RESUMEN
BACKGROUND: A balanced intake of protein and constituent amino acids (AAs) requires adjustments to total food intake (protein leverage [PL]) and food selection to balance deficits and excesses (complementary feeding). We provided mice with choices of casein and whey, 2 protein sources that are complementary in AA balance, across a range of protein concentrations (P%) of digestible energy (DE). OBJECTIVES: We aimed to determine if: 1) PL operates similarly for casein and whey; 2) one protein source is preferred at control P%; 3) the preference changes as P% falls; and 4) AA intakes under control and low P% levels identify AAs that drive changes in protein selection. METHODS: Food intake and plasma fibroblast growth factor-21 (FGF21) concentrations were measured in mice at various P% (P7.5%-P33%). For direct comparisons, defined diets were used in which the protein source was either casein or whey. In food choice studies, mice had access to foods in which both casein and whey were provided at the same P% level at the same time. RESULTS: PL operated at different P% thresholds in casein (13%)- and whey (10%)-based diets, and the magnitude of PL was greater for casein. Although mice preferred casein under control conditions (P23%), a pronounced preference shift to whey occurred as P% fell to P13% and P10%. At low P%, increases in food intake were accompanied by increases in plasma FGF21, a protein hunger signal. Among AAs deficient in casein and enriched in whey, the intake of Cys was the most invariant as P% changed between P23% and P10%, appearing to drive the switch in protein preference. CONCLUSIONS: Mice selected between complementary protein sources, casein and whey, achieving stable total energy intake and regulated intake of AAs as P% varied. Supplementation of low P% casein diets with one whey-enriched AA, Cys, suppressed plasma FGF21 and total food intake.
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Aminoácidos , Caseínas , Proteínas en la Dieta , Ingestión de Energía , Factores de Crecimiento de Fibroblastos , Animales , Ratones , Aminoácidos/sangre , Aminoácidos/metabolismo , Proteínas en la Dieta/administración & dosificación , Proteínas en la Dieta/farmacología , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/sangre , Caseínas/administración & dosificación , Masculino , Ratones Endogámicos C57BL , Preferencias Alimentarias , Proteína de Suero de Leche/administración & dosificación , DietaRESUMEN
The relationships between parathyroid hormone (PTH) secretion and parathyroid cell membrane potential, including the identities and roles of K+ channels that regulate and/or modulate membrane potential are not well defined. Here we have used Western blot/immunohistochemistry as well as patch-clamp and perifusion techniques to identify and localize specific K+ channels in parathyroid cells and to investigate their roles in the control of membrane potential and PTH secretion. We also re-investigated the relationship between membrane potential and exocytosis. We showed that in single human parathyroid cells K+ current is dependent on at least two types of Ca2+-activated K+ channels: a small-conductance Ca2+-activated K+ channel (KSK) and a large-conductance voltage and Ca2+-activated K+ channel (KBK). These channels were sensitive to specific peptide blocking toxins including apamin, charybdotoxin, and iberiotoxin. These channels confer sensitivity of the membrane potential in single cells to high extracellular K+, TEA, and peptide toxins. Blocking of KBK potently inhibited K+ channel current, and KBK was shown to be expressed in the plasma membrane of parathyroid cells. In addition, when using the capacitance technique as an indicator of exocytosis, clamping the parathyroid cell at -60 mV prevented exocytosis, whereas holding the membrane potential at 0 mV facilitated it. Taken together, the results show that human parathyroid cells have functional KBK and KSK channels but the data presented herein suggest that KBK/KSK channels likely contribute to the maintenance of the membrane potential, and that membrane potential, per se, modulates exocytosis independently of [Ca2+]i.
