Cadmium, a toxic environmental pollutant, affects the function of different organs such as lungs, liver and kidney. Less is known about its toxic effects on the gastric mucosa. The aim of this study was to investigate the mechanisms by which cadmium impacts on the physiology of gastric mucosa. To this end, intact amphibian mucosae were mounted in Ussing chambers and the rate of acid secretion, short circuit current (Isc), transepithelial potential (Vt) and resistance (Rt) were recorded in the continuous presence of cadmium. Addition of cadmium (20 μM to 1 mM) on the serosal but not luminal side of the mucosae resulted in inhibition of acid secretion and increase in NPPB-sensitive, chloride-dependent short circuit current. Remarkably, cadmium exerted its effects only on histamine-stimulated tissues. Experiments with TPEN, a cell-permeant chelator for heavy metals, showed that cadmium acts from the intracellular side of the acid secreting cells. Furthermore, cadmium-induced inhibition of acid secretion and increase in Isc cannot be explained by an action on: 1) H2 histamine receptor, 2) Ca2+ signalling 3) adenylyl cyclase or 4) carbonic anhydrase. Conversely, cadmium was ineffective in the presence of the H+/K+-ATPase blocker omeprazole suggesting that the two compounds likely act on the same target. Our findings suggest that cadmium affects the functionality of histamine-stimulated gastric mucosa by inhibiting the H+/K+-ATPase from the intracellular side. These data shed new light on the toxic effect of this dangerous environmental pollutant and may result in new avenues for therapeutic intervention in acute and chronic intoxication.

Mutations in the lamin A/C gene (LMNA) were associated with dilated cardiomyopathy (DCM) and, recently, were related to severe forms of arrhythmogenic right ventricular cardiomyopathy (ARVC). Both genetic and phenotypic overlap between DCM and ARVC was observed; molecular pathomechanisms leading to the cardiac phenotypes caused by LMNA mutations are not yet fully elucidated. This study involved a large Italian family, spanning 4 generations, with arrhythmogenic cardiomyopathy of different phenotypes, including ARVC, DCM, system conduction defects, ventricular arrhythmias, and sudden cardiac death. Mutation screening of LMNA and ARVC-related genes PKP2, DSP, DSG2, DSC2, JUP, and CTNNA3 was performed. We identified a novel heterozygous mutation (c.418_438dup) in LMNA gene exon 2, occurring in a highly conserved protein domain across several species. This newly identified variant was not found in 250 ethnically-matched control subjects. Genotype-phenotype correlation studies suggested a co-segregation of the LMNA mutation with the disease phenotype and an incomplete and age-related penetrance. Based on clinical, pedigree, and molecular genetic data, this mutation was considered likely disease-causing. To clarify its potential pathophysiologic impact, functional characterization of this LMNA mutant was performed in cultured cardiomyocytes expressing EGFP-tagged wild-type and mutated LMNA constructs, and indicated an increased nuclear envelope fragility, leading to stress-induced apoptosis as the main pathogenetic mechanism. This study further expands the role of the LMNA gene in the pathogenesis of cardiac laminopathies, suggesting that LMNA should be included in mutation screening of patients with suspected arrhythmogenic cardiomyopathy, particularly when they have ECG evidence for conduction defects. The combination of clinical, genetic, and functional data contribute insights into the pathogenesis of this form of life-threatening arrhythmogenic cardiac laminopathy.

Secretory granules of pancreatic β-cells contain high concentrations of Ca2+ ions that are co-released with insulin in the extracellular milieu upon activation of exocytosis. As a consequence, an increase in the extracellular Ca2+ concentration ([Ca2+]ext) in the microenvironment immediately surrounding β-cells should be expected following the exocytotic event. Using Ca2+-selective microelectrodes we show here that both high glucose and non-nutrient insulinotropic agents elicit a reversible increase of [Ca2+]ext within rat insulinoma (INS-1E) β-cells pseudoislets. The glucose-induced increases in [Ca2+]ext are blocked by pretreatment with different Ca2+ channel blockers. Physiological agonists acting as positive or negative modulators of the insulin secretion and drugs known to intersect the secretory machinery at different levels also induce [Ca2+]ext changes as predicted on the basis of their described action on insulin secretion. Finally, the glucose-induced [Ca2+]ext increase is strongly inhibited after disruption of the actin web, indicating that the dynamic [Ca2+]ext changes recorded in INS-1E pseudoislets by Ca2+-selective microelectrodes occur mainly as a consequence of exocytosis of Ca2+-rich granules. In conclusion, our data directly demonstrate that the extracellular spaces surrounding β-cells constitute a restricted domain where Ca2+ is co-released during insulin exocytosis, creating the basis for an autocrine/paracrine cell-to-cell communication system via extracellular Ca2+ sensors

