Calcitonin receptors are ancient modulators for rhythms of preferential temperature in insects and body temperature in mammals.

Daily body temperature rhythm (BTR) is essential for maintaining homeostasis. BTR is regulated separately from locomotor activity rhythms, but its molecular basis is largely unknown. While mammals internally regulate BTR, ectotherms, including Drosophila, exhibit temperature preference rhythm (TPR) behavior to regulate BTR. Here, we demonstrate that the diuretic hormone 31 receptor (DH31R) mediates TPR during the active phase in Drosophila DH31R is expressed in clock cells, and its ligand, DH31, acts on clock cells to regulate TPR during the active phase. Surprisingly, the mouse homolog of DH31R, calcitonin receptor (Calcr), is expressed in the suprachiasmatic nucleus (SCN) and mediates body temperature fluctuations during the active phase in mice. Importantly, DH31R and Calcr are not required for coordinating locomotor activity rhythms. Our results represent the first molecular evidence that BTR is regulated distinctly from locomotor activity rhythms and show that DH31R/Calcr is an ancient specific mediator of BTR during the active phase in organisms ranging from ectotherms to endotherms.

The Dual Amylin and Calcitonin Receptor Agonist, KBP-066, Induces an Equally Potent Weight Loss Across a Broad Dose Range While Higher Doses May Further Improve Insulin Action.

Pharmacological treatment with dual amylin and calcitonin receptor agonists (DACRAs) cause significant weight loss and improvement of glucose homeostasis. In this study, the maximally efficacious dose of the novel DACRA, KeyBiosciencePeptide (KBP)-066, was investigated. Two different rat models were used: high-fat diet (HFD)-fed male Sprague-Dawley rats and male Zucker diabetic fatty (ZDF, fa/fa) rats to determine the maximum weight loss and glucose homeostatic effect, respectively. One acute study and one chronic study was performed in HFD rats. Two chronic studies were performed in ZDF rats: a preventive and an interventive. All studies covered a dose range of 5, 50, and 500 µg/kg KBP-066 delivered by subcutaneous injection. Treatment with KBP-066 resulted in a significant weight reduction of 13%-16% and improved glucose tolerance in HFD rats, which was independent of dose concentration. Dosing with 50 and 500 µg/kg led to a transient but significant increase in blood glucose, both in the acute and the chronic study in HFD rats. All doses of KBP-066 significantly improved glucose homeostasis in ZDF rats, both in the preventive and interventive study. Moreover, dosing with 50 and 500 µg/kg preserved insulin secretion to a greater extent than 5 µg/kg when compared with ZDF vehicle rats. Taken together, these results show that maximum weight loss is achieved with 5 µg/kg, which is within the range of previously reported DACRA dosing, whereas increasing dosing concentration to 50 and 500 µg/kg may further improve preservation of insulin secretion compared with 5 µg/kg in diabetic ZDF rats. SIGNIFICANCE STATEMENT: Here we show that KeyBiosciencePeptide (KBP)-066 induces an equally potent body weight loss across a broad dose range in obese rats. However, higher dosing of KBP-066 may improve insulin action in diabetic rats both as preventive and interventive treatment.

Calcium-containing crystals enhance receptor activator of nuclear factor κB ligand/macrophage colony-stimulating factor-mediated osteoclastogenesis via extracellular-signal-regulated kinase and p38 pathways.

