L-type amino acids stimulate gastric acid secretion by activation of the calcium-sensing receptor in parietal cells

Parietal cells are the primary acid secretory cells of the stomach. We have previously shown that activation of the calcium-sensing receptor (CaSR) by divalent (Ca(2+)) or trivalent (Gd(3+)) ions stimulates acid production in the absence of secretagogues by increasing H(+),K(+)-ATPase activity. When overexpressed in HEK-293 cells, the CaSR can be allosterically activated by L-amino acids in the presence of physiological concentrations of extracellular Ca(2+) (Ca(o)(2+); 1.5-2.5 mM). To determine whether the endogenously expressed parietal cell CaSR is allosterically activated by L-amino acids, we examined the effect of the amino acids L-phenylalanine (L-Phe), L-tryptophan, and L-leucine on acid secretion. In ex vivo whole stomach preparations, exposure to L-Phe resulted in gastric luminal pH significantly lower than controls. Studies using D-Phe (inactive isomer) failed to elicit a response on gastric pH. H(+)-K(+)-ATPase activity was monitored by measuring the intracellular pH (pH(i)) of individual parietal cells in isolated rat gastric glands and calculating the rate of H(+) extrusion. We demonstrated that increasing Ca(o)(2+) in the absence of secretagogues caused a dose-dependent increase in H(+) extrusion. These effects were amplified by the addition of amino acids at various Ca(o)(2+) concentrations. Blocking the histamine-2 receptor with cimetidine or inhibiting system L-amino acid transport with 2-amino-2-norbornane-carboxylic acid did not affect the rate of H(+) extrusion in the presence of L-Phe. These data support the conclusion that amino acids, in conjunction with a physiological Ca(o)(2+) concentration, can induce acid secretion independent of hormonal stimulation via allosteric activation of the stomach CaSR.     

The intact strontium ranelate complex stimulates osteoblastogenesis and suppresses osteoclastogenesis by antagonizing NF-κB activation

Strontium ranelate, a pharmaceutical agent shown in clinical trials to be effective in managing osteoporosis and reducing fracture risk in postmenopausal women, is relatively unique in its ability to both blunt bone resorption and stimulate bone formation. However, its mechanisms of action are largely unknown. As the nuclear factor-kappa B (NF-κB) activation antagonists both stimulate osteoblastic bone formation and repress osteoclastic bone resorption, we hypothesized that strontium ranelate may achieve its anabolic and anti-catabolic activities by modulating NF-κB activation in bone cells. In this study, osteoclast and osteoblast precursors were treated with intact strontium ranelate or its individual components sodium ranelate and/or strontium chloride, and its effect on in vitro osteoclastogenesis and osteoblastogenesis and on NF-κB activation quantified. Although the activity of strontium ranelate has been attributed to the release of strontium ions, low dose intact strontium ranelate complex, but not sodium ranelate and/or strontium chloride, potently antagonized NF-κB activation in osteoclasts and osteoblasts in vitro, and promoted osteoblast differentiation while suppressing osteoclast formation. Taken together, our data suggest a novel centralized mechanism by which strontium ranelate promotes osteoblast activity and suppresses osteoclastogenesis, based on suppression of NF-κB signal transduction. We further demonstrate that the biological actions of strontium ranelate may be related to low dose of the intact molecule rather than dissociation and release of strontium ions, as previously thought. These data may facilitate the development of additional novel pharmacological agents for the amelioration of osteoporosis, based on NF-κB blockade.     