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Calcio , Canales de Potasio , Humanos , Potenciales de la Membrana , Calcio/metabolismo , Péptidos/metabolismo , ExocitosisRESUMEN
Whether GPCRs support the sensing of temperature as well as other chemical and physical modalities is not well understood. Introduction: Extracellular Ca2+ concentration (Ca2+ o) modulates core body temperature and the firing rates of temperature-sensitive CNS neurons, and hypocalcemia provokes childhood seizures. However, it is not known whether these phenomena are mediated by Ca2+ o-sensing GPCRs, including the calcium-sensing receptor (CaSR). In favor of the hypothesis, CaSRs are expressed in hypothalamic regions that support core temperature regulation, and autosomal dominant hypocalcemia, due to CaSR activating mutations, is associated with childhood seizures. Methods: Herein, we tested whether CaSR-dependent signaling is temperature sensitive using an established model system, CaSR-expressing HEK-293 cells. Results: We found that the frequency of Ca2+ o-induced Ca2+ i oscillations but not the integrated response was linearly dependent on temperature in a pathophysiologically relevant range. Chimeric receptor analysis showed that the receptor's C-terminus is required for temperature-dependent modulation and experiments with the PKC inhibitor GF109203X and CaSR mutants T888A and T888M, which eliminate a key phosphorylation site, demonstrated the importance of repetitive phosphorylation and dephosphorylation. Discussion and Conclusion: CaSRs mediate temperature-sensing and the mechanism, dependent upon repetitive phosphorylation and dephosphorylation, suggests that GPCRs more generally contribute to temperature-sensing.
RESUMEN
Cold ischemia and hence travel time can adversely affect outcomes of islet isolation. The aim of this study was to compare the isolation and transplant outcomes of donor pancreata according to the distance from islet isolation facility. Principally, those within a 50 km radius of the centre were compared with those from regional areas within the state and those from interstate donors within Australia. Organ donors were categorised according to distance from National Pancreas Transplant Unit Westmead (NPTU). Donor characteristics were analysed statistically against islet isolation outcomes. These were age, BMI, cause and mechanism of death, days in ICU, gender, inotrope and steroid use, cold ischemia time (CIT) and retrieval surgical team. Between March 2007 and December 2020, 297 islet isolations were performed at our centre. A total of 149 donor pancreata were local area, and 148 non-local regions. Mean distance from the isolation facility was 780.05 km. Mean pancreas CIT was 401.07 ± 137.71 min and was significantly different between local and non-local groups (297.2 vs. 487.5 min, p < 0.01). Mean age of donors was 45.22 years, mean BMI was 28.82, sex ratio was 48:52 F:M and mean time in ICU was 3.07 days. There was no significant difference between local and non-local for these characteristics. The mean CIT resulting in islet transplantation was 297.1 ± 91.5 min and longest CIT resulting in transplantation was 676 min. There was no significant difference in islet isolation outcomes between local and non-local donors for characteristics other than CIT. There was also no significant effect of distance from the isolation facility on positive islet transplant outcomes (C-peptide > 0.2 at 1 month post-transplant). Conclusions: Distance from the isolation centre did not impact on isolation or transplant outcomes supporting the ongoing nationwide use of shipping pancreata for islet isolation and transplantation.
RESUMEN
Extracellular phosphate regulates its own renal excretion by eliciting concentration-dependent secretion of parathyroid hormone (PTH). However, the phosphate-sensing mechanism remains unknown and requires elucidation for understanding the aetiology of secondary hyperparathyroidism in chronic kidney disease (CKD). The calcium-sensing receptor (CaSR) is the main controller of PTH secretion and here we show that raising phosphate concentration within the pathophysiologic range for CKD significantly inhibits CaSR activity via non-competitive antagonism. Mutation of residue R62 in anion binding site-1 abolishes phosphate-induced inhibition of CaSR. Further, pathophysiologic phosphate concentrations elicit rapid and reversible increases in PTH secretion from freshly-isolated human parathyroid cells consistent with a receptor-mediated action. The same effect is seen in wild-type murine parathyroid glands, but not in CaSR knockout glands. By sensing moderate changes in extracellular phosphate concentration, the CaSR represents a phosphate sensor in the parathyroid gland, explaining the stimulatory effect of phosphate on PTH secretion.