OBJECTIVE: Diseases associated with calcium-containing crystal deposition can lead to local bone erosion. We aimed to determine whether calcium-containing crystal-hydroxyapatite, ß-tricalcium phosphate and CPPD enhanced osteoclastogenesis and to define underlying mechanisms of action. METHODS: Osteoclastogenesis was studied by culturing murine RAW 264.7 osteoclast precursor cells with RANK ligand (RANKL)/ M-CSF and/or calcium-containing crystals, and observing the tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and TRAP activity. Resorption pit formation was used to evaluate osteoclast activity. Real-time RT-PCR analysis revealed osteoclast marker genes, including TRAP, cathepsin K and calcitonin receptor (CTR). Western blotting was used to analyse the phosphorylation levels of signal transduction molecules. RESULTS: Three kinds of calcium-containing crystal significantly enhanced RANKL/M-CSF-induced osteoclastogenesis in RAW 264.7 cells, as evidenced by the increased number of TRAP-positive multinucleated cells, TRAP activity and resorption pit formation in a dose-dependent manner. Hydroxyapatite, ß-tricalcium phosphate and CPPD treatments significantly enhanced RANKL/M-CSF-induced mRNA expression of TRAP, cathepsin K and CTR. Moreover, the three kinds of calcium-containing crystal enhanced the phosphorylation of extracellular-signal-regulated kinase and p38 in RANKL/M-CSF-treated cells. CONCLUSION: We concluded that calcium-containing crystals can promote osteoclastogenesis and bone resorption through the extracellular-signal-regulated kinase and p38 pathways. Together with synovial activation, this mechanism may be important in the pathogenesis of destructive arthropathies triggered by calcium-containing crystals.

[Odontoclasts and calcitonin].

Although it is believed that odontoclasts, which mediated root resorption of deciduous teeth, possess common properties to osteoclasts, these regulatory mechanisms differ from osteoclastic bone resorption. It is well established that calcitonin receptor is an important osteoclast marker and that calcitonin is a potent inhibitory hormone of osteoclastic bone resorption. However, the presence and function of calcitonin receptors in human odontoclasts are still controversial. We summarize the physiological properties and differentiation mechanisms of odontoclasts, and the effects of calcitonin on root resorption, including our recent results using human odontoclasts and periodontal ligament cells freshly isolated from deciduous tooth roots.

The calcitonin receptor regulates osteocyte lacunae acidity during lactation in mice.

The physiological role of calcitonin, and its receptor, the CTR (or Calcr), has long been debated. We previously provided the first evidence for a physiological role of the CTR to limit maternal bone loss during lactation in mice by a direct action on osteocytes to inhibit osteocytic osteolysis. We now extend these findings to show that CTR gene expression is upregulated two- to three-fold in whole bone of control mice at the end of pregnancy (E18) and lactation (P21) compared to virgin controls. This was associated with an increase in osteoclast activity evidenced by increases in osteoclast surface/bone surface and Dcstamp gene expression. To investigate the mechanism by which the CTR inhibits osteocytic osteolysis, in vivo acidification of the osteocyte lacunae during lactation (P14 days) was assessed using a pH indicator dye. A lower pH was observed in the osteocyte lacunae of lactating Global-CTRKOs compared to controls and was associated with an increase in the gene expression of ATPase H+ transporting V0 subunit D2 (Atp6v0d2) in whole bone of Global-CTRKOs at the end of lacation (P21). To determine whether the CTR is required for the replacement of mineral within the lacunae post-lactation, lacunar area was determined 3 weeks post-weaning. Comparison of the largest 20% of lacunae by area did not differ between Global-CTRKOs and controls post-lactation. These results provide evidence for CTR activation to inhibit osteocytic osteolysis during lactation being mediated by regulating the acidity of the lacunae microenvironment, whilst the CTR is dispensable for replacement of bone mineral within lacunae by osteocytes post-lactation.

Paraventricular Calcitonin Receptor-Expressing Neurons Modulate Energy Homeostasis in Male Mice.