The calcium-sensing receptor acts as a modulator of gastric acid secretion in freshly isolated human gastric glands

Gastric acid secretion is activated by two distinct pathways: a neuronal pathway via the vagus nerve and release of acetylcholine and an endocrine pathway involving gastrin and histamine. Recently, we demonstrated that activation of H(+)-K(+)-ATPase activity in parietal cells in freshly isolated rat gastric glands is modulated by the calcium-sensing receptor (CaSR). Here, we investigated if the CaSR is functionally expressed in freshly isolated gastric glands from human patients undergoing surgery and if the CaSR is influencing histamine-induced activation of H(+)-K(+)-ATPase activity. In tissue samples obtained from patients, immunohistochemistry demonstrated the expression in parietal cells of both subunits of gastric H(+)-K(+)-ATPase and the CaSR. Functional experiments using the pH-sensitive dye 2',7'-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein and measurement of intracellular pH changes allowed us to estimate the activity of H(+)-K(+)-ATPase in single freshly isolated human gastric glands. Under control conditions, H(+)-K(+)-ATPase activity was stimulated by histamine (100 microM) and inhibited by omeprazole (100 microM). Reduction of the extracellular divalent cation concentration (0 Mg(2+), 100 microM Ca(2+)) inactivated the CaSR and reduced histamine-induced activation of H(+)-K(+)-ATPase activity. In contrast, activation of the CaSR with the trivalent cation Gd(3+) caused activation of omeprazole-sensitive H(+)-K(+)-ATPase activity even in the absence of histamine and under conditions of low extracellular divalent cations. This stimulation was not due to release of histamine from neighbouring enterochromaffin-like cells as the stimulation persisted in the presence of the H(2) receptor antagonist cimetidine (100 microM). Furthermore, intracellular calcium measurements with fura-2 and fluo-4 showed that activation of the CaSR by Gd(3+) led to a sustained increase in intracellular Ca(2+) even under conditions of low extracellular divalent cations. These experiments demonstrate the presence of a functional CaSR in the human stomach and show that this receptor may modulate the activity of acid-secreting H(+)-K(+)-ATPase in parietal cells. Furthermore, our results show the viability of freshly isolated human gastric glands and may allow the use of this preparation for experiments investigating the physiological regulation and properties of human gastric glands in vitro.     

N1-Arylsulfonyl-N2-(1-aryl)ethyl-3-phenylpropane-1,2-diamines as novel calcimimetics acting on the calcium sensing receptor

The synthesis and calcimimetic properties of N1-arylsulfonyl-N2-(1-aryl)ethyl-3-phenylpropane-1,2-diamines are described. The most active compound of the series (3n, used at 10 microM) produced 97+/-11% of the maximal stimulation of [3H]IP production obtained by 10 mM Ca2+ in CHO cells expressing the calcium sensing receptor (CaSR). This calcimimetic activity was due to a specific interaction of this compound with the CaSR.     

Calcium-sensing receptor modulates extracellular Ca(2+) entry via TRPC-encoded receptor-operated channels in human aortic smooth muscle cells

Ca-sensing receptor (CaSR), a member of the G protein-coupled receptor family, regulates the synthesis of parathyroid hormone in response to changes in serum Ca(2+) concentrations. The functions of CaSR in human vascular smooth muscle cells are largely unknown. Here we sought to study CaSR activation and the underlying molecular mechanisms in human aortic smooth muscle cells (HASMC). Extracellular Ca(2+) ([Ca(2+)](o)) dose-dependently increased free cytosolic Ca(2+) ([Ca(2+)](cyt)) in HASMC, with a half-maximal response (EC(50)) of 0.52 mM and a Hill coefficient of 5.50. CaSR was expressed in HASMC, and the [Ca(2+)](o)-induced [Ca(2+)](cyt) rise was abolished by dominant negative mutants of CaSR. The CaSR-mediated increase in [Ca(2+)](cyt) was also significantly inhibited by pertussis toxin, the phospholipase C inhibitor U-73122, or the general protein kinase C (PKC) inhibitor chelerythrine, but not by the conventional PKC inhibitor, G?6976. Depletion of membrane cholesterol by pretreatment with methyl-β-cyclodextrin markedly decreased CaSR-induced increase in [Ca(2+)](cyt). Blockade of TRPC channels with 2-aminoethoxydiphenyl borate, SKF-96365, or La(3) significantly inhibited [Ca(2+)](o) entry, whereas activation of TRPC6 channels with flufenamic acid potentiated [Ca(2+)](o) entry. Neither cyclopiazonic acid nor caffeine or ionomycin had any effect on [Ca(2+)](cyt) in [Ca(2+)](o)-free solutions. TRPC6 and PKCε mRNA and proteins were detected in HASMC, and [Ca(2+)](o) induced PKCε phosphorylation, which could be prevented by chelerythrine. Our data suggest that CaSR activation mediates [Ca(2+)](o) entry, likely through TRPC6-encoded receptor-operated channels that are regulated by a PLC/PKCε cascade. Our study therefore provides evidence not only for functional expression of CaSR, but also for a novel pathway whereby it regulates [Ca(2+)](o) entry in HASMC.     