The antidiuretic hormone vasopressin regulates water reabsorption in the nephron by inducing apical plasma membrane exocytosis of the water channel aquaporin 2 in the kidney collecting duct principal cells. Disruption of this physiological mechanism by genetic alteration of either the vasopressin type 2 receptor gene or the aquaporin 2 gene, results in a rare genetic disorder known as nephrogenic diabetes insipidus, which main hallmark are polyuria and polydipsia. Over the last decades, analysis of patients affected by this disease helped genetists, clinicians, cell and molecular biologists and pharmacologists to better understand the physiology of water reabsorption in the kidney, the molecular basis of the disease and to propose protocols for rapid diagnosis and pharmacological handling of the disease. Much still remains to be done in terms of targeted therapy to make sure that these patients benefit from an improved quality of life. In this article we provide an overview on the most recent strategies under investigation for rescuing the mutated gene products activity or for bypassing defective vasopressin receptor signaling.

The mutation gain-of-function A843E of the Calcium Sensing Receptor (CASR) causes a Bartter syndrome type V. Patients carrying this CASR variant showed a remarkable reduced renal NaCl reabsorption in Thick Ascendent Limb (TAL) resulting in renal loss of NaCl in the absence of mutations in renal Na+ and Cl- ion transporters. The molecular mechanisms underlying this clinical phenotype are still unknown. Indeed, we investigated, in LLC-PK1 epithelial renal cells, the possible functional cross-talk of CASR-A843E mutant with NKCC2, the main transporter involved in the NaCl reabsorption in the TAL. The steady-state NKCC2 phosphorylation and activity were inhibited by about 60% in cells transfected with CASR-A843E mutant compared to CASR WT-transfected cells, used as control. Of note, the low-chloride-dependent NKCC2 activation was also strongly inhibited upon the expression of CASR-A843E mutant. Interestingly the maximal activation of CASR WT mimicked the effect of CASR-A843E on NKCC2 regulation. The expression of the CASR mutant did not alter the apical localization of NKCC2 in LLC-PK1 cells, suggesting that the CASR-A843E affected intracellular pathway/s modulating NKCC2 activity rather than NKCC2 intracellular trafficking. Our findings opened new avenues not only to gain more insight in the role of CASR in the physiopathology of the kidney, but also to conceive novel therapeutic strategies for the treatment of the Bartter syndrome type V.

Lamin A/C is a structural protein of the nuclear envelope (NE) and cardiac involvement in Lamin A/C mutations was one of the first phenotypes to be reported in humans, suggesting a crucial role of this protein in the cardiomyocytes function. Mutations in LMNA gene cause a class of pathologies generically named 'Lamanopathies' mainly involving heart and skeletal muscles. Moreover, the well-known disease called Hutchinson-Gilford Progeria Syndrome due to extensive mutations in LMNA gene, in addition to the systemic phenotype of premature aging, is characterised by the death of patients at around 13 typically for a heart attack or stroke, suggesting again the heart as the main site sensitive to Lamin A/C disfunction. Indeed, the identification of the roles of the Lamin A/C in cardiomyocytes function is a key area of exploration. One of the primary biological roles recently conferred to Lamin A/C is to affect contractile cells lineage determination and senescence. Then, in differentiated adult cardiomyocytes both the 'structural' and 'gene expression hypothesis' could explain the role of Lamin A in the function of cardiomyocytes. In fact, recent advances in the field propose that the structural weakness/stiffness of the NE, regulated by Lamin A/C amount in NE, can 'consequently' alter gene expression. © 2014 Société Française des Microscopies and Société de Biologie Cellulaire de France.

Aquaporin 2 (AQP2) is the vasopressin-regulated water channel palying a crucial role in urine concentration in the kidney. Rosiglitazone (RGZ), an agonist of the peroxisome proliferator-activated receptor gamma (PPARγ), increases AQP2 expression in normal rats. Here we tested whether RGZ could also modulate AQP2 intracellular trafficking in renal cells. Wild type C57BL-6 mice were treated for 3 days with 20mg/Kg RGZ to confirm the transcriptional effect on AQP2 in mice. In parallel, we used mouse MCD4 renal collecting duct cells to study the effect of RGZ on AQP2 trafficking to the apical membrane. Apical surface biotinylation and confocal analysis were used to semiquantify the effect. Single cell live microfluorimetry and FRET were used to measure changes in Ca2+ and cAMP intracellular concentrations, respectively. In mice, RGZ induced a twofold increase of AQP2 compared to control mice. In MCD4 cells, stimulation with 50µM RGZ for 30 min robustly increased AQP2 trafficking to the apical plasma membrane to an extent comparable to that elicited by forskolin stimulation. Interestingly, RGZ was able to increasely AQP2 phosphorylation at Ser256 concomitant with intracellular Ca++ increase and without apparent intracellular cAMP elevation. Taken together these results suggest that RGZ might be useful to increase AQP2 transcription and apical trafficking in renal cells bypassing the stimulation of vasopressin receptor which is defective in pathological conditions as in Nephrogenic Diabetes Insipidus.

