NO hyperpolarizes pulmonary artery smooth muscle cells and decreases the intracellular Ca2+ concentration by activating voltage-gated K+ channels.

NO causes pulmonary vasodilation in patients with pulmonary hypertension. In pulmonary arterial smooth muscle cells, the activity of voltage-gated K+ (Kv) channels controls resting membrane potential. In turn, membrane potential is an important regulator of the intracellular free calcium concentration ([Ca2+]i) and pulmonary vascular tone. We used patch clamp methods to determine whether the NO-induced pulmonary vasodilation is mediated by activation of Kv channels. Quantitative fluorescence microscopy was employed to test the effect of NO on the depolarization-induced rise in [Ca2+]i. Blockade of Kv channels by 4-aminopyridine (5 mM) depolarized pulmonary artery myocytes to threshold for initiation of Ca2+ action potentials, and thereby increased [Ca2+]i. NO (approximately 3 microM) and the NO-generating compound sodium nitroprusside (5-10 microM) opened Kv channels in rat pulmonary artery smooth muscle cells. The enhanced K+ currents then hyperpolarized the cells, and blocked Ca(2+)-dependent action potentials, thereby preventing the evoked increases in [Ca2+]i. Nitroprusside also increased the probability of Kv channel opening in excised, outside-out membrane patches. This raises the possibility that NO may act either directly on the channel protein or on a closely associated molecule rather than via soluble guanylate cyclase. In isolated pulmonary arteries, 4-aminopyridine significantly inhibited NO-induced relaxation. We conclude that NO promotes the opening of Kv channels in pulmonary arterial smooth muscle cells. The resulting membrane hyperpolarization, which lowers [Ca2+]i, is apparently one of the mechanisms by which NO induces pulmonary vasodilation.

Increase of intracellular Ca2+ and relocation of E-cadherin during experimental decompaction of mouse embryos

To determine the role of intracellular Ca2+ in compaction, the first morphogenetic event in embryogenesis, we analyzed preimplantation mouse embryos under several decompacting conditions, including depletion of extracellular Ca2+, blocking of Ca2+ channels, and inhibition of microfilaments, calmodulin, and intracellular Ca2+ release. Those treatments induced decompaction of mouse morulae and simultaneously induced changes in cytosolic free Ca2+ concentration and deregionalization of E-cadherin and fodrin. When morulae were allowed to recompact, the location of both proteins recovered. In contrast, actin did not change its cortical location with compaction nor with decompaction-recompaction. Calmodulin localized in areas opposite to cell–cell contacts in eight-cell stage embryos before and after compaction. Inhibition of calmodulin with trifluoperazine induced its delocalization while morulae decompacted. A nonspecific rise of intracellular free Ca2+ provoked by ionomycin did not affect the compacted shape. Moreover, the same decompacting treatments when applied to uncompacted embryos did not produce any change in intracellular Ca2+. Our results demonstrate that in preimplantation mouse embryos experimentally induced stage-specific changes of cell shape are accompanied by changes of intracellular free Ca2+ and redistribution of the cytoskeleton-related proteins E-cadherin, fodrin, and calmodulin. We conclude that intracellular Ca2+ specifically is involved in compaction and probably regulates the function and localization of cytoskeleton elements.

Effect of β-amyloid block of the fast-inactivating K+ channel on intracellular Ca2+ and excitability in a modeled neuron

β-Amyloid peptide (Aβ), one of the primary protein components of senile plaques found in Alzheimer disease, is believed to be toxic to neurons by a mechanism that may involve loss of intracellular calcium regulation. We have previously shown that Aβ blocks the fast-inactivating potassium (A) current. In this work, we show, through the use of a mathematical model, that the Aβ-mediated block of the A current could result in increased intracellular calcium levels and increased membrane excitability, both of which have been observed in vitro upon acute exposure to Aβ. Simulation results are compared with experimental data from the literature; the simulations quantitatively capture the observed concentration dependence of the neuronal response and the level of increase in intracellular calcium.

Glucose stimulation of insulin release in the absence of extracellular Ca2+ and in the absence of any increase in intracellular Ca2+ in rat pancreatic islets.