The calcium-sensing receptor is involved in strontium ranelate-induced osteoclast apoptosis. New insights into the associated signaling pathways

Strontium ranelate exerts both an anti-catabolic and an anabolic effect on bone cells. To further investigate the molecular mechanism whereby strontium ranelate inhibits bone resorption, we focused our attention on the effects of strontium ranelate on osteoclast apoptosis and on the underlying mechanism(s). Using primary mature rabbit osteoclasts, we demonstrated that strontium (Sro2+) dose-dependently stimulates the apoptosis of mature osteoclasts. As shown previously for calcium (Cao2+), the Sro2+-induced effect on mature osteoclasts is mediated by the Cao2+-sensing receptor, CaR, which in turn stimulates a phospholipase C-dependent signaling pathway and nuclear translocation of NF-kappaB. Unlike Cao2+, however, Sro2+-induced osteoclast apoptosis was shown to depend on PKCbetaII activation and to be independent of inositol 1,4,5-trisphosphate action. As a consequence of these differences in their intracellular signaling pathways, Sro2+ and Cao2+ in combination were shown to exert a greater effect on mature osteoclast apoptosis than did either divalent cation by itself. Altogether, our results show that Sro2+ acts through the CaR and induces osteoclast apoptosis through a signaling pathway similar to but different in certain respects from that of Cao2+. This difference in the respective signaling cascades enables Sro2+ to potentiate Cao2+-induced osteoclast apoptosis and vice versa. In this manner, it is conceivable that Sro2+ and Cao2+ act together to inhibit bone resorption in strontium ranelate-treated patients.     

Induction of a program gene expression during osteoblast differentiation with strontium ranelate

Strontium ranelate, a new agent for the treatment of osteoporosis, has been shown stimulate bone formation in various experimental models. This study examines the effect of strontium ranelate on gene expression in osteoblasts, as well as the formation of mineralized (von Kossa-positive) colony-forming unit-osteoblasts (CFU-obs). Bone marrow-derived stromal cells cultured for 21 days under differentiating conditions, when exposed to strontium ranelate, displayed a significant time- and concentration-dependent increase in the expression of the master gene, Runx2, as well as bone sialoprotein (BSP), but interestingly without effects on osteocalcin. This was associated with a significant increase in the formation of CFU-obs at day 21 of culture. In U-33 pre-osteoblastic cells, strontium ranelate significantly enhanced the expression of Runx2 and osteocalcin, but not BSP. Late, more mature osteoblastic OB-6 cells showed significant elevations in BSP and osteocalcin, but with only minimal effects on Runx2. In conclusion, strontium ranelate stimulates osteoblast differentiation, but the induction of the program of gene expression appears to be cell type-specific. The increased osteoblastic differentiation is the likely basis underlying the therapeutic bone-forming actions of strontium ranelate.     

Polyvalent cation-sensing mechanism increased Na(+)-independent Mg(2+) transport in renal epithelial cells