The paraventricular nucleus of the hypothalamus (PVH) is a heterogeneous collection of neurons that play important roles in modulating feeding and energy expenditure. Abnormal development or ablation of the PVH results in hyperphagic obesity and defects in energy expenditure whereas selective activation of defined PVH neuronal populations can suppress feeding and may promote energy expenditure. Here, we characterize the contribution of calcitonin receptor-expressing PVH neurons (CalcRPVH) to energy balance control. We used Cre-dependent viral tools delivered stereotaxically to the PVH of CalcR2Acre mice to activate, silence, and trace CalcRPVH neurons and determine their contribution to body weight regulation. Immunohistochemistry of fluorescently-labeled CalcRPVH neurons demonstrates that CalcRPVH neurons are largely distinct from several PVH neuronal populations involved in energy homeostasis; these neurons project to regions of the hindbrain that are implicated in energy balance control, including the nucleus of the solitary tract and the parabrachial nucleus. Acute activation of CalcRPVH neurons suppresses feeding without appreciably augmenting energy expenditure, whereas their silencing leads to obesity that may be due in part due to loss of PVH melanocortin-4 receptor signaling. These data show that CalcRPVH neurons are an essential component of energy balance neurocircuitry and their function is important for body weight maintenance. A thorough understanding of the mechanisms by which CalcRPVH neurons modulate energy balance might identify novel therapeutic targets for the treatment and prevention of obesity.

Calcitonin receptor-like receptor guides arterial differentiation in zebrafish.

The calcitonin receptor-like receptor (crlr) is a major endothelial cell receptor for adrenomedullin, a peptide vasodilator involved in cardiovascular development, homeostasis, and disease. Here, we used the zebrafish (Danio rerio) model to characterize the role of crlr in vascular development. Crlr is expressed within somites from the 4- to the 13-somite stage and by arterial progenitors and axial vessels during zebrafish development. Loss of crlr results in profound alterations in vascular development and angiogenesis, including atrophic trunk dorsal aorta and interruption of anterior aortic bifurcation, delay in intersomitic vessel development, and lack of blood circulation. Remarkably, crlr morphants are characterized by the loss of arterial endothelial cell identity in dorsal aorta, as shown by the lack of expression of the arterial markers ephrin-B2a, DeltaC, and notch5. Down-regulation of crlr affects vascular endothelial growth factor (vegf) expression, whereas vegf overexpression is sufficient to rescue arterial differentiation in crlr morphants. Finally, genetic and biochemical evidences indicate that somitic crlr expression is under the control of sonic hedgehog. These data demonstrate that crlr plays a nonredundant role in arterial differentiation, representing a novel element of the sonic hedgehog-vegf-notch signaling cascade that controls arterial/venous fate.

G protein-coupled receptors as potential drug targets for lymphangiogenesis and lymphatic vascular diseases.

G protein-coupled receptors (GPCRs) are widely expressed cell surface receptors that have been successfully exploited for the treatment of a variety of human diseases. Recent studies in genetically engineered mouse models have led to the identification of several GPCRs important for lymphatic vascular development and function. The adrenomedullin receptor, which consists of an oligomer between calcitonin receptor-like receptor and receptor activity modifying protein 2, is required for normal lymphatic vascular development and regulates lymphatic capillary permeability in mice. Numerous studies also suggest that lysophospholipid receptors are involved in the development of lymphatic vessels and lymphatic endothelial cell permeability. Given our current lack of pharmacological targets for the treatment of lymphatic vascular diseases like lymphedema, the continued identification and study of GPCRs in lymphatic endothelial cells may eventually lead to major breakthroughs and new pharmacological strategies for the treatment of lymphedema.

Calcitonin Receptor Neurons in the Mouse Nucleus Tractus Solitarius Control Energy Balance via the Non-aversive Suppression of Feeding.

To understand hindbrain pathways involved in the control of food intake, we examined roles for calcitonin receptor (CALCR)-containing neurons in the NTS. Ablation of NTS Calcr abrogated the long-term suppression of food intake, but not aversive responses, by CALCR agonists. Similarly, activating CalcrNTS neurons decreased food intake and body weight but (unlike neighboring CckNTS cells) failed to promote aversion, revealing that CalcrNTS neurons mediate a non-aversive suppression of food intake. While both CalcrNTS and CckNTS neurons decreased feeding via projections to the PBN, CckNTS cells activated aversive CGRPPBN cells while CalcrNTS cells activated distinct non-CGRP PBN cells. Hence, CalcrNTS cells suppress feeding via non-aversive, non-CGRP PBN targets. Additionally, silencing CalcrNTS cells blunted food intake suppression by gut peptides and nutrients, increasing food intake and promoting obesity. Hence, CalcrNTS neurons define a hindbrain system that participates in physiological energy balance and suppresses food intake without activating aversive systems.