Asunto(s)
Glándulas Paratiroides/metabolismo , Hormona Paratiroidea/metabolismo , Fosfatos/metabolismo , Receptores Sensibles al Calcio/metabolismo , Animales , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Hiperparatiroidismo Secundario/etiología , Hiperparatiroidismo Secundario/metabolismo , Ratones , Mutación , Receptores Sensibles al Calcio/genética , Insuficiencia Renal Crónica/complicaciones , Insuficiencia Renal Crónica/metabolismoRESUMEN
Human calcitonin release is promoted by elevated extracellular Ca2+ (Ca2+o) concentration acting, at least in part, via the calcium-sensing receptor (CaSR). The CaSR is positively modulated by L-amino acids, including the aromatic amino acids L-phenylalanine (Phe) and L-tryptophan (Trp). To investigate the effect of L-amino acids on human calcitonin secretion, we selected thyroid TT cells and exposed them to various Ca2+o concentrations in the absence or presence of L-Phe, plasma-like mixtures of L-amino acids, or the clinically effective positive modulator (calcimimetic) cinacalcet. In the presence of L-Phe or plasma-like mixtures of amino acids, TT cells exhibited enhanced Ca2+o sensitivity in assays of calcitonin release and intracellular Ca2+ mobilization. Furthermore, the effect of elevated Ca2+o and L-Phe on calcitonin release was markedly suppressed by the calcilytic NPS-2143. These effects were dependent on CaSR-mediated activation of Gq/11 as revealed by the specific inhibitor YM-254890. The findings support the hypothesis that calcitonin release is stimulated by increases in plasma L-amino acid levels as well as elevated Ca2+o concentration. They also demonstrate that stimulated calcitonin release as well as basal levels of calcitonin secretion are mediated by a CaSR:Gq/11 signaling mechanism.
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Calcio/farmacología , Fenilalanina/farmacología , Receptores Sensibles al Calcio/metabolismo , Transducción de Señal/fisiología , Células Epiteliales Tiroideas/metabolismo , Triptófano/farmacología , Calcitonina/metabolismo , Humanos , Transducción de Señal/efectos de los fármacos , Células Epiteliales Tiroideas/efectos de los fármacosRESUMEN
Negative allosteric modulators (NAMs) of the human calcium-sensing receptor (CaSR) have previously failed to show efficacy in human osteoporosis clinical trials, but there is now significant interest in repurposing these drugs for hypocalcemic disorders and inflammatory lung diseases. However, little is known about how CaSR NAMs inhibit the response to endogenous activators. An improved understanding of CaSR negative allosteric modulation may afford the opportunity to develop therapeutically superior CaSR-targeting drugs. In an attempt to elucidate the mechanistic and structural basis of allosteric modulation mediated by the previously reported NAM, calhex231, we herein demonstrate that calhex231 actually potentiates or inhibits the activity of multiple CaSR agonists depending on whether it occupies one or both protomers in a CaSR dimer. These findings reveal a novel mechanism of mode-switching at a Class C G protein-coupled receptor that has implications for drug discovery and potential clinical utility.
RESUMEN
Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor (GPCR) that maintains extracellular Ca(2+) homeostasis through the regulation of parathyroid hormone secretion. It functions as a disulfide-tethered homodimer composed of three main domains, the Venus Flytrap module, cysteine-rich domain, and seven-helix transmembrane region. Here, we present the crystal structures of the entire extracellular domain of CaSR in the resting and active conformations. We provide direct evidence that L-amino acids are agonists of the receptor. In the active structure, L-Trp occupies the orthosteric agonist-binding site at the interdomain cleft and is primarily responsible for inducing extracellular domain closure to initiate receptor activation. Our structures reveal multiple binding sites for Ca(2+) and PO4(3-) ions. Both ions are crucial for structural integrity of the receptor. While Ca(2+) ions stabilize the active state, PO4(3-) ions reinforce the inactive conformation. The activation mechanism of CaSR involves the formation of a novel dimer interface between subunits.
