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The calcium-sensing receptor (CaSR) drives essential calcium ion (Ca 2+ ) and E-cadherin‒mediated processes in the epidermis, including differentiation, cell-to-cell adhesion, and epidermal barrier homeostasis in cells and in young adult mice. We now report that decreased CaSR expression leads to impaired Ca 2+ signal propagation in aged mouse (aged >22 months) epidermis and human (aged >79 years, donor age) keratinocytes. Baseline cytosolic Ca 2+ concentrations were higher, and capacitive Ca 2+ entry was lower in aged than in young keratinocytes. As in Casr-knockout mice ( Epid CaSR -/- ), decreased CaSR expression led to decreased E-cadherin and phospholipase C-γ expression and to a compensatory upregulation of STIM1. Pretreatment with the CaSR agonist N-(3-[2-chlorophenyl]propyl)-(R)-alpha-methyl-3-methoxybenzylamine normalized Ca 2+ propagation and E-cadherin organization after experimental wounding. These results suggest that age-related defects in CaSR expression dysregulate normal keratinocyte and epidermal Ca 2+ signaling, leading to impaired E-cadherin expression, organization, and function. These findings show an innovative mechanism whereby Ca 2+ - and E-cadherin‒dependent functions are impaired in aging epidermis and suggest a new therapeutic approach by restoring CaSR function., (Published by Elsevier Inc.)

High-intensity sound often leads to the dysfunction and impairment of central nervous system (CNS), but the underlying mechanism is unclear. The present study was aimed to investigate the related mechanisms of CNS lesions in Bama miniature pig model treated with high-intensity sound. The pigs with normal hearing were divided into control and high-intensity sound (900 Hz-142 dB SPL, 15 min) groups. After the treatment, hippocampi were collected immediately. Fluo-4 was used to indicate intracellular Ca 2+ concentration ([Ca 2+ ] i ) change. Real-time PCR and Western blot were used to detect mRNA and protein expressions of calcium-sensing receptor, L-Ca 2+ channel α2/δ1 subunit, PKC and PI3K, respectively. DAPI staining was used to identify nuclear features. The result showed that high-intensity sound exposure resulted in significantly swollen cell nucleus and increased [Ca 2+ ] i in hippocampal cells. Compared with control group, high-intensity sound group showed increased levels of PI3K, PKC and L-Ca 2+ channel α2/δ1 subunit mRNA expressions, as well as up-regulated PKC and calcium-sensing receptor protein expressions. These results suggest that the high-intensity sound activates PKC signaling pathway and induces calcium overload, eventually leads to hippocampal injury, which would supply a novel strategy to prevent nervous system from high-intensity sound-induced injury.

The calcium-sensing receptor (CaSR) couples to signalling pathways via intracellular loops 2 and 3, and the C-terminus. However, the requirements for signalling are largely undefined. We investigated the impacts of selected point mutations in iL-2 (F706A) and iL-3 (L797A and E803A), and a truncation of the C-terminus (R866X) on extracellular Ca(2+) (Ca(2+)o)-stimulated phosphatidylinositol-specific phospholipase-C (PI-PLC) and various other signalling responses. CaSR-mediated activation of PI-PLC was markedly attenuated in all four mutants and similar suppressions were observed for Ca(2+)o-stimulated ERK1/2 phosphorylation. Ca(2+)o-stimulated intracellular Ca(2+) (Ca(2+)i) mobilization, however, was relatively preserved for the iL-2 and iL-3 mutants and suppression of adenylyl cyclase was unaffected by either E803A or R866X. The CaSR selects for specific signalling pathways via the proximal C-terminus and key residues in iL-2, iL-3., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The calcium-sensing receptor (CaSR) has played an important role as a target in the treatment of a variety of disease states over the past 20 plus years. In this review, we give an overview of the receptor at the cellular level and then provide details as to how this receptor has been targeted to modulate cellular ion transport mechanisms. As a member of the G protein-coupled receptor (GPCR) family, it has a high degree of homology with a variety of other members in this class, which could explain why this receptor has been identified in so many different tissues throughout the body. This diversity of locations sets it apart from other members of the family and may explain how the receptor interacts with so many different organ systems in the body to modulate the physiology and pathophysiology. The receptor is unique in that it has two large exofacial lobes that sit in the extracellular environment and sense changes in a wide variety of environmental cues including salinity, pH, amino acid concentration, and polyamines to name just a few. It is for this reason that there has been a great deal of research associated with normal receptor physiology over the past 20 years. With the ongoing research, in more recent years a focus on the pathophysiology has emerged and the effects of receptor mutations on cellular and organ physiology have been identified. We hope that this review will enhance and update the knowledge about the importance of this receptor and stimulate future potential investigations focused around this receptor in cellular, organ, and systemic physiology and pathophysiology., (Copyright © 2014 the American Physiological Society.)