Investigation of salmon calcitonin in regulating fibrosis-related molecule production and cell-substrate adhesion in frozen shoulder synovial/capsular fibroblasts.

The purpose of this study was to evaluate the effect of salmon calcitonin (sCT) on improving fibrosis-related indicators in frozen shoulder synovial/capsular fibroblasts (SCFs) and detect the potential downstream pathway. Quantitative real-time polymerase chain reaction and cell-substrate adhesion assays were used to measure alterations in fibrosis-related molecule expression and the cell adhesion ability of frozen shoulder SCFs after treatment with range concentrations of sCT. The presence of calcitonin receptors (CTRs) in shoulder joint synovial/capsular tissue samples was detected by immunohistochemistry (IHC). The downstream pathways of sCT in SCFs were further explored by utilizing three classical pathway inhibitors. With the addition of sCT to the culture medium of frozen shoulder SCFs, the messenger RNA (mRNA) expression of collagen type I (COL1A1), COL3A1, fibronectin 1, laminin 1, transforming growth factor-ß1 (TGF-ß1), and interleukin-1α (IL-1α) showed a descending trend as the sCT concentration increased. Treatment with sCT increased the expression of vascular endothelial growth factor and IL-6 in a dose-dependent manner. The enhanced adhesion ability of frozen shoulder SCFs gradually diminished with increasing concentrations of sCT. By using IHC, the CTR was detected extensively in the frozen shoulder joint synovium and capsule. Blocking the protein kinase C (PKC) pathway reversed the sCT-mediated suppression of COL1A1 production. Blocking the PKC or protein kinase A (PKA) pathway eliminated the sCT-induced inhibition of TGF-ß1 production. This study demonstrated that sCT effectively improved the mRNA expression of fibrosis-related molecules and decreased the enhanced cell-substrate adhesion ability of frozen shoulder SCFs. sCT might achieve these effects by interacting with the CTR that is expressed on the SCF surface and by activating the downstream PKC or PKA pathway.

Calcitonin directly increases adrenocorticotropic hormone-stimulated corticosterone production in the hen adrenal gland.

The present study was performed to elucidate whether the receptor for calcitonin (CT) exists in the adrenocortical cells of hens and to determine the effect of CT on adrenocorticotropic hormone (ACTH)-stimulated corticosterone production in its cell. The binding site of CT in the membrane fraction of the adrenal gland in hens was determined using a [125I]CT binding assay system. The binding properties in the adrenal gland satisfied the criteria of a receptor-ligand interaction in terms of specificity, reversibility, and saturation. When the cortical cells were incubated in vitro with chicken ACTH in the presence of CT, greater corticosterone production was observed. The result suggested that CT acts directly on the adrenocortical cells via its receptor binding and increases responsiveness of ACTH on corticosterone production in the laying hen.

Procalcitonin has bioactivity at calcitonin receptor family complexes: potential mediator implications in sepsis.

CONTEXT: Sepsis is a major cause of death in the United States and accounts for approximately 50% of the fatalities in intensive care units. Serum procalcitonin (ProCT) levels are markedly elevated in sepsis and correlate positively with severity of the illness and mortality, however, little is known about the biological activity of ProCT. OBJECTIVE: To explore the biological activity of purified human ProCT at the calcitonin (CT) family of receptors. DESIGN: Human ProCT was purified from the TT medullary thyroid carcinoma cell line. Human CTa receptor or human CT receptor-like receptor (CLR) was transiently expressed in COS-7 cells alone or together with individual receptor activity-modifying proteins (RAMPs) to generate the CTa (CT) receptor, the AMY1 (amylin) receptor, the CGRP1 (CT gene-related peptide) receptor, and the AM1 and AM2 (adrenomedullin) receptors. Biological activity of ProCT was assessed by measurement of cAMP accumulation. RESULTS: ProCT was effectively inert at CTa, AM1, and AM2 receptors. In contrast, it was a potent partial agonist (50-60% of the CGRP efficacy) of the CGRP1 receptor with an EC50 as high as 0.56 nM, although the potency was batch dependent. ProCT also displayed weak partial agonist activity at the AMY1 receptor with an EC50 of approximately 100 nM. Moreover, ProCT also robustly inhibited CGRP-dependent cyclic adenosine monophosphate responses at the CGRP1 receptor. CONCLUSIONS: Our data provide a potential molecular mechanism for the observation that ProCT appears to be toxic while CGRP treatment appears to be beneficial in animal models of sepsis.