Extracellular Ca(2+)/polyvalent cation-sensing receptor (CaSR) is capable of monitoring changes in extracellular polyvalent cation concentrations. In the present study, we investigated whether CaSR agonists reinforce the decrease of intracellular free Mg(2+) concentration ([Mg(2+)](i)) induced by extracellular Mg(2+) plus Na(+) removal. Interestingly, exposure of NRK-52E renal epithelial cells to increasing extracellular Mg(2+) concentrations from 0.8 to 15 mM for 1-2 days resulted in a twofold increase in the levels of CaSR mRNA and protein. By fluorophotometer (with mag-fura 2 fluorescent dye) and atomic absorption spectrophotometer, we confirmed that activation of CaSR by neomycin (0.5 mM) or gadolinium (1 mM) reinforced the decrease of [Mg(2+)](i) induced by Mg(2+) removal in the cells cultured in 10 mM Mg(2+)-containing medium. The neomycin-induced [Mg(2+)](i) decrease was inhibited by nicardipine (50 microM), but not by verapamil (50 microM) or amiloride (0.1 mM). These results indicate that CaSR monitors extracellular Mg(2+) concentration, and probably cause activation of Na(+)-independent Mg(2+)-transport system.     

2-aminoethoxydiphenyl borate reveals heterogeneity in receptor-activated Ca(2+) discharge and store-operated Ca(2+) influx.

We have investigated Ca(2+) release and receptor- and store-operated Ca(2+) influxes in Chinese hamster ovary-K1 (CHO) cells, SH-SY5Y human neuroblastoma cells and RBL-1 rat basophilic leukemia cells using Fura-2 and patch-clamp measurements. Ca(2+) release and subsequent Ni(2+)-sensitive, store-operated influx were induced by thapsigargin and stimulation of G protein-coupled receptors. The alleged noncompetitive IP3 receptor inhibitor,2-aminoethoxydiphenyl borate (2-APB) rapidly blocked a major part of the secondary influx response in CHO cells in a reversible manner. It also reduced Mn(2+) influx in response to thapsigargin. Inhibition of Ca(2+) release was also seen but this was less complete, slower in onset, less reversible, and required higher concentration of 2-APB. In RBL-1 cells, I(CRAC) activity was rapidly blocked by extracellular 2-APB whereas intracellular 2-APB was less effective. Store-operated Ca(2+) influxes were only partially blocked by 2-APB. In SH-SY5Y cells, Ca(2+) influxes were insensitive to 2-APB. Ca(2+) release in RBL-1 cells was partially sensitive but in SH-SY5Y cells the release was totally resistant to 2-APB. The results suggest, that 2-APB (1) may inhibit distinct subtypes of IP3 receptors with different sensitivity, and (2) that independently of this, it also inhibits some store-operated Ca(2+) channels via a direct, extracellular action.     

Stimulatory pathways of the Calcium-sensing receptor on acid secretion in freshly isolated human gastric glands.

Gastric acid secretion is not only stimulated via the classical known neuronal and hormonal pathways but also by the Ca(2+)-Sensing Receptor (CaSR) located at the basolateral membrane of the acid-secretory gastric parietal cell. Stimulation of CaSR with divalent cations or the potent agonist Gd(3+) leads to activation of the H(+)/K(+)-ATPase and subsequently to gastric acid secretion. Here we investigated the intracellular mechanism(s) mediating the effects of the CaSR on H(+)/K(+)-ATPase activity in freshly isolated human gastric glands. Inhibition of heterotrimeric G-proteins (G(i) and G(o)) with pertussis toxin during stimulation of the CaSR with Gd(3+) only partly reduced the observed stimulatory effect. A similar effect was observed with the PLC inhibitor U73122. The reduction of the H(+)/K(+)-ATPase activity measured after incubation of gastric glands with BAPTA-AM, a chelator of intracellular Ca(2+), showed that intracellular Ca(2+) plays an important role in the signalling cascade. TMB-8, a ER Ca(2+)store release inhibitor, prevented the stimulation of H(+)/K(+)-ATPase activity. Also verapamil, an inhibitor of L-type Ca(2+)-channels reduced stimulation suggesting that both the release of intracellular Ca(2+) from the ER as well as Ca(2+) influx into the cell are involved in CaSR-mediated H(+)/K(+)-ATPase activation. Chelerythrine, a general inhibitor of protein kinase C, and Go 6976 which selectively inhibits Ca(2+)-dependent PKC(alpha) and PKC(betaI)-isozymes completely abolished the stimulatory effect of Gd(3+). In contrast, Ro 31-8220, a selective inhibitor of the Ca(2+)-independent PKCepsilon and PKC-delta isoforms reduced the stimulatory effect of Gd(3+) only about 60 %. On the other hand, activation of PKC with DOG led to an activation of H(+)/K(+)-ATPase activity which was only about 60 % of the effect observed with Gd(3+). Incubation of the parietal cells with PD 098059 to inhibit ERK1/2 MAP-kinases showed a significant reduction of the Gd(3+) effect. Thus, in the human gastric parietal cell the CaSR is coupled to pertussis toxin sensitive heterotrimeric G-Proteins and requires calcium to enhance the activity of the proton-pump. PLC, ERK 1/2 MAP-kinases as well as Ca(2+) dependent and Ca(2+)-independent PKC isoforms are part of the down-stream signalling cascade.     

