulin antisera and were sensitive to tubulin-disruptive agents. Tubulin-disruptive agents also prevented calcitriol-induced alignment and intranuclear accumulation of VDR and cGMP, but did not prevent the initial cGMP accumulation in the cytoplasm. Then we studied the effect of molybdate on VDR reorganization and on cGMP accumulation. Sodium molybdate inhibits steroid receptor transformation into a DNA binding form through interaction with the steroid binding region of the receptor. The mechanism of molybdate effect on steroid receptors is not well understood and the interaction of molybdate with guanylate cyclase has not been investigated. We found in cells pretreated with molybdate that the addition of calcitriol resulted in a prolonged and accentuated association of VDR and cGMP with the microtubules. Furthermore, both immunocytology and radioimmunoassay demonstrated that molybdate is a highly potent inducer of guanylate cyclase. Neither calcitriol nor molybdate effect on guanylate cyclase were prevented by methylene blue pretreatment, suggesting that they activate particulate guanylate cyclase. Pretreatment of cells with dibutyryl-cGMP mimicked molybdate effect on VDR reorganization. The effect of molybdate on cGMP may participate in molybdate stabilization of steroid receptors. We suggest that rapid cGMP accumulation after steroid exposure plays a role in facilitation of intracellular transport of the steroid receptor through interaction with microtubules.

Rapid effects of insulin on cyclic GMP location in an intact protozoan.

We studied rapid changes in location of cyclic GMP in Tetrahymena pyriformis. Insulin caused cGMP localization in cilia and near the plasma membrane (0.5-1 min). Later (1-5 min) cGMP localization was diffuse in cytoplasm with perinuclear accentuation. Inactive insulin analogs did not elicit these changes.

Rapid accumulation of cyclic GMP near activated vitamin D receptors.

The mechanisms of early calcitriol (1 alpha,25-dihydroxycholecalciferol) effects, including its receptor activation process as well as its "nongenomic" effects, are poorly understood. Calcitriol causes a rapid accumulation of cGMP, dependent on the presence of normal vitamin D receptors (VDRs). We recently developed an immunocytology method based on rapid microwave fixation suitable to detect the locations of agonist-induced intracellular cGMP accumulation. With the same technique we found that calcitriol induces stepwise and rapid reorganization of VDRs. Here we used this technique to study the subcellular compartmentalization of cGMP accumulation after exposure of cells to various steroid-related agonists and to study the spatial relationship between cGMP accumulation and VDRs. Calcitriol (10 nM) within 15 sec caused clumping of VDRs and accumulation of cGMP around VDR clumps; thereafter (up to 5 min), the cGMP accumulation surrounded VDRs throughout their stepwise reorganization. In fibroblasts from subjects with mutations affecting VDR function, we found disruptions of the calcitriol-induced patterns of cGMP accumulation analogous to the disruptions of VDR reorganization. The colocalization of cGMP accumulation with reorganizing VDRs at early moments after calcitriol addition indicates transduction of the cGMP increase by VDRs inside the cell, rather than by components in the plasma membrane. Other steroid-related agonists caused compartmentalized and sequential changes in cGMP accumulation that seemed specific for each class of agonist. Our findings suggest that compartmentalized cGMP accumulation is an early and common step during activation of steroid-related receptors.

Immunocytology with microwave-fixed fibroblasts shows 1 alpha,25-dihydroxyvitamin D3-dependent rapid and estrogen-dependent slow reorganization of vitamin D receptors.

Prior studies have given no evidence for regulation of vitamin D receptor (VDR) compartmentalization or subcellular organization. Microwave fixation (9-15 s) and an indirect immunodetection system of avidin-biotin enhancement and phycoerythrin fluorophore resulted in sufficient spatial and temporal resolution to allow analysis of these processes. We studied cultured fibroblasts from normals or from patients with four different types of hereditary defect compromising VDR function (mutant cells). Compartmentalization of VDRs in the absence of 1,25-dihydroxyvitamin D3 (calcitriol) was regulated by serum or estrogen. VDRs were mainly cytoplasmic in cells cultured without serum and phenol red, but VDRs were mainly intranuclear after addition of serum or an estrogen to cells for at least 18 h (slow regulation). Calcitriol initiated a rapid and multistep process (rapid regulation) of reorganization in a portion of VDRs: clumping within 15-45 s, alignment of clumps along fibrils within 30-45 s, perinuclear accumulation of clumps within 45-90 s, and intranuclear accumulation of clumps within 1-3 min. We found similar rapid effects of calcitriol on VDRs in various other types of cultured cells. These sequential VDR pattern changes showed calcitriol dose dependency and calcitriol analogue specificity characteristic for the VDR. In mutant fibroblasts VDR pattern changes after calcitriol were absent or severely disturbed at selected steps. Treatment of normal cells with wheat germ agglutinin, which blocks protein transport through nuclear pores, also blocked calcitriol-dependent translocation of VDRs. We conclude that immunocytology after microwave fixation provides evidence for regulation of VDR organization and localization.

Immunocytology on microwave-fixed cells reveals rapid and agonist-specific changes in subcellular accumulation patterns for cAMP or cGMP.