Osteoclast receptors and signaling.

Osteoclasts are bone-resorbing cells derived from hematopoietic precursors of the monocyte-macrophage lineage. Besides the well known Receptor Activator of Nuclear factor-kappaB (RANK), RANK ligand and osteoprotegerin axis, a variety of factors tightly regulate osteoclast formation, adhesion, polarization, motility, resorbing activity and life span, maintaining bone resorption within physiological ranges. Receptor-mediated osteoclast regulation is rather complex. Nuclear receptors, cell surface receptors, integrin receptors and cell death receptors work together to control osteoclast activity and prevent both reduced or increased bone resorption. Here we will discuss the signal transduction pathways activated by the main osteoclast receptors, integrating their function and mechanisms of action.

Transgenic mice with ocular overexpression of an adrenomedullin receptor reflect human acute angle-closure glaucoma.

Glaucoma, frequently associated with high IOP (intra-ocular pressure), is a leading cause of blindness, characterized by a loss of retinal ganglion cells and the corresponding optic nerve fibres. In the present study, acutely and transiently elevated IOP, characteristic of acute angle-closure glaucoma in humans, was observed in CLR (calcitonin receptor-like receptor) transgenic mice between 1 and 3 months of age. Expression of CLR under the control of a smooth muscle alpha-actin promoter in these mice augmented signalling of the smooth-muscle-relaxing peptide adrenomedullin in the pupillary sphincter muscle and resulted in pupillary palsy. Elevated IOP was prevented in CLR transgenic mice when mated with hemizygote adrenomedullin-deficient mice with up to 50% lower plasma and organ adrenomedullin concentrations. This indicates that endogenous adrenomedullin of iris ciliary body origin causes pupillary palsy and angle closure in CLR transgenic mice overexpressing adrenomedullin receptors in the pupillary sphincter muscle. In human eyes, immunoreactive adrenomedullin has also been detected in the ciliary body. Furthermore, the CLR and RAMP2 (receptor-activity-modifying protein 2), constituting adrenomedullin receptor heterodimers, were identified in the human pupillary sphincter muscle. Thus, in humans, defective regulation of adrenomedullin action in the pupillary sphincter muscle, provoked in the present study in CLR transgenic mice, may cause acute and chronic atony and, thereby, contribute to the development of angle-closure glaucoma. The CLR transgenic mice used in the present study provide a model for acute angle-closure glaucoma.

Enhanced vascular responses to adrenomedullin in mice overexpressing receptor-activity-modifying protein 2.

Adrenomedullin (AM) levels are elevated in cardiovascular disease, but little is known of the role of specific receptor components. AM acts via the calcitonin receptor-like receptor (CLR) interacting with a receptor-activity-modifying protein (RAMP). The AM1 receptor is composed of CLR and RAMP2, and the calcitonin gene-related peptide (CGRP) receptor of CLR and RAMP1, as determined by molecular and cell-based analysis. This study examines the relevance of RAMP2 in vivo. Transgenic (TG) mice that overexpress RAMP2 in smooth muscle were generated. The role of RAMP2 in the regulation of blood pressure and in vascular function was investigated. Basal blood pressure, acute angiotensin II-raised blood pressure, and cardiovascular properties were similar in wild-type (WT) and TG mice. However, the hypotensive effect of IV AM, unlike CGRP, was enhanced in TG mice (P<0.05), whereas a negative inotropic action was excluded by left-ventricular pressure-volume analysis. In aorta relaxation studies, TG vessels responded in a more sensitive manner to AM (EC50, 8.0+/-1.5 nmol/L) than WT (EC50, 17.9+/-3.6 nmol/L). These responses were attenuated by the AM receptor antagonist, AM(22-52), such that residual responses were identical in all mice. Remaining relaxations were further inhibited by CGRP receptor antagonists, although neither affected AM responses when given alone. Mesenteric and cutaneous resistance vessels were also more sensitive to AM in TG than WT mice. Thus RAMP2 plays a key role in the sensitivity and potency of AM-induced hypotensive responses via the AM1 receptor, providing evidence that this receptor is a selective target for novel therapeutic approaches.