This paper aims to investigate the effect of store-operated calcium channels (SOC) and receptor-operated calcium channels (ROC) on Ca(2+)-sensing receptor (CaR)-induced extracellular Ca(2+) influx and nitric oxide (NO) generation in human umbilical vein endothelial cells (HUVEC). SOC blocker, non-selective cation channel blocker, ROC agonist and ROC blocker were used separately and combined. Intracellular Ca(2+) concentration ([Ca(2+)]i) was measured by Fura-2/AM loading. The activity of endothelial nitric oxide synthase (eNOS) and the production of NO were determined by the DAF-FM diacetate (DAF-FM DA). The results showed that increases of [Ca(2+)]i, eNOS activity and NO generation induced by CaR agonist Spermine were all reduced after single blocking the SOC or ROC, respectively (P < 0.05). ROC agonist can partially abolish the ROC blocker's effect (P < 0.05). The above mentioned effects evoked by CaR agonist Spermine were further reduced when blocking both SOC and ROC than single blocking SOC or ROC in HUVEC (P < 0.05). In conclusion, these results suggest that the SOC and ROC participate in the processes of CaR-evoked extracellular Ca(2+) influx and NO generation by a synergistic manner in HUVEC.

Maintaining a constant level of blood Ca(2+) is essential because of calcium's myriad intracellular and extracellular roles. The CaSR plays key roles in maintaining [Formula: see text] homeostasis by detecting small changes in blood Ca(2+) and modulating the production/secretion of the Ca(2+)-regulating hormones, PTH, CT, FGF23 and 1,25(OH)2D3, so as to appropriately regulate Ca(2+) transport into or out of blood via kidney, intestine, and/or bone. When Ca(2+) is high, the CaSR suppresses PTH synthesis and secretion, promotes its degradation, and inhibits parathyroid cellular proliferation. It has just the opposite effects on the C-cell, stimulating CT when [Formula: see text] is high. In bone, Ca(2+), acting via the CaSR, stimulates recruitment and proliferation of preosteoblasts, their differentiation to mature osteoblasts, and synthesis and mineralization of bone proteins. Conversely, [Formula: see text] inhibits the formation and activity and promotes apoptosis of osteoclasts, likely via the CaSR. These actions tend to mobilize skeletal Ca(2+) during [Formula: see text] deficiency and retain it when Ca(2+) is plentiful., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The calcium sensing receptor (CaSR) is expressed by subpopulations of neuronal and glial cells throughout the brain and is activated by extracellular calcium [Formula: see text] . During development, the CaSR regulates neuronal cell growth and migration as well as oligodendroglial maturation and function. Emerging evidence suggests that in nerve terminals, CaSR is implicated in synaptic plasticity and neurotransmission. In this review, we analyze the roles attributed to CaSR in regulating diverse brain functions, including central regulation of body fluid composition and blood pressure. We also discuss the potential relevance of Ca(2+)-sensing in brain by other family C G protein-coupled receptors. Finally, evidence that the CaSR contributes to the pathogenesis of various brain disorders raises the possibility that pharmacological modulators of the CaSR may have therapeutic benefit., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The epidermis is a stratified squamous epithelium composed of proliferating basal and differentiated suprabasal keratinocytes. It serves as the body's major physical and chemical barrier against infection and harsh environmental insults, as well as preventing excess water loss from the body into the atmosphere. Calcium is a key regulator of the proliferation and differentiation in keratinocytes. Elevated extracellular Ca(2+) concentration ([Ca(2+)]o) raises the levels of intracellular free calcium ([Ca(2+)]i), promotes cell-cell adhesion, and activates differentiation-related genes. Keratinocytes deficient in the calcium-sensing receptor fail to respond to [Ca(2+)]o stimulation and to differentiate, indicating a role for the calcium-sensing receptor in transducing the [Ca(2+)]o signal during differentiation. The concepts derived from in vitro gene knockdown experiments have been evaluated and confirmed in three mouse models in vivo., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In fetal mammals, serum levels of both total and ionized calcium significantly exceed those in