We developed a method for cAMP and cGMP immunocytology based upon fixation by microwave irradiation. Fixation by microwave irradiation prevented three problems found with other fixation methods: nucleotide loss from cells, nucleotide diffusion within cells, and chemical modification of immunologic epitopes. Six agonists (four that stimulate adenylate cyclase and two that stimulate guanylate cyclase) produced cAMP or cGMP accumulation patterns that were agonist-specific, dose-dependent, detectable at physiologic concentrations of hormone, and time-dependent within 15 sec to 30 min. cAMP accumulation after 1 mM forskolin was greatest in the nucleus. Isoproterenol, prostaglandin E2, or calcitonin caused initial accumulation of cAMP along the plasma membrane, but later accumulation was greater in the cytoplasm. With calcitonin the later accumulation of cAMP was selectively perinuclear and along the nuclear membrane. Sodium nitroprusside stimulated cGMP accumulation diffusely throughout the cytoplasm. Atrial natriuretic peptide initiated cGMP accumulation near the plasma membrane, and cGMP accumulation moved from there into the cytoplasm. In conclusion, microwave irradiation preserved cell structure and allowed visualization of expected as well as unsuspected changes in intracellular accumulation patterns of cAMP and cGMP.

Selective expression of a normal action of the 1,25-dihydroxyvitamin D3 receptor in human skin fibroblasts with hereditary severe defects in multiple actions of that receptor.

We evaluated three actions of 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] in human skin fibroblasts to test for heterogeneity in hormone-response coupling. In fibroblasts from normal subjects the 1,25-(OH)2D3 concentrations for half-maximal effect (EC50) were: for mitogenic effect 0.0001-0.0005 nM, for antimitogenic effect 1 nM, and for induction of 25-OHD3 24-hydroxylase (24-OHase) 5 nM. To evaluate the effects of mutations presumed to be in the gene for the 1,25-(OH)2D3 receptor we examined cell lines representing four kindreds with hereditary resistance to 1,25-(OH)2D3 ("mutant" cell lines). In one mutant cell line all three 1,25-(OH)2D3 actions were severely abnormal. In one mutant cell line 24-OHase induction and mitogenic action were undetectable, but EC50 and maximal effect were normal for antimitogenic action of 1,25-(OH)2D3. In two mutant cell lines 24-OHase induction and antimitogenic actions were undetectable or severely impaired but mitogenic action were undetectable or severely impaired but mitogenic action was normal in EC50 and normal or increased in maximal effect. The mitogenic and antimitogenic actions in normal cells showed a similar profile of potency ratios for 1,25-(OH)2D3 and six analogues. Whenever a mutant cell showed a normal or even an abnormal mitogenic or antimitogenic effect of 1,25-(OH)2D3, these effects showed potency ratios similar to wild type, suggesting mediation by a similar 1,25-(OH)2D3 receptor. We conclude that three 1,25-(OH)2D3 actions show important differences in hormone response coupling indicated by differences in EC50 for 1,25-(OH)2D3 and by different consequences of receptor mutations.

Ongoing protein synthesis needed for 1,25-(OH)2D3-mediated rapid increase of cyclic GMP in human skin fibroblasts.

Recently we reported that 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) through interaction with its specific receptor rapidly (within 1 min) stimulated intracellular cGMP production in cultured human skin fibroblasts. Here we show that this effect of 100 nM 1,25-(OH)2D3 is prevented by brief (30 min) inhibition of RNA synthesis (with actinomycin D or alpha-amanitin) or by brief inhibition of protein synthesis (with cycloheximide or diphtheria toxin). The protein synthesis inhibitors also blocked stimulation of cGMP by other steroids (testosterone or dexamethasone at 100 nM) but did not block cGMP stimulation by sodium nitroprusside. Since the time for the 1,25-(OH)2D3 receptor to increase cGMP seems too short to require de novo protein synthesis, we conclude that the 1,25-(OH)2D3 receptor acts together with rapidly turning over protein(s) to stimulate cGMP synthesis.

Dual effects of calcitonin and calcitonin gene-related peptide on intracellular cyclic 3',5'-monophosphate in a human breast cancer cell line.

The effects of salmon calcitonin (sCT) and human calcitonin (hCT) and of rat (r) and human (h) calcitonin gene-related peptide (CGRP) on intracellular cAMP accumulation were tested in human breast cancer cells (MCF7). In addition to the well known stimulatory effect, each showed a significant inhibitory effect on cAMP accumulation at low doses. cAMP concentrations in response to sCT, hCT, and rCGRP decreased to 47 +/- 2, 45 +/- 4, and 56 +/- 2% (mean +/- 1 SE) of baseline. The potency ratios for the inhibitory action of sCT, hCT, and rCGRP (1:0.25:0.005, respectively) were similar to the potency ratios for stimulatory action (1:0.3:0.005). The inhibition of cAMP accumulation developed at 300-fold lower peptide concentrations than the stimulation. Preincubation with pertussis toxin or with manganese completely abolished the inhibitory effect of the peptides, suggesting that this is mediated by an inhibitory adenylate cyclase regulatory protein. sCT, hCT, and CGRP each showed unique patterns with regard to time course of inhibition of cAMP accumulation. We conclude that 1) CT can activate an inhibitory adenylate cyclase regulatory protein and a stimulatory adenylate cyclase regulatory protein, and 2) CT effect on an inhibitory adenylate cyclase regulatory protein in MCF 7 cells is evident at far lower hormone concentrations than its effect on a stimulatory adenylate cyclase regulatory protein.