Immortalization of osteoclast precursors by targeting Bcl -XL and Simian virus 40 large T antigen to the osteoclast lineage in transgenic mice.

Cellular and molecular characterization of osteoclasts (OCL) has been extremely difficult since OCL are rare cells, and are difficult to isolate in large numbers. We used the tartrate-resistant acid phosphatase promoter to target the bcl-XL and/or Simian Virus 40 large T antigen (Tag) genes to cells in the OCL lineage in transgenic mice as a means of immortalizing OCL precursors. Immunocytochemical studies confirmed that we had targeted Bcl-XL and/or Tag to OCL, and transformed and mitotic OCL were readily apparent in bones from both Tag and bcl-XL/Tag mice. OCL formation in primary bone marrow cultures from bcl-XL, Tag, or bcl-XL/Tag mice was twofold greater compared with that of nontransgenic littermates. Bone marrow cells from bcl-XL/Tag mice, but not from singly transgenic bcl-XL or Tag mice, have survived in continuous culture for more than a year. These cells form high numbers of bone-resorbing OCL when cultured using standard conditions for inducing OCL formation, with approximately 50% of the mononuclear cells incorporated into OCL. The OCL that form express calcitonin receptors and contract in response to calcitonin. Studies examining the proliferative capacity and the resistance of OCL precursors from these transgenic mice to apoptosis demonstrated that the increased numbers of OCL precursors in marrow from bcl-XL/Tag mice was due to their increased survival rather than an increased proliferative capacity compared with Tag, bcl-XL, or normal mice. Histomorphometric studies of bones from bcl-XL/Tag mice also confirmed that there were increased numbers of OCL precursors (TRAP + mononuclear cells) present in vivo. These data demonstrate that by targeting both bcl-XL and Tag to cells in the OCL lineage, we have immortalized OCL precursors that form bone-resorbing OCL with an efficiency that is 300-500 times greater than that of normal marrow.

Calcitonin receptor-stimulated migration of prostate cancer cells is mediated by urokinase receptor-integrin signaling.

Abundance of calcitonin (CT) and calcitonin receptor (CTR) mRNA in primary prostate tumors positively correlates with tumor grade, and exogenously added CT increases the invasion of prostate cancer cell lines. We examined acute and chronic actions of CT on migration of highly metastatic PC-3M cells and poorly invasive LNCaP cells on several extracellular matrices in a spheroid disaggregation/migration assay. While PC-3M spheroids displayed maximum disaggregation/migration on vitronectin (VN), LNCaP spheroids preferred collagen but also migrated significantly on VN. Up-regulation of CT significantly enhanced disaggregation/migration of PC-3M spheroids on VN, but not on fibronectin. In contrast, down-regulation of CT, CTR, protein kinase A or urokinase-type plasminogen activator receptor (uPAR) led to amelioration of PC-3M spheroid disaggregation/migration. CT selectively increased surface activity of alpha v beta 3 or alpha 6 beta 5 integrins in PC-3M and LNCaP cell lines, respectively, and uPAR-integrin association. Finally, either CT or urokinase could completely restore migration of CT-knock-down PC-3M spheroids. But, only forced expression of urokinase receptor coupled with exogenous addition of urokinase restored migration of CTR-knock-down spheroids. These results support our hypothesis that up-regulation of CT biosynthesis and activation of CT-CTR axis in primary prostate tumors may have direct relevance in their progression to the metastatic phenotype.