the adult. This relative fetal hypercalcemia is crucial for skeletal development and is maintained irrespectively of maternal serum calcium levels. Elegant studies by Kovacs and Kronenberg have previously addressed the role of the CaSR in creating and maintaining this relative fetal hypercalcemia, through the regulation of parathyroid hormone-related peptide secretion. More recently we have shown that the CaSR is widely distributed throughout the developing fetus, where the receptor plays major, unexpected roles in ensuring growth and maturation of several organs. In this article, we present evidence for a role of the CaSR in the control of skeletal development, and how fetal hypercalcemia, acting through the CaSR, regulates lung development., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Rationale: An increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for PASMC proliferation and pulmonary vascular remodeling. The dihydropyridine Ca(2+) channel blockers, such as nifedipine, have been used for treatment of idiopathic pulmonary arterial hypertension (IPAH)., Objective: Our previous study demonstrated that the Ca(2+)-sensing receptor (CaSR) was upregulated and the extracellular Ca(2+)-induced increase in [Ca(2+)](cyt) was enhanced in PASMC from patients with IPAH and animals with experimental pulmonary hypertension. Here, we report that the dihydropyridines (eg, nifedipine) increase [Ca(2+)](cyt) by activating CaSR in PASMC from IPAH patients (in which CaSR is upregulated), but not in normal PASMC., Methods and Results: The nifedipine-mediated increase in [Ca(2+)](cyt) in IPAH-PASMC was concentration dependent with a half maximal effective concentration of 0.20 µmol/L. Knockdown of CaSR with siRNA in IPAH-PASMC significantly inhibited the nifedipine-induced increase in [Ca(2+)](cyt), whereas overexpression of CaSR in normal PASMC conferred the nifedipine-induced rise in [Ca(2+)](cyt). Other dihydropyridines, nicardipine and Bay K8644, had similar augmenting effects on the CaSR-mediated increase in [Ca(2+)](cyt) in IPAH-PASMC; however, the nondihydropyridine blockers, such as diltiazem and verapamil, had no effect on the CaSR-mediated rise in [Ca(2+)](cyt)., Conclusions: The dihydropyridine derivatives increase [Ca(2+)](cyt) by potentiating the activity of CaSR in PASMC independently of their blocking (or activating) effect on Ca(2+) channels; therefore, it is possible that the use of dihydropyridine Ca(2+) channel blockers (eg, nifedipine) to treat IPAH patients with upregulated CaSR in PASMC may exacerbate pulmonary hypertension.

The extracellular calcium ([Formula: see text])-sensing receptor (CaSR) was the first GPCR identified whose principal physiological ligand is an ion, namely extracellular Ca(2+). It maintains the near constancy of [Formula: see text] that complex organisms require to ensure normal cellular function. A wealth of information has accumulated over the past two decades about the CaSR's structure and function, its role in diseases and CaSR-based therapeutics. This review briefly describes the CaSR and key features of its structure and function, then discusses the extracellular signals modulating its activity, provides an overview of the intracellular signaling pathways that it controls, and, finally, briefly describes CaSR signaling both in tissues participating in [Formula: see text] homeostasis as well as those that do not. Factors controlling CaSR signaling include various factors affecting the expression of the CaSR gene as well as modulation of its trafficking to and from the cell surface. The dimeric cell surface CaSR, in turn, links to various heterotrimeric and small molecular weight G proteins to regulate intracellular second messengers, lipid kinases, various protein kinases, and transcription factors that are part of the machinery enabling the receptor to modulate the functions of the wide variety of cells in which it is expressed. CaSR signaling is impacted by its interactions with several binding partners in addition to signaling elements per se (i.e., G proteins), including filamin-A and caveolin-1. These latter two proteins act as scaffolds that bind signaling components and other key cellular elements (e.g., the cytoskeleton). Thus CaSR signaling likely does not take place randomly throughout the cell, but is compartmentalized and organized so as to facilitate the interaction of the receptor with its various signaling pathways.