ERK binds, phosphorylates InsP3 type 1 receptor and regulates intracellular calcium dynamics in DT40 cells

Modulation on the duration of intracellular Ca(2+) transients is essential for B-cell activation. We have previously shown that extracellular-signal-regulated kinase (ERK) can phosphorylate inositol 1,4,5-trisphosphate receptor type 1 (IP(3)R1) at serine 436 and regulate its calcium channel activity. Here we investigate the potential physiological interaction between ERK and IP(3)R1 using chicken DT40 B-cell line in which different mutants are expressed. The interaction between ERK and IP(3)R1 is confirmed by co-immunoprecipitation and fluorescence resonance energy transfer (FRET) assays. This constitutive interaction is independent of either ERK kinase activation or IP(3)R1 phosphorylation status. Back phosphorylation analysis further shows that type 1 IP(3)R (IP(3)R1) is phosphorylated by ERK in anti-IgM-activated DT40 cells. Finally, our data show that the phosphorylation of Ser 436 in the IP(3)-binding domain of IP(3)R1 leads to less Ca(2+) release from endoplasmic reticulum (ER) microsomes and accelerates the declining of calcium increase in DT40 cells in response to anti-IgM stimulation.     

Mechanisms of neurotransmitter release facilitation in strontium solutions

Mechanisms of neurotransmitter release facilitation were studied using electrophysiological recording of end-plate currents (EPC) and nerve ending (NE) responses after substitution of extracellular Ca ions with Sr ions at the frog neuromuscular junction. The solutions with 0.5 mM concentration of Ca ions (calcium solution) or 1 mM concentration of Sr ions (strontium solution) were used where baseline neurotransmitter release (at low-frequency stimulation) is equal. Decay of paired-pulse facilitation of EPC at calcium solutions with increase of interpulse interval from 5 to 500 ms was well described by three-exponential function consisting of early, first and second components. Facilitation at strontium solutions was significantly diminished due mainly to decrease of early and first components. At the same time, EPC facilitation with rhythmic stimulation (10 or 50 imp/s) at strontium solutions was significantly increased. Also more pronounced decrease of NE response 3rd phase, reflecting potassium currents was detected under rhythmic stimulation of 50 imp/s at strontium solutions comparing to calcium solutions. It was concluded that facilitation sites underlying first and early components had lower affinity to Sr ions than to Ca ions. The enhancement of frequency facilitation at strontium solutions is mediated by two mechanisms: more pronounced broadening of NE action potential and increase of bivalent cation influx due to feebly marked activation of Ca(2+)-dependent potassium current by Sr ions, and slower dynamics of Sr(2+) removal from NE axoplasm comparing to Ca(2+).     

Minireview: the intimate link between calcium sensing receptor trafficking and signaling: implications for disorders of calcium homeostasis