Insulin secretion has been studied in isolated rat pancreatic islets under stringent Ca(2+)-depleted, Ca(2+)-free conditions. Under these conditions, the effect of 16.7 mM glucose to stimulate insulin release was abolished. Forskolin, which activates adenylyl cyclase, also failed to stimulate release in the presence of either low or high glucose concentrations. A phorbol ester (phorbol 12-myristate 13-acetate; PMA) increased the release rate slightly and this was further increased by 16.7 mM glucose. Remarkably, in the presence of both forskolin and PMA, 16.7 mM glucose strongly augmented insulin release. The augmentation was concentration dependent and monophasic and had a temporal profile similar to the "second phase" of glucose-stimulated insulin release, which is seen under normal conditions when Ca2+ is present. Metabolism is required for the effect because mannoheptulose abolished the glucose response. Other nutrient secretagogues, alpha-ketoisocaproate, and the combination of leucine and glutamine augmented release under the same conditions. Norepinephrine, a physiological inhibitor of insulin secretion, totally blocked the stimulation of release by forskolin and PMA and the augmentation of release by glucose. Thus, under the stringent Ca(2+)-free conditions imposed, the stimulation of insulin release by forskolin and PMA, as well as the augmentation of release by glucose, is under normal physiological control. As no increase in intracellular [Ca2+] was observed, the results demonstrate that glucose can increase the rate of exocytosis and insulin release by pancreatic islets in a Ca(2+)-independent manner. This interesting pathway of stimulus-secretion coupling for glucose appears to exert its effect at a site beyond the usual elevation of intracellular [Ca2+] and is not due to an activation by glucose of protein kinase A or C.

The cystic fibrosis transmembrane conductance regulator (CFTR) inhibits ENaC through an increase in the intracellular Cl- concentration

Activation of the CFTR Cl- channel inhibits epithelial Na+ channels (ENaC), according to studies on epithelial cells and overexpressing recombinant cells. Here we demonstrate that ENaC is inhibited during stimulation of the cystic fibrosis trans-membrance conductance regulator (CFTR) in Xenopus oocytes, independent of the experimental set-up and the magnitude of the whole-cell current. Inhibition of ENaC is augmented at higher CFTR Cl- currents. Similar to CFTR, ClC-0 Cl- currents also inhibit ENaC, as well as high extracellular Na+ and Cl- in partially permeabilized oocytes. Thus, inhibition of ENaC is not specific to CFTR and seems to be mediated by Cl-.

Muscarinic and nicotinic ACh receptor activation differentially mobilize Ca2+ in rat intracardiac ganglion neurons

The origin of intracellular Ca2+ concentration ([Ca2+](i)) transients stimulated by nicotinic ( nAChR) and muscarinic ( mAChR) receptor activation was investigated in fura-2-loaded neonatal rat intracardiac neurons. ACh evoked [Ca2+](i) increases that were reduced to similar to 60% of control in the presence of either atropine ( 1 muM) or mecamylamine ( 3 muM) and to < 20% in the presence of both antagonists. Removal of external Ca2+ reduced ACh-induced responses to 58% of control, which was unchanged in the presence of mecamylamine but reduced to 5% of control by atropine. The nAChR-induced [Ca2+](i) response was reduced to 50% by 10 μM ryanodine, whereas the mAChR-induced response was unaffected by ryanodine, suggesting that Ca2+ release from ryanodine-sensitive Ca2+ stores may only contribute to the nAChR-induced [Ca2+](i) responses. Perforated-patch whole cell recording at - 60 mV shows that the rise in [Ca2+](i) is concomitant with slow outward currents on mAChR activation and with rapid inward currents after nAChR activation. In conclusion, different signaling pathways mediate the rise in [Ca2+](i) and membrane currents evoked by ACh binding to nicotinic and muscarinic receptors in rat intracardiac neurons.

Obesity Resistance Promotes Mild Contractile Dysfunction Associated with Intracellular Ca2+ Handling

Abstract Background: Diet-induced obesity is frequently used to demonstrate cardiac dysfunction. However, some rats, like humans, are susceptible to developing an obesity phenotype, whereas others are resistant to that. Objective: To evaluate the association between obesity resistance and cardiac function, and the impact of obesity resistance on calcium handling. Methods: Thirty-day-old male Wistar rats were distributed into two groups, each with 54 animals: control (C; standard diet) and obese (four palatable high-fat diets) for 15 weeks. After the experimental protocol, rats consuming the high-fat diets were classified according to the adiposity index and subdivided into obesity-prone (OP) and obesity-resistant (OR). Nutritional profile, comorbidities, and cardiac remodeling were evaluated. Cardiac function was assessed by papillary muscle evaluation at baseline and after inotropic maneuvers. Results: The high-fat diets promoted increase in body fat and adiposity index in OP rats compared with C and OR rats. Glucose, lipid, and blood pressure profiles remained unchanged in OR rats. In addition, the total heart weight and the weight of the left and right ventricles in OR rats were lower than those in OP rats, but similar to those in C rats. Baseline cardiac muscle data were similar in all rats, but myocardial responsiveness to a post-rest contraction stimulus was compromised in OP and OR rats compared with C rats. Conclusion: Obesity resistance promoted specific changes in the contraction phase without changes in the relaxation phase. This mild abnormality may be related to intracellular Ca2+ handling.