The kidney handles calcium by filtration and reabsorption. About 60% of the plasma calcium is filterable, and 99% is reabsorbed in the tubule. In the proximal tubule, the reabsorption is passive and paracellular, but in the distal tubule is active and transcellular. Thus, renal tubular cells are exposed to very high concentrations of calcium in both, the extracellular and the intracellular compartments. Extracellular calcium signaling is transmitted by the calcium sensing receptor, located both in the luminal and basolateral sides of tubular cells. This receptor is able to control levels of extracellular calcium and acts in consequence to maintain calcium homeostasis. Furthermore, renal tubular cells possess several calcium channels that regulate some of the cell functions. Among those, voltage gated calcium channels, transient receptor potential channels and N-methyl-D-aspartate receptor channels have been reported to control several functions. Those functions include survival, apoptosis, differentiation, epithelial-mesenchymal transition, and active vitamin D and renin synthesis.

Spontaneous release of glutamate is important for maintaining synaptic strength and controlling spike timing in the brain. Mechanisms regulating spontaneous exocytosis remain poorly understood. Extracellular calcium concentration ([Ca(2+)](o)) regulates Ca(2+) entry through voltage-activated calcium channels (VACCs) and consequently is a pivotal determinant of action potential-evoked vesicle fusion. Extracellular Ca(2+) also enhances spontaneous release, but via unknown mechanisms. Here we report that external Ca(2+) triggers spontaneous glutamate release more weakly than evoked release in mouse neocortical neurons. Blockade of VACCs has no effect on the spontaneous release rate or its dependence on [Ca(2+)](o). Intracellular [Ca(2+)] slowly increases in a minority of neurons following increases in [Ca(2+)](o). Furthermore, the enhancement of spontaneous release by extracellular calcium is insensitive to chelation of intracellular calcium by BAPTA. Activation of the calcium-sensing receptor (CaSR), a G-protein-coupled receptor present in nerve terminals, by several specific agonists increased spontaneous glutamate release. The frequency of spontaneous synaptic transmission was decreased in CaSR mutant neurons. The concentration-effect relationship for extracellular calcium regulation of spontaneous release was well described by a combination of CaSR-dependent and CaSR-independent mechanisms. Overall these results indicate that extracellular Ca(2+) does not trigger spontaneous glutamate release by simply increasing calcium influx but stimulates CaSR and thereby promotes resting spontaneous glutamate release.

The calcium-sensing receptor (CaR) is the major sensor and regulator of extracellular Ca(2+), whose activity is allosterically regulated by amino acids and pH. Recently, CaR has been identified in the stomach and intestinal tract, where it has been proposed to function in a non-Ca(2+) homeostatic capacity. Luminal nutrients, such as Ca(2+) and amino acids, have been recognized for decades as potent stimulants for gastrin and acid secretion, although the molecular basis for their recognition remains unknown. The expression of CaR on gastrin-secreting G cells in the stomach and their shared activation by Ca(2+), amino acids, and elevated pH suggest that CaR may function as the elusive physiologic sensor regulating gastrin and acid secretion. The genetic and pharmacologic studies presented here comparing CaR-null mice and wild-type littermates support this hypothesis. Gavage of Ca(2+), peptone, phenylalanine, Hepes buffer (pH 7.4), and CaR-specific calcimimetic, cinacalcet, stimulated gastrin and acid secretion, whereas the calcilytic, NPS 2143, inhibited secretion only in the wild-type mouse. Consistent with known growth and developmental functions of CaR, G-cell number was progressively reduced between 30 and 90 d of age by more than 65% in CaR-null mice. These studies of nutrient-regulated G-cell gastrin secretion and growth provide definitive evidence that CaR functions as a physiologically relevant multimodal sensor. Medicinals targeting diseases of Ca(2+) homeostasis should be reviewed for effects outside traditional Ca(2+)-regulating tissues in view of the broader distribution and function of CaR.