Preliminary results by our group showed that exposure to Rosiglitazone (RGZ) induces phosphorylation and apical translocation of AQP2 in mouse collecting duct clone 4 (MCD4) cells. Here we studied the effect of short-term exposure to 50 µM RGZ on cAMP- and Ca2+-mediated signaling pathways, both key players for AQP2-mediated water reabsorption in the collecting duct. Cytosolic Ca2+ and cAMP levels were imaged in real-time in MCD4 cells loaded with Fura-2 or transiently transfected with the EPAC-based fluorescent probe H90, respectively. Physiologically, AQP2 phosphorylation/translocation depends on cytosolic cAMP levels. Nonetheless, cAMP measurements showed that RGZ did not induce significant changes in cAMP levels. Conversely, 20 minutes RGZ stimulation of Fura-2 loaded MCD4 cells induced a large, transient cytosolic Ca2+ peak that was not the result of direct blockade of the SERCA pump since the rate of store empting elicited by CPA in the absence of external Ca2+ was not significantly different in the presence of RGZ. Importantly, removal of external Ca2+ and inhibition of Ca2+ channels with ruthenium red prevented the RGZ-induced increase in cytosolic Ca2+ indicating a prominent role for Ca2+ entry at the plasma membrane. In conclusion, RGZ-induced AQP2 phosphorylation/translocation process is likely initiated by a fast, large extracellular Ca2+ influx most likely via Ca2+-dependent transient receptor potential channels. Further studies to ascertain which cascade of kinases is involved in this scenario are in progress.

The current interest of the scientific community for research in the field of calcium sensing in general and on the calcium-sensing Receptor (CaR) in particular is demonstrated by the still increasing number of papers published on this topic. The extracellular calcium-sensing receptor is the best-known G-protein-coupled receptor (GPCR) able to sense external Ca2+changes. Widely recognized as a fundamental player in systemic Ca2+homeostasis, the CaR is ubiquitously expressed in the human body where it activates multiple signalling pathways. In this review, old and new notions regarding the mechanisms by which extracellular Ca2+microdomains are created and the tools available to measure them are analyzed. After a survey of the main signalling pathways triggered by the CaR, a special attention is reserved for the emerging concepts regarding CaR function in the heart, CaR trafficking and pharmacology. Finally, an overview on other Ca2+sensors is provided.

Mutations in the Lamin A/C gene (LMNA), which encodes A-type nuclear Lamins, represent the most frequent genetic cause of dilated cardiomyopathy (DCM). This study is focused on a LMNA nonsense mutation (R321X) identified in several members of an Italian family that produces a truncated protein isoform, which co-segregates with a severe form of cardiomyopathy with poor prognosis. However, no molecular mechanisms other than nonsense mediated decay of the messenger and possible haploinsufficiency were proposed to explain DCM. Aim of this study was to gain more insights into the disease-causing mechanisms induced by the expression of R321X at cellular level. We detected the expression of R321X by Western blotting from whole lysate of a mutation carrier heart biopsy. When expressed in HEK293 cells, GFP- (or mCherry)-tagged R321X mislocalized in the endoplasmic reticulum (ER) inducing the PERK-CHOP axis of the ER stress response. Of note, confocal microscopy showed phosphorylation of PERK in sections of the mutation carrier heart biopsy. ER mislocalization of mCherry-R321X also induced impaired ER Ca(2+) handling, reduced capacitative Ca(2+) entry at the plasma membrane and abnormal nuclear Ca(2+) dynamics. In addition, expression of R321X by itself increased the apoptosis rate. In conclusion, R321X is the first LMNA mutant identified to date, which mislocalizes into the ER affecting cellular homeostasis mechanisms not strictly related to nuclear functions.