The calcium-sensing receptor (CaSR) regulates organismal Ca(2+) homeostasis. Dysregulation of CaSR expression or mutations in the CASR gene cause disorders of Ca(2+) homeostasis and contribute to the progression or severity of cancers and cardiovascular disease. This brief review highlights recent findings that define the CaSR life cycle, which controls the cellular abundance of CaSR and CaSR signaling. A novel mechanism, termed agonist-driven insertional signaling (ADIS), contributes to the unique hallmarks of CaSR signaling, including the high degree of cooperativity and the lack of functional desensitization. Agonist-mediated activation of plasma membrane-localized CaSR increases the rate of insertion of CaSR at the plasma membrane without altering the constitutive endocytosis rate, thereby acutely increasing the maximum signaling response. Prolonged CaSR signaling requires a large intracellular ADIS-mobilizable pool of CaSR, which is maintained by signaling-mediated increases in biosynthesis. This model provides a rational framework for characterizing the defects caused by CaSR mutations and the altered functional expression of wild-type CaSR in disease states. Mechanistic dissection of ADIS of CaSR should lead to optimized pharmacological approaches to normalize CaSR signaling in disorders of Ca(2+) homeostasis.     

The functional expression of calcium-sensing receptor in the differentiated THP-1 cells

The expression and function of calcium-sensing receptor (CaSR) in differentiated THP-1 (human acute monocytic leukemia cell line) cells are unknown currently. This study investigated above-mentioned issues using TRAP staining, immunofluorescence staining, Western blotting, ELISA, and Laser Confocal Scanning Microscopy techniques. We found that CaSR protein was expressed, and mainly located in the membrane and cytoplasm in differentiated THP-1 cells. Elevated extracellular calcium or GdCl₃ (an agonist of CaSR) raised intracellular calcium concentration. And this increase was inhibited or abolished by NPS2390 (an inhibitor of CaSR), U73122 (a specific inhibitor of phospholipase C, PLC) or thapsigargin (a Ca²⁺-ATPase inhibitor). The extracellular GdCl₃ elevation stimulated both of IL-1β and TNFα release, and this effect of GdCl₃ was inhibited by NPS2390. In conclusion, CaSR is functionally expressed in differentiated THP-1 cells, and the activated CaSR contributes to intracellular calcium increment through Gq-PLC- inositol triphosphate (IP3) pathway and commits to cytokine secretion. These results suggest that CaSR might be involved in a variety of pathological processes mediated by activated monocyte-macrophages.     

A novel recombinant hyperaffinity inositol 1,4,5-trisphosphate (IP(3)) absorbent traps IP(3), resulting in specific inhibition of IP(3)-mediated calcium signaling.

We have developed a novel recombinant hyperaffinity inositol 1,4,5-trisphosphate (IP(3)) absorbent, called the "IP(3) sponge," which we constructed on the basis of the ligand-binding site of the mouse type 1 IP(3) receptor (IP(3)R1). The IP(3) sponge exhibited approximately 1000-fold higher affinity for IP(3) than the parental IP(3)R1 and specifically competed with the endogenous IP(3)R for binding to IP(3). Trapping IP(3) with the IP(3) sponge inhibited IP(3)-induced Ca(2+) release (IICR) from cerebellar microsomes in a dose-dependent manner. The IP(3) sponge expressed in HEK293 cells also inhibited IICR in response to stimulation with carbachol or ATP. Its inhibitory effects were dependent upon the level of its expression over the increased IP(3) contents. Moreover, the IP(3) sponge significantly reduced the carbachol-induced phosphorylation of cAMP-response element-binding protein in HEK293 cells, indicating that the activation of cAMP-response element-binding protein by Ca(2+)-dependent phosphorylation may be partly attributable to IICR.     

Type 1 inositol 1,4,5-trisphosphate receptors mediate UTP-induced cation currents, Ca2+ signals, and vasoconstriction in cerebral arteries