Breast cancer is the most frequent form of cancer in women, with the highest incidence of metastasis to the bone. The reason for the preferential destination to the bone is believed to be due to chemoattractant factors released during bone resorption, which act on the cancer cells facilitating their metastasis. One of the factors released during osteolysis that may mediate breast cancer bone localization is Ca2+. Here, we show that extracellular Ca2+ (Ca2+(o)) acting via the calcium-sensing receptor (CaSR), greatly promotes the migration of bone-preferring breast cancer cells. In Boyden Chamber and Scratch Wound migration assays, an increase in breast cancer cell migration was observed at 2.5 mM and 5 mM Ca2+(o) compared to basal levels for three of the four breast cancer cell lines tested. However, a significantly greater migratory response was observed for the highly bone metastatic MDA-MB-231 cells, compared to the MCF7 and T47D, which have a lower metastatic potential in vivo. The BT474 cells, which do not metastasize to the bone, did not respond to elevated concentrations of Ca2+(o) in the migration assays. Inhibition of either ERK1/2 MAPK or phospholipase Cbeta (PLCbeta) led to an abolition of the Ca2+(o)-induced migration, implicating these pathways in the migratory response. Knockdown of the CaSR by siRNA resulted in an inhibition of the Ca2+(o)-induced migration, demonstrating the involvement of this receptor in the effect. These results suggest that the activation of the CaSR by elevated Ca2+(o) concentrations, such as those found near resorbing bone, produces an especially strong chemoattractant effect on bone metastatic breast cancer cells toward the Ca2+-rich environment.

The extracellular calcium-sensing receptor (CaR) is a class III G-protein coupled receptor that coordinates cellular responses to changes in extracellular free Ca(2+) or amino acid concentrations as well as ionic strength and pH. It regulates signalling cascades via recruiting and controlling the activities of various heterotrimeric G-proteins, including G(q/11), G(i/0), and G(12/13), even G(s) in some "unusual" circumstances, thereby inducing changes in the metabolism of membrane lipids, the phosphorylation state of protein kinases and their targets, the activation state of monomeric G-proteins and the levels of intracellular second messengers including cAMP, Ca(2+) ions, fatty acids and other small molecules. According to its site(s) of expression and available signalling pathways, the CaR modulates cell proliferation and survival, differentiation, peptide hormone secretion, ion and water transport and various other processes. In this article we consider the complex intracellular mechanisms by which the CaR elicits its cellular functions. We also consider some of the better understood CaR-regulated cell functions and the nature of the signalling mechanisms that support them.

The past several decades of research into calcium signaling have focused on intracellular calcium (Ca(i)(2+)), revealing both exquisite spatial and dynamic control of this potent second messenger. Our understanding of Ca(i)(2+) signaling has benefited from the evolution of cell culture methods, development of high affinity fluorescent calcium indicators (both membrane-permeant small molecules and genetically encoded proteins), and high-resolution fluorescence microscopy. As our understanding of single cell calcium dynamics has increased, translational efforts have attempted to push calcium signaling studies back into tissues, organs and whole animals. Emerging results from these more complicated, diffusion-limited systems have begun to define a role for extracellular calcium (Ca(o)(2+)) as an agonist, spurred by the cloning and characterization of a G protein-coupled receptor activated by Ca(o)(2+) (the calcium sensing receptor, CaR). Here, we review the current state-of-the art for measurement of Ca(o)(2+) fluctuations, and the evidence that fluctuations in Ca(o)(2+) can act as primary signals regulating cell function. Current results suggest that Ca(o)(2+) in bone and epidermis may act as a chemotactic homing signal, targeting cells to the appropriate tissue locations prior to initiation of the differentiation program. Ca(i)(2+) signaling-mediated Ca(o)(2+) fluctuations in interstitial spaces may integrate cell signaling responses in multicellular networks through activation of CaR. Appreciation of the importance of Ca(o)(2+) fluctuations in coordinating cell function will likely spur identification of additional, niche-specific Ca(2+) sensors, and provide unique insights into the regulation of multicellular signaling networks.

The study by Tu et al. in this issue details the surprising finding that not only is the CaR active intracellularly but also its intracellular localization includes both the ER, traditionally thought of as the main intracellular Ca2+ signaling store, and the Golgi, more recently shown to be important in keratinocyte signaling.

Calcium transport and calcium signalling mechanisms in bone cells have, in many cases, been discovered by study of diseases with disordered bone metabolism. Calcium matrix deposition is driven primarily by phosphate production, and disorders in bone deposition include abnormalities in membrane phosphate transport such as in chondrocalcinosis, and defects in phosphate-producing enzymes such as in hypophosphatasia. Matrix removal is driven by acidification, which dissolves the mineral. Disorders in calcium removal from bone matrix by osteoclasts cause osteopetrosis. On the other hand, although bone is central to management of extracellular calcium, bone is not a major calcium sensing organ, although calcium sensing proteins are expressed in both osteoblasts and osteoclasts. Intracellular calcium signals are involved in secondary control including cellular motility and survival, but the relationship of these findings to specific diseases is not clear. Intracellular calcium signals may regulate the balance of cell survival versus proliferation or anabolic functional response as part of signalling cascades that integrate the response to primary signals via cell stretch, estrogen, tyrosine kinase, and tumor necrosis factor receptors.