Store-Operated Cyclic AMP Signaling (SOcAMPS) represents a novel signaling mechanism in which depletion of Ca2+ in the endoplasmic reticulum (ER) leads to a STIM1- dependent (Stromal Interaction Molecule 1) increase in cAMP levels, independently of cytosolic Ca2+. Here we aimed to evaluate whether SOcAMPS was manifest in neonatal rat ventricular myocytes (NRVM) and human "iCardiomyocytes" and exploit its potential role in cardiac cell hypertrophy. cAMP levels and ER [Ca2+]were monitored by live cell fluorescence imaging after transfection with the EPAC H30 and D1ER cameleon probes, respectively. The existence of SOcAMPS in NRVM was first assessed by using the low affinity Ca2+ chelator TPEN, able to induce a reduction of SR Ca2+ levels without affecting cytosolic [Ca2+]. TPEN (1mM) was shown to induce significant cAMP increases both in the absence and presence of 5 M Forskolin (FRSK). Depletion of SR by ionomycin (10 M) was found to exert similar effects. Similar data were obtained in human "iCardiomyocytes". The participation of STIM1 in the observed phenomenon was proven in NRVM by the 47% reduction of the [cAMP] response obtained after shRNA-mediated knockdown of STIM1. Interestingly, a significant increase of the TPEN+FRSK induced response was found after "in vitro" induced cell hypertrophy. These data establish, for the first time, the existence of SOcAMPS in the two cardiac cell models analyzed and suggest a potential role for this new signaling mechanism in cardiac cell hypertrophy.

Background and Aims: Store-Operated Cyclic AMP Signaling (SOcAMPS) represents a recently identified mechanism of cross-talk between Ca2+ and cAMP signals. In this process, depletion of Ca2+ in the endoplasmic reticulum (ER) leads to increases in cAMP levels, independently of cytosolic Ca2+ changes. Expression and functionality of STIM1 (Stromal Interaction Molecule 1), a transmembrane ER Ca2+ sensor protein, is necessary for SOcAMPS to occur. Interestingly, recent reports have demonstrated a critical role for STIM1 in the development of cardiac hypertrophy, a process notoriously controlled both by Ca2+ and cAMP signaling. Here we aimed to evaluate whether SOcAMPS was manifest in neonatal rat cardiomyocytes and its potential role in cardiac cell hypertrophy. Methods: To monitor changes in cAMP levels, real time imaging experiments were performed on neonatal rat cardiomyocytes transiently transfected with an EPAC-based fluorescent probe for [cAMP], EPAC H30. Fura-2 and Fluo-4 were used to monitor cytosolic Ca2+ levels and an ER/SR targeted probe, D1ERcameleon, was used to measure ER [Ca2+]. Long term incubation (48h) of cardiomyocytes with angiotensin II (1 μM) and aldosterone (1 μM) was used to induce "in vitro" cell hypertrophy. Increases in cell size and/or sarcomere alignment were monitored microscopically after labeling with phalloidin-TRITC. Results: To verify the existence of SOcAMPS in neonatal rat cardiomyocytes, cells were stimulated in Ca2+-free Ringer's solutions with the low affinity membrane permeant Ca2+ chelator TPEN (1mM), able to induce a reduction of SR Ca2+ levels ([Ca2+]SR) without affecting cytosolic [Ca2+]. SR Ca2+ measurements demonstrated that under these experimental conditions, 1 mM TPEN led to a reduction in intraluminal [Ca2+] that was 50,5±2,4% (8 exp, 11 cells, p<0.001) of the maximal store depletion. Parallel experiments performed with the EPAC H30 cAMP sensor showed increases in [cAMP] that were 26,5±3% (13 exp, 13 cells, p<0.001) of the maximum delta ratio. In the presence of 5 μM Forskolin (FRSK) the TPEN-induced cAMP augmentation resulted 63,7±3,9% of the maximal response (16 exp, 19 cells, p<0.001). Also depletion of SR by the Ca2+ ionophore ionomycin (10 μM) was found to induce significant cAMP increases both in the absence and presence of FRSK. The participation of STIM1 in the observed phenomenon was proven by the 47 % reduction of the TPEN+FRSK induced [cAMP] signal after transfection of cells with a shRNA against STIM1 (6 exp, p<0,01). To evaluate the putative role of SOcAMPS in cardiac hypertrophy, cAMP measurements were performed on angio+aldo treated cells and compared to control cardiomyocytes. Under these experimental conditions a 20% increase of the TPEN+FRSK induced response was observed in hypertrophic myocytes (16 exp, p<0,01). Conclusions: These data straightforwardly establish, for the first time, the existence of SOcAMPS in the neonatal cardiomyocyte cell model. Also, a significantly increased SOcAMP signalling was shown to exist in hypertrophic cardiomyocytes. Further experiments to ascertain whether a causeand- effect relationship exists between SOcAMPS and cardiac cell hypertrophy are in progress.