Adrenomedullin in the kidney-renal physiological and pathophysiological roles.

Adrenomedullin (AM) is a potent vasodilatory peptide originally discovered in human pheochromocytoma tissue. AM and AM gene expression are widely distributed in the cardiovascular system, including the kidney. The co-localization of AM and its receptor components such as calcitonin receptor-like receptor (CRLR), receptor activity modifying protein (RAMP)2 and RAMP3 in the kidney, heart, and vasculature suggests an important role for the peptide as a regulator of renal, cardiac, and vascular function. Indeed, in addition to its cardiovascular effects, AM has renal vasodilatory, natriuretic, and diuretic actions. Consistent with these observations, immunohistochemical studies revealed that AM is stained in the collecting duct, distal convoluted tubules, vessels, and glomerular mesangial cells, endothelial cells and podocytes. Plasma AM levels are increased in patients with renal impairment in proportion to the severity of the disease. Previously we and other investigators showed that two molecular forms of AM, AM-glycine, an inactive form, and AM-mature, an active form, circulate in human plasma. Urine also contains both forms of AM; however, the AM-mature/AM-glycine ratio is higher in urine than in plasma. Interestingly, plasma AM-glycine and AM-mature levels are increased in renal failure, whereas urinary AM-glycine and AM-mature are decreased in this condition. These results indicate that the origin of urinary AM is different from that of plasma AM. Experimental studies showed that the renal tissue AM-mature/AM-glycine ratio is higher than that in plasma and urine. In addition, renal tissue concentrations of AM are increased in severely hypertensive rats. Considering that AM has antiapoptotic, antifibrotic, and antiproliferative effects, the increase of AM in renal disease may be a protective mechanism. In fact, AM gene delivery or long-term AM infusion significantly improved glomerular sclerosis, interstitial fibrosis, and renal arteriosclerosis in several malignant hypertensive models. This review describes the biochemistry, physiology, and circulating levels of AM and also discusses what is known about the pathophysiological role of AM in renal disease.

Receptors as microprocessors: pharmacological nuance on metabotropic glutamate receptors 1alpha.

G protein-coupled receptors have revealed themselves to be complex information-processing units that may be exploited for subtle therapeutic signaling effects. Thus, ligands may not only turn receptors on and off, but may also select from their repertoire of signaling effects to further refine drug response.

Calcitonin gene-related peptide receptor activation by receptor activity-modifying protein-1 gene transfer to vascular smooth muscle cells.

The neuropeptide calcitonin gene-related peptide (CGRP) is a potent vasodilator that plays a protective role in the cardiovascular system. The receptor for CGRP is an unusual complex of the G protein-coupled calcitonin-like receptor and an obligate receptor activity modifying protein-1 (RAMP1). In this report we provide the first evidence that RAMP1 is rate limiting in vascular smooth muscle cells. Although cultured rat aorta smooth muscle cells express calcitonin like-receptor and RAMP1, we found that CGRP is not a potent activator of the receptor. After overexpression of RAMP1 by adenoviral gene transfer, there was a striking increase in CGRP-induced production of cAMP, with a 75-fold decrease in the EC(50) and a 1.5-fold increase in the maximal response. The biological consequence of this increased receptor activity was observed in three different paradigms. First, RAMP1 gene transfer caused a CGRP-dependent decrease in cell proliferation. Second, RAMP1 and CGRP treatment led to a 3-fold greater free radical-induced reduction in cell number. Finally, RAMP1 gene transfer resulted in a 5-fold CGRP-dependent increase in terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling-positive apoptotic cells upon serum withdrawal. The mechanisms underlying these effects involved cAMP-dependent pathways. We propose that RAMP1 gene transfer may be an effective strategy for increasing the effectiveness of CGRP-induced decrease in restenosis after aortic angioplasty.