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Calcio/metabolismo , Receptores Sensibles al Calcio/agonistas , Receptores Sensibles al Calcio/química , Triptófano/química , Triptófano/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Fosfatos/metabolismo , Unión Proteica , Conformación Proteica , Multimerización de ProteínaRESUMEN
The calcium-sensing receptor (CaR) modulates renal calcium reabsorption and parathyroid hormone (PTH) secretion and is involved in the etiology of secondary hyperparathyroidism in CKD. Supraphysiologic changes in extracellular pH (pHo) modulate CaR responsiveness in HEK-293 (CaR-HEK) cells. Therefore, because acidosis and alkalosis are associated with altered PTH secretion in vivo, we examined whether pathophysiologic changes in pHo can significantly alter CaR responsiveness in both heterologous and endogenous expression systems and whether this affects PTH secretion. In both CaR-HEK and isolated bovine parathyroid cells, decreasing pHo from 7.4 to 7.2 rapidly inhibited CaR-induced intracellular calcium (Ca(2+)i) mobilization, whereas raising pHo to 7.6 potentiated responsiveness to extracellular calcium (Ca(2+)o). Similar pHo effects were observed for Ca(2+)o-induced extracellular signal-regulated kinase phosphorylation and actin polymerization and for L-Phe-induced Ca(2+)i mobilization. Intracellular pH was unaffected by acute 0.4-unit pHo changes, and the presence of physiologic albumin concentrations failed to attenuate the pHo-mediated effects. None of the individual point mutations created at histidine or cysteine residues in the extracellular domain of CaR attenuated pHo sensitivity. Finally, pathophysiologic pHo elevation reversibly suppressed PTH secretion from perifused human parathyroid cells, and acidosis transiently increased PTH secretion. Therefore, pathophysiologic pHo changes can modulate CaR responsiveness in HEK-293 and parathyroid cells independently of extracellular histidine residues. Specifically, pathophysiologic acidification inhibits CaR activity, thus permitting PTH secretion, whereas alkalinization potentiates CaR activity to suppress PTH secretion. These findings suggest that acid-base disturbances may affect the CaR-mediated control of parathyroid function and calcium metabolism in vivo.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glándulas Paratiroides/metabolismo , Hormona Paratiroidea/metabolismo , Receptores Sensibles al Calcio/metabolismo , Acidosis/metabolismo , Alcalosis/metabolismo , Animales , Bovinos , Cisteína/genética , Cisteína/metabolismo , Células HEK293 , Histidina/genética , Histidina/metabolismo , Humanos , Concentración de Iones de Hidrógeno , FosforilaciónRESUMEN
Calcium-sensing receptors (CaSRs) are class C G protein-coupled receptors that respond to physiological activators, including extracellular Ca2+ (Cao2+) and L-amino acids as well as the pharmaceutical calcimimetic, cinacalcet. Unlike Cao2+, which is an orthosteric agonist, L-amino acids and cinacalcet are positive allosteric modulators. CaSR expression levels vary considerably between tissues, but the physiological significance of these differences in expression for the effects of its activators is unknown. To investigate the impact of receptor expression on CaSR-mediated signaling we used a tetracycline-inducible expression system and focused on intracellular Ca2+ (Cai2+) responses in single cells and considered both population and single-cell behavior. Increased receptor expression positively modulated CaSR-mediated Cai2+ mobilization in response to elevated Cao2+, the amino acid L-phenylalanine, or the calcimimetic cinacalcet. It lowered threshold concentrations for the initiation of Cai2+ oscillations and for their transformation to sustained Cai2+ elevations, and it increased the proportions of responding cells. It also positively modulated the frequency of Cai2+ oscillations with the order of effectiveness: cinacalcet equal to or greater than Cao2+ greater than L-phenylalanine. The results indicate that receptor expression modulates key characteristics of the Cai2+ response at the single-cell level as well as the amplitude of whole-tissue CaSR-mediated responses by recruiting quiescent cells into the active pool of responding cells. By lowering the threshold concentrations for Cao2+- and L-amino acid-induced responses below the physiological levels of these nutrients in plasma, mechanisms that up-regulate receptor expression can control tissue function in the absence of dynamic changes in ligand concentration.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Calcio/metabolismo , Receptores Sensibles al Calcio/metabolismo , Calcio/farmacología , Cinacalcet , Citoplasma/metabolismo , Células HEK293 , Humanos , Naftalenos/farmacología , Fenilalanina/farmacología , Receptores Sensibles al Calcio/genéticaRESUMEN
In addition to its acute effects on hormone secretion, epithelial transport, and shape change, the calcium-sensing receptor (CaSR) modulates the expression of genes that control cell survival, proliferation, and differentiation as well as the synthesis of peptide hormones and enzymes. In the present study, we investigated the impacts of a CaSR agonist and several CaSR modulators on phosphorylation of transcription factor CREB residue Ser(133) in CaSR-expressing HEK293 (HEK-CaSR) cells and human adenomatous parathyroid cells. Elevated Ca(2+)o concentration had no effect on CREB phosphorylation (p-CREB) in control HEK293 cells but stimulated p-CREB in both HEK-CaSR cells and human parathyroid cells. In addition, p-CREB was stimulated by the positive modulator cinacalcet and inhibited by the negative modulator NPS 2143 in both CaSR-expressing cell types. Two positive modulators that bind in the receptor's Venus Fly Trap domain, l-phenylalanine and S-methylglutathione, had no effect on p-CREB in HEK-CaSR cells, demonstrating the existence of pronounced signaling bias. Analysis of the signaling pathways using specific inhibitors demonstrated that phosphoinositide-specific phospholipase C and conventional protein kinase C isoforms make major contributions to Ca(2+)o-induced p-CREB in both cell-types, suggesting key roles for Gq/11. In addition, in parathyroid cells but not HEK-CaSR cells, activation of p-CREB was dependent on Gi/o, demonstrating the existence of cell type-specific signaling.