The mammary gland is subjected to extensive calcium loads during lactation to support the requirements of milk calcium enrichment. Despite the indispensable nature of calcium homeostasis and signaling in regulating numerous biological functions, the mechanisms by which systemic calcium is transported into milk by the mammary gland are far from completely understood. Furthermore, the implications of calcium signaling in terms of regulating proliferation, differentiation and apoptosis in the breast are currently uncertain. Deregulation of calcium homeostasis and signaling is associated with mammary gland pathophysiology and as such, calcium transporters, channels and binding proteins represent potential drug targets for the treatment of breast cancer.

Termination of cyclic adenosine monophosphate (cAMP) signaling via the extracellular Ca(2+)-sensing receptor (CaR) was visualized in single CaR-expressing human embryonic kidney (HEK) 293 cells using ratiometric fluorescence resonance energy transfer-dependent cAMP sensors based on protein kinase A and Epac. Stimulation of CaR rapidly reversed or prevented agonist-stimulated elevation of cAMP through a dual mechanism involving pertussis toxin-sensitive Galpha(i) and the CaR-stimulated increase in intracellular [Ca2+]. In parallel measurements with fura-2, CaR activation elicited robust Ca2+ oscillations that increased in frequency in the presence of cAMP, eventually fusing into a sustained plateau. Considering the Ca2+ sensitivity of cAMP accumulation in these cells, lack of oscillations in [cAMP] during the initial phases of CaR stimulation was puzzling. Additional experiments showed that low-frequency, long-duration Ca2+ oscillations generated a dynamic staircase pattern in [cAMP], whereas higher frequency spiking had no effect. Our data suggest that the cAMP machinery in HEK cells acts as a low-pass filter disregarding the relatively rapid Ca2+ spiking stimulated by Ca(2+)-mobilizing agonists under physiological conditions.

Calcium acts as a universal signal that is responsible for controlling a spectrum of cellular processes ranging from fertilization to apoptosis. For a long time, calcium was regarded solely as an intracellular second messenger. However, the discovery that calcium can also act as an external ligand together with the molecular cloning of its cell surface receptor, the Calcium Sensing Receptor (CaSR), demonstrated that calcium also acts as an important extracellular or first messenger. Here, we give an overview of the main structural, pharmacological and physiological features of the CaSR and provide an assessment of its functions and cellular and molecular mechanisms of action. In addition, we propose possible avenues for future research into the trafficking of CaSR and the role(s) of this receptor in the central nervous system.

Secretion of alpha-melanophore-stimulating hormone (alpha-MSH) from the neuroendocrine melanotrope cells in the intermediate lobe of the pituitary gland of the clawed frog Xenopus laevis is regulated by various inhibitory, stimulatory and autocrine factors. The neuropeptide sauvagine stimulates alpha-MSH secretion by changing the pattern of intracellular Ca2+ oscillations and the electrical properties of the cell membrane. In the present study we investigated whether another secreto-stimulator, the extracellular Ca2+-sensing receptor (CaR), also affects the Ca2+ oscillatory pattern and electrical membrane properties. Using high-speed dynamic video-imaging we show that activation of the CaR with the specific agonist l-phenylalanine (l-Phe) changes the Ca2+ oscillatory pattern by increasing the number of Ca2+ steps, which are the "building blocks" of the oscillations. Moreover, using patch-clamp electrophysiology it is demonstrated that l-Phe affects membrane properties by increasing frequency and duration of action currents. Compared to sauvagine, the CaR has different effects on the action current parameters, suggesting that multiple mechanisms regulate the electrical properties of the melanotrope cell membrane and, thereby, the Ca2+ oscillation-dependent level of alpha-MSH secretion.