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) regulate diverse physiological functions, including contraction and proliferation. There are three IP(3)R isoforms, but their functional significance in arterial smooth muscle cells is unclear. Here, we investigated relative expression and physiological functions of IP(3)R isoforms in cerebral artery smooth muscle cells. We show that 2-aminoethoxydiphenyl borate and xestospongin C, membrane-permeant IP(3)R blockers, reduced Ca(2+) wave activation and global intracellular Ca(2+) ([Ca(2+)](i)) elevation stimulated by UTP, a phospholipase C-coupled purinergic receptor agonist. Quantitative PCR, Western blotting, and immunofluorescence indicated that all three IP(3)R isoforms were expressed in acutely isolated cerebral artery smooth muscle cells, with IP(3)R1 being the most abundant isoform at 82% of total IP(3)R message. IP(3)R1 knockdown with short hairpin RNA (shRNA) did not alter baseline Ca(2+) wave frequency and global [Ca(2+)](i) but abolished UTP-induced Ca(2+) wave activation and reduced the UTP-induced global [Ca(2+)](i) elevation by approximately 61%. Antibodies targeting IP(3)R1 and IP(3)R1 knockdown reduced UTP-induced nonselective cation current (I(cat)) activation. IP(3)R1 knockdown also reduced UTP-induced vasoconstriction in pressurized arteries with both intact and depleted sarcoplasmic reticulum (SR) Ca(2+) by approximately 45%. These data indicate that IP(3)R1 is the predominant IP(3)R isoform expressed in rat cerebral artery smooth muscle cells. IP(3)R1 stimulation contributes to UTP-induced I(cat) activation, Ca(2+) wave generation, global [Ca(2+)](i) elevation, and vasoconstriction. In addition, IP(3)R1 activation constricts cerebral arteries in the absence of SR Ca(2+) release by stimulating plasma membrane I(cat).     

Amino acids stimulate cholecystokinin release through the Ca2+-sensing receptor

Cholecystokinin (CCK) is produced by discrete endocrine cells in the proximal small intestine and is released following the ingestion of food. CCK is the primary hormone responsible for gallbladder contraction and has potent effects on pancreatic secretion, gastric emptying, and satiety. In addition to fats, digested proteins and aromatic amino acids are major stimulants of CCK release. However, the cellular mechanism by which amino acids affect CCK secretion is unknown. The Ca(2+)-sensing receptor (CaSR) that was originally identified on parathyroid cells is not only sensitive to extracellular Ca(2+) but is activated by extracellular aromatic amino acids. It has been postulated that this receptor may be involved in gastrointestinal hormone secretion. Using transgenic mice expressing a CCK promoter driven/enhanced green fluorescent protein (GFP) transgene, we have been able to identify and purify viable intestinal CCK cells. Intestinal mucosal CCK cells were enriched >200-fold by fluorescence-activated cell sorting. These cells were then used for real-time PCR identification of CaSR. Immunohistochemical staining with an antibody specific for CaSR confirmed colocalization of CaSR to CCK cells. In isolated CCK cells loaded with a Ca(2+)-sensitive dye, the amino acids phenylalanine and tryptophan, but not nonaromatic amino acids, caused an increase in intracellular Ca(2+) ([Ca(2+)](i)). The increase in [Ca(2+)](i) was blocked by the CaSR inhibitor Calhex 231. Phenylalanine and tryptophan stimulated CCK release from intestinal CCK cells, and this stimulation was also blocked by CaSR inhibition. Electrophysiological recordings from isolated CCK-GFP cells revealed these cells to possess a predominant outwardly rectifying potassium current. Administration of phenylalanine inhibited basal K(+) channel activity and caused CCK cell depolarization, consistent with changes necessary for hormone secretion. These findings indicate that amino acids have a direct effect on CCK cells to stimulate CCK release by activating CaSR and suggest that CaSR is the physiological mechanism through which amino acids regulate CCK secretion.     

A minimal generic model of bacteria-induced intracellular Ca2+ oscillations in epithelial cells

The toxin alpha-hemolysin expressed by uropathogenic Escherichia coli bacteria was recently shown as the first pathophysiologically relevant protein to induce oscillations of the intracellular Ca(2+) concentration in target cells. Here, we propose a generic three-variable kinetic model describing the Ca(2+) oscillations induced in single rat renal epithelial cells by this toxin. Specifically, we take into account the interplay between 1), the cytosolic Ca(2+) concentration; 2), IP(3)-sensitive Ca(2+) channels located in the membrane separating the cytosol and endoplasmic reticulum; and 3), toxin-related activation of production of IP(3) by phospholipase C. With these ingredients, the predicted response of cells exposed to the toxin is in good agreement with the results of experiments.