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Proteína de Unión a CREB/metabolismo , Calcio/metabolismo , Glándulas Paratiroides/metabolismo , Receptores Sensibles al Calcio/metabolismo , Cinacalcet , Glutatión/análogos & derivados , Glutatión/farmacología , Células HEK293 , Humanos , Naftalenos/farmacología , Fenilalanina/farmacología , Fosforilación , Transducción de SeñalRESUMEN
Since their introduction to the toad-free Australian continent cane toads (Bufo marinus) have caused a dramatic increase in naïve varanid mortality when these large lizards attempt to feed on this toxic amphibian. In contrast Asian-African varanids, which have coevolved with toads, are resistant to toad toxin. Toad toxins, such as Bufalin target the H1-H2 domain of the α(1) subunit of the sodium-potassium-ATPase enzyme. Sequencing of this domain revealed identical nucleotide sequences in four Asian as well as in three African varanids, and identical sequences in all 11 Australian varanids. However, compared to the Asian-African varanids, the Australian varanids showed four-base-pair substitutions, resulting in the alteration in three of the 12 amino acids representing the H1-H2 domain. The phenotypic effect of the substitutions was investigated in human embryonic kidney (HEK) 293 cells stably transfected with the Australian and the Asian-African H1-H2 domains. The transfections resulted in an approximate 3000-fold reduction in resistance to Bufalin in the Australian HEK293 cells compared to the Asian-African HEK293 cells, demonstrating the critical role of this minor mutation in providing Bufalin resistance. Our study hence presents a clear link between genotype and phenotype, a critical step in understanding the evolution of phenotypic diversity.
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Bufanólidos/toxicidad , Bufonidae/genética , Cardiotónicos/toxicidad , Lagartos/genética , Mutación , ATPasa Intercambiadora de Sodio-Potasio/genética , Sustitución de Aminoácidos , Animales , Australia , Ecosistema , Evolución Molecular , Células HEK293 , Humanos , Islas , Estructura Terciaria de Proteína , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/químicaRESUMEN
OBJECTIVE: Activating mutations of the calcium-sensing receptor (CASR) gene are associated with autosomal dominant hypocalcemia (ADH) characterized by benign hypocalcemia, inappropriately low (PTH) levels and mostly hypercalciuria. Herein, we report a novel activating mutation in the CASR gene in a Korean family with ADH. METHOD: The CASR gene was sequenced in the patient with ADH. The identified mutations were also evaluated in the patient's family members by PCR-based sequencing. For functional studies, we examined phosphorylation of ERK1/2. In addition, intracellular Ca(2+) mobilization and the effects of the calcilytic, AXT914 were measured using fluorophore Fura-2 dye. RESULT: Direct sequencing analysis of the CASR gene showed that the proband and her daughter possess a novel mutation c.1230T>A, resulting in a D410E missense mutation on exon 4 of the CASR gene. Escalation of the extracellular Ca(2+) concentration resulted in stronger phosphorylation of ERK1/2 and higher levels of intracellular Ca(2+) in HEK293 cells expressing mutant CASR, compared with wild-type CASR. The increase in intracellular Ca(2+) signalling via CASR was successively blunted by treatment with AXT914. CONCLUSIONS: Over 60 activating mutations in the CASR gene have been identified to cause ADH so far. Here, we add one more activating mutation that causes ADH. The novel activating mutation (D410E) occurred in the loop 3 region of CASR, where its function was believed to be of little importance; therefore, this mutation may be of interest. Further clinical study will be needed to validate the effectiveness of calcilytics in treatment of ADH in vivo.