Genetically encoded signaling proteins provide remarkable opportunities to design and target the expression of molecules that can be used to report critical cellular events in vivo, thereby markedly extending the scope and physiological relevance of studies of cell function. Here we report the development of a transgenic mouse expressing such a reporter and its use to examine postsynaptic signaling in smooth muscle. The circularly permutated, Ca2+-sensing molecule G-CaMP (Nakai, J., Ohkura, M., and Imoto, K. (2001) Nat. Biotechnol. 19, 137-141) was expressed in vascular and non-vascular smooth muscle and functioned as a lineage-specific intracellular Ca2+ reporter. Detrusor tissue from these mice was used to identify two separate types of postsynaptic Ca2+ signals, mediated by distinct neurotransmitters. Intrinsic nerve stimulation evoked rapid, whole-cell Ca2+ transients, or "Ca2+ flashes," and slowly propagating Ca2+ waves. We show that Ca2+ flashes occur through P2X receptor stimulation and ryanodine receptor-mediated Ca2+ release, whereas Ca2+ waves arise from muscarinic receptor stimulation and inositol trisphosphate-mediated Ca2+ release. The distinct ionotropic and metabotropic postsynaptic Ca2+ signals are related at the level of Ca2+ release. Importantly, individual myocytes are capable of both postsynaptic responses, and a transition between Ca2+ -induced Ca2+ release and inositol trisphosphate waves occurs at higher synaptic inputs. Ca2+ signaling mice should provide significant advantages in the study of processive biological signaling.

This study documents the detailed biochemical, structural, and functional identity of a novel Ca(2+)-modulated membrane guanylate cyclase transduction system in the inner retinal neurons. The guanylate cyclase is the previously characterized ROS-GC1 from the photoreceptor outer segments (PROS), and its new modulator is neurocalcin delta. At the membrane, the myristoylated form of neurocalcin delta senses submicromolar increments in free Ca(2+), binds to its specific ROS-GC1 domain, and stimulates the cyclase. Neurocalcin delta is not present in PROS, indicating the absence of the pathway in the outer segments and the dissociation of its linkage with phototransduction. Thus, the pathway is linked specifically with the visual transduction machinery in the secondary neurons of the retina. With the inclusion of this pathway, the findings broaden the understanding of the existing mechanisms showing how ROS-GC1 is able to receive and transduce diverse Ca(2+) signals into the cell-specific generation of second-messenger cyclic GMP in the retinal neurons.

Ca2+ influx through L-type Cav1.2 (alpha1C) Ca2+ channels is a critical step in the activation of cardiac ryanodine receptors (RyRs) and release of Ca2+ via Ca2+-induced Ca2+ release(CICR). The released Ca2+, in turn, is the dominant determinant of inactivation of the Ca2+ current (ICa) and termination of release. Although Ca2+ cross-signalling is mediated by high Ca2+ fluxes in the microdomains of alpha1C-RyR complexes, ICa-gated Ca2+ cross-signalling is surprisingly resistant to intracellular Ca2+ buffering and has steeply voltage-dependent gain, inconsistent with a strict CICR mechanism, suggesting the existence of additional regulatory step(s). To explore the possible regulatory role of the carboxyl (C)-terminal tail of alpha1C in modulating Ca2+ signalling, we tested the effects of introducing two alpha1C C-terminal peptides, LA (1571-1599) and K (1617-1636) on the central alpha1C-unassociated Ca2+-release sites of atrial myocytes, using rapid (240 Hz) two-dimensional confocal Ca2+ imaging. The frequency of spontaneously activating central sparks increased by approximately fourfold on dialysing LA- but not K-peptide into myocytes voltage-clamped at -80 mV. The rate but not the magnitude of caffeine (10 mM)-triggered central Ca2+ release was significantly accelerated by LA- but not K-peptide. Individual Ca2+ spark size and flux were larger in LA- but not in K-peptide-dialysed myocytes. Although LA-peptide did not change the amplitude or inactivation kinetics of ICa, LA-peptide did strongly enhance the central Ca2+ transients triggered by ICa at -30 mV (small ICa) but not at +20 mV (large ICa). In contrast, K-peptide had no effect on either ICa or the local Ca2+ transients. LA-peptide with a deleted calmodulin-binding region (LM1-peptide) had no significant effects on the central spark frequency but suppressed spontaneous spark frequency in the periphery. Our results indicate that the calmodulin-binding LA motif of the alpha1C C-terminal tail may sensitize the RyRs, thereby increasing their open probability and providing for both the voltage-dependence of CICR and the higher frequency of spark occurrence in the periphery of atrial myocytes where the native alpha1C-RyR complexes are intact.