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Hipocalcemia/genética , Receptores Sensibles al Calcio/genética , Adulto , Femenino , Células HEK293 , Humanos , Hipocalcemia/metabolismo , Masculino , Persona de Mediana Edad , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Mutación , Fosforilación , Receptores Sensibles al Calcio/metabolismoRESUMEN
γ-Glutamyl peptides were identified previously as novel positive allosteric modulators of Ca(2+)(o)-dependent intracellular Ca(2+) mobilization in HEK-293 cells that bind in the calcium-sensing receptor VFT domain. In the current study, we investigated whether γ-glutamyl-tripeptides including γ-Glu-Cys-Gly (glutathione) and its analogs S-methylglutathione and S-propylglutathione, or dipeptides including γ-Glu-Ala and γ-Glu-Cys are positive allosteric modulators of Ca(2+)(o)-dependent Ca(2+)(i) mobilization and PTH secretion from normal human parathyroid cells as well as Ca(2+)(o)-dependent suppression of intracellular cAMP levels in calcium-sensing receptor (CaR)-expressing HEK-293 cells. In addition, we compared the effects of the potent γ-glutamyl peptide S-methylglutathione, and the amino acid L-Phe on HEK-293 cells that stably expressed either the wild-type CaR or the double mutant T145A/S170T, which exhibits selectively impaired responses to L-amino acids. We find that γ-glutamyl peptides are potent positive allosteric modulators of the CaR that promote Ca(2+)(o)-dependent Ca(2+)(i) mobilization, suppress intracellular cAMP levels and inhibit PTH secretion from normal human parathyroid cells. Furthermore, we find that the double mutant T145A/S170T exhibits markedly impaired Ca(2+)(i) mobilization and cAMP suppression responses to S-methylglutathione as well as L-Phe indicating that γ-glutamyl peptides and L-amino acids activate the CaR via a common mechanism.
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Aminoácidos/metabolismo , Calcio/metabolismo , AMP Cíclico/metabolismo , Oligopéptidos/metabolismo , Hormona Paratiroidea/metabolismo , Receptores Sensibles al Calcio/metabolismo , Aminoácidos/genética , Aminoácidos/farmacología , AMP Cíclico/genética , Células HEK293 , Humanos , Mutación , Oligopéptidos/genética , Oligopéptidos/farmacología , Estructura Terciaria de Proteína , Receptores Sensibles al Calcio/genéticaRESUMEN
The calcium-sensing receptor (CaR) elicits oscillatory Ca(2+)(i) mobilization associated with dynamic, inhibitory protein kinase C-mediated phosphorylation of CaR(T888). While modest CaR stimulation elicits Ca(2+)(i) oscillations, greater stimulation either increases oscillation frequency or elicits sustained responses by an unknown mechanism. Here, moderate CaR stimulation (2.5 mm Ca(2+)(o), 10 min) increased CaR(T888) phosphorylation (160-kDa mature receptor) 5-fold in CaR stably transfected HEK-293 cells, whereas 3-5 mm Ca(2+)(o) treatments were without apparent effect. Treatment with 2 mm Ca(2+)(o) caused sustained CaR(T888) phosphorylation (> or = 20 min) and oscillatory Ca(2+)(i) mobilization. However, 5 mm Ca(2+)(o) increased CaR(T888) phosphorylation only briefly while eliciting sustained Ca(2+)(i) mobilization, suggesting that greater CaR activation induces rapid CaR(T888) dephosphorylation, thus permitting sustained Ca(2+)(i) responses. Indeed, 5 mm Ca(2+)(o) stimulated protein phosphatase 2A activity and induced CaR(T888) dephosphorylation following acute phorbol ester pretreatment, the latter effect being mimicked by CaR-positive allosteric modulators (NPS-R467 and l-Phe). Finally, the phosphatase inhibitor calyculin-A reversed CaR-induced inhibition of parathyroid hormone secretion from bovine parathyroid slices and normal human parathyroid cells, demonstrating the physiological importance of phosphorylation status on parathyroid function. Therefore, high Ca(2+)(o)-stimulated protein kinase C acts in concert with high Ca(2+)(o)-induced phosphatase activity to generate and maintain CaR-induced Ca(2+)(i) oscillations via the dynamic phosphorylation and dephosphorylation of CaR(T888).
Asunto(s)
Calcio/metabolismo , Glándulas Paratiroides/metabolismo , Hormona Paratiroidea/metabolismo , Proteína Quinasa C/metabolismo , Receptores Sensibles al Calcio/metabolismo , Animales , Señalización del Calcio , Bovinos , Células Cultivadas , Humanos , Immunoblotting , Riñón/citología , Riñón/metabolismo , Fosforilación , Proteína Fosfatasa 2/metabolismo , Transducción de SeñalRESUMEN
Oscillations of the intracellular concentration of Ca(2+) in cultured HEK-293 cells, which heterologously expressed the calcium-sensing receptor, were recorded with the fluorophore Fura-2 using fluorescence microscopy. HEK-293 cells are extremely sensitive to small perturbations in extracellular calcium concentrations. Resting cells were attached to cover slips and perifused with saline solution containing physiologically relevant extracellular Ca(2+) concentrations in the range 0.5-5 mM. Acquired digitized images of the cells showed oscillatory fluctuations in the intracellular Ca(2+) concentration over the time course, and were processed as a function of the change in Fura-2 excitation ratio and frequency at 12-37 degrees C. Newly developed data processing techniques with wavelet analysis were used to estimate the frequency at which the rectified sinusoidal oscillations occurred; we estimated ~4 min(-1) under normal conditions. Temperature variations revealed an Arrhenius relationship in oscillation frequency. A critical Ca(2+) concentration of ~2 mM was estimated, below which oscillations did not occur. These data were used to develop a kinetic model of the system that was simulated using Mathematica; kinetic parameter values were adjusted to match the experimentally observed oscillations of intracellular Ca(2+) concentration as a function of extracellular Ca(2+) concentration, and temperature; and from these, limit cycles were obtained and control coefficients were estimated for all parameters.
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Señalización del Calcio , Calcio/metabolismo , Simulación por Computador , Modelos Biológicos , Línea Celular , Humanos , Cinética , Receptores Sensibles al Calcio/metabolismo , TemperaturaRESUMEN
The calcium-sensing receptor (CaR) is recognized as a member of class 3 of the G-protein coupled receptor superfamily. Members of this subgroup, which have large N-terminal extracellular domains, include receptors that respond specifically to the amino acid glutamate; receptors that respond to the glutamate analogue, gamma-amino butyric acid; and several receptors that act as broad-spectrum amino acid sensors. The CaR is one of these broad-spectrum amino acid sensors that, along with several other members of the subgroup, also responds to extracellular Ca2+. In this mini-review, we consider evidence that the CaR is a sensor of aromatic amino acids, that it has broad-spectrum amino acid sensing properties, that it provides an amino acid binding site in its extracellular N-terminal Venus Fly Trap domain, and that amino acids have a physiological impact on systems in which the CaR is expressed.
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Aminoácidos Aromáticos/farmacología , Calcio/fisiología , Receptores Sensibles al Calcio/fisiología , Papilas Gustativas/fisiología , Animales , Células CHO , Línea Celular , Cricetinae , Cricetulus , Humanos , Cinética , Glándulas Paratiroides/efectos de los fármacos , Glándulas Paratiroides/fisiología , Ratas , Receptores Sensibles al Calcio/efectos de los fármacos , Papilas Gustativas/efectos de los fármacosRESUMEN
The calcium-sensing receptor (CaR) mediates feedback control of Ca2+o (extracellular Ca2+) concentration. Although the mechanisms are not fully understood, the CaR couples to several important intracellular signalling enzymes, including PI-PLC (phosphoinositide-specific phospholipase C), leading to Ca2+i (intracellular Ca2+) mobilization, and ERK1/2 (extracellular-signal-regulated kinase 1/2). In addition to Ca2+o, the CaR is activated allosterically by several subclasses of L-amino acids, including the aromatics L-phenylalanine and L-tryptophan. These amino acids enhance the Ca2+o-sensitivity of Ca2+i mobilization in CaR-expressing HEK-293 (human embryonic kidney) cells and normal human parathyroid cells. Furthermore, on a background of a physiological fasting serum L-amino acid mixture, they induce a small, but physiologically significant, enhancement of Ca2+o-dependent suppression of PTH (parathyroid hormone) secretion. The impact of amino acids on CaR-stimulated ERK1/2, however, has not been determined. In the present study, we examined the effects of L-amino acids on Ca2+o-stimulated ERK1/2 phosphorylation as determined by Western blotting and a newly developed quantitative assay (SureFire). L-Amino acids induced a small, but significant, enhancement of Ca2+o-stimulated ERK1/2. In CaR-expressing HEK-293 cells, 10 mM L-phenylalanine lowered the EC50 for Ca2+o from approx. 2.3 to 2.0 mM in the Western blot assay and from 3.4 to 2.9 mM in the SureFire assay. The effect was stereoselective (L>D), and another aromatic amino acid, L-tryptophan, was also effective. The effects of amino acids were investigated further in HEK-293 cells that expressed the CaR mutant S169T. L-Phenylalanine normalized the EC50 for Ca2+o-stimulated Ca2+i mobilization from approx. 12 mM to 5.0 mM and ERK1/2 phosphorylation from approx. 4.6 mM to 2.6 mM. Taken together, the data indicate that L-phenylalanine and other amino acids enhance the Ca2+o-sensitivity of CaR-stimulated ERK1/2 phosphorylation; however, the effect is comparatively small and operates in the form of a fine-tuning mechanism.
Asunto(s)
Aminoácidos/metabolismo , Canales de Calcio/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Regulación Alostérica , Sustitución de Aminoácidos , Línea Celular , Humanos , Riñón , Fenilalanina/metabolismo , Fosforilación , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND: With minimally invasive parathyroidectomy (MIP) not all enlarged parathyroid glands are necessarily removed, and intraoperative measurement of parathyroid hormone levels (IO-PTH) does not necessarily predict multiple enlarged glands. The aim of this study was to compare morphology with function, using Ca(2+)-regulated PTH secretion. METHODS: PTH secretion was determined by perifusion: (1) cells from 12 normal parathyroids were compared with 14 parathyroid adenomas; (2) functional characteristics (PTH secretion, sestamibi uptake, IO-PTH decrease) were correlated with morphologic characteristics; (3) PTH secretion as a predictor of IO-PTH decrease was determined in 7 patients with 2 enlarged parathyroids. RESULTS: (1) There were significant differences between normal and pathological parathyroid cells consistent with reduced sensitivity to Ca(2+). Maximum secretion rates for normal and adenomatous cells were, respectively, 3.9 +/- 0.4 fg min(-1) cell(-1) and 2.0 +/- 0.4 fg min(-1) cell(-1) (P = .002) and minimum secretion rates, 0.7 +/- 0.1 fg min(-1) cell(-1) and 0.4 +/- 0.1 fg min(-1) cell(-1) (P = .008). However, the IC(50) value for Ca(2+) was elevated in adenomatous cells indicating an apparent loss of extracellular Ca(2+) sensitivity being 1.1 +/- 0.02 mmol/L for normal and 1.2 +/- 0.02 mmol/L for adenomatous cells (P = .02). (2) There was no overall correlation between PTH secretion and gland morphology. (3) In 5 of 7 cases, PTH secretion correctly predicted the decrease in IO-PTH. CONCLUSION: Parathyroid adenomas generally exhibit abnormal PTH secretory function; however, enlarged parathyroid glands that do not contribute to the biochemical changes of hyperparathyroidism do exist, and, in these cases, cellular secretory function is a useful predictor of IO-PTH dynamics.
Asunto(s)
Adenoma/metabolismo , Adenoma/patología , Glándulas Paratiroides/metabolismo , Glándulas Paratiroides/patología , Hormona Paratiroidea/metabolismo , Neoplasias de las Paratiroides/metabolismo , Neoplasias de las Paratiroides/patología , Adenoma/cirugía , Estudios de Casos y Controles , Humanos , Hiperparatiroidismo/etiología , Hiperparatiroidismo/cirugía , Procedimientos Quirúrgicos Mínimamente Invasivos , Neoplasias de las Paratiroides/cirugía , Paratiroidectomía , Técnicas de Cultivo de TejidosRESUMEN
The extracellular Ca(2+)-sensing receptor is activated allosterically by l-amino acids, and recent molecular analysis indicates that amino acids are likely to bind in the receptor's Venus flytrap domain. In the current study we set out to identify residues in the VFT domain that specifically support amino acid binding and/or amino acid-dependent receptor activation. Herein we describe two mutations of the Ca(2+)-sensing receptor (CaR) Venus Flytrap domain, T145A and S170T, that specifically impair amino acid sensing, leaving Ca2+ sensing intact, as determined by receptor-dependent activation of intracellular Ca2+ mobilization in fura-2-loaded HEK293 cells. With respect to the wild-type CaR, T145A and S170T exhibited reduced sensitivity to l-Phe, and T145A also exhibited markedly impaired l/d selectivity. When combined, the double mutant T145A/S170T exhibited normal or near-normal sensitivity to extracellular Ca2+ but was resistant to l-Phe at concentrations up to 100 mm. We conclude that T145A/S170T selectively disables l-amino acid sensing and that the Ca2+ and l-amino acid-sensing functions of the CaR can be dissociated.