T-type Ca2+ channels, SK2 channels and SERCAs gate sleep-related oscillations in thalamic dendrites.

T-type Ca2+ channels (T channels) underlie rhythmic burst discharges during neuronal oscillations that are typical during sleep. However, the Ca2+-dependent effectors that are selectively regulated by T currents remain unknown. We found that, in dendrites of nucleus reticularis thalami (nRt), intracellular Ca2+ concentration increases were dominated by Ca2+ influx through T channels and shaped rhythmic bursting via competition between Ca2+-dependent small-conductance (SK)-type K+ channels and Ca2+ uptake pumps. Oscillatory bursting was initiated via selective activation of dendritically located SK2 channels, whereas Ca2+ sequestration by sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) and cumulative T channel inactivation dampened oscillations. Sk2-/- (also known as Kcnn2) mice lacked cellular oscillations, showed a greater than threefold reduction in low-frequency rhythms in the electroencephalogram of non-rapid-eye-movement sleep and had disrupted sleep. Thus, the interplay of T channels, SK2 channels and SERCAs in nRt dendrites comprises a specialized Ca2+ signaling triad to regulate oscillatory dynamics related to sleep.

Interactions between intracellular Ca2+ stores: Ca2+ released from the NAADP pool potentiates cADPR-induced Ca2+ release

Cells possess multiple intracellular Ca2+-releasing systems. Sea urchin egg homogenates are a well-established model to study intracellular Ca2+ release. In the present study the mechanism of interaction between three intracellular Ca2+ pools, namely the nicotinic acid adenine dinucleotide phosphate (NAADP), the cyclic ADP-ribose (cADPR) and the inositol 1',4',5'-trisphosphate (IP3)-regulated Ca2+ stores, is explored. The data indicate that the NAADP Ca2+ pool could be used to sensitize the cADPR system. In contrast, the IP3 pool was not affected by the Ca2+ released by NAADP. The mechanism of potentiation of the cADPR-induced Ca2+ release, promoted by Ca2+ released from the NAADP pool, is mediated by the mechanism of Ca2+-induced Ca2+ release. These data raise the possibility that the NAADP Ca2+ store may have a role as a regulator of the cellular sensitivity to cADPR.

Juice of Bryophyllum pinnatum (Lam.) inhibits oxytocin-induced increase of the intracellular calcium concentration in human myometrial cells

http://www.ncbi.nlm.nih.gov/pubmed/20381326

Characterization of the Effect of the Mitochondrial Protein Hint2 on Intracellular Ca2+ dynamics

Hint2, one of the five members of the superfamily of the histidine triad AMP-lysine hydrolase proteins, is expressed in mitochondria of various cell types. In human adrenocarcinoma cells, Hint2 modulates Ca2+ handling by mitochondria. As Hint2 is highly expressed in hepatocytes, we investigated if this protein affects Ca2+ dynamics in this cell type. We found that in hepatocytes isolated from Hint2−/− mice, the frequency of Ca2+ oscillations induced by 1 μM noradrenaline was 150% higher than in the wild-type. Using spectrophotometry, we analyzed the rates of Ca2+ pumping in suspensions of mitochondria prepared from hepatocytes of either wild-type or Hint2−/− mice; we found that Hint2 accelerates Ca2+ pumping into mitochondria. We then resorted to computational modeling to elucidate the possible molecular target of Hint2 that could explain both observations. On the basis of a detailed model for mitochondrial metabolism proposed in another study, we identified the respiratory chain as the most probable target of Hint2. We then used the model to predict that the absence of Hint2 leads to a premature opening of the mitochondrial permeability transition pore in response to repetitive additions of Ca2+ in suspensions of mitochondria. This prediction was then confirmed experimentally.

Effects of extracellular Ca2+ concentration on hair-bundle stiffness and gating-spring integrity in hair cells

When a hair cell is stimulated by positive deflection of its hair bundle, increased tension in gating springs opens transduction channels, permitting cations to enter stereocilia and depolarize the cell. Ca2+ is thought to be required in mechanoelectrical transduction, for exposure of hair bundles to Ca2+ chelators eliminates responsiveness by disrupting tip links, filamentous interstereociliary connections that probably are the gating springs. Ca2+ also participates in adaptation to stimuli by controlling the activity of a molecular motor that sets gating-spring tension. Using a flexible glass fiber to measure hair-bundle stiffness, we investigated the effect of Ca2+ concentration on stiffness before and after the disruption of gating springs. The stiffness of intact hair bundles depended nonmonotonically on the extracellular Ca2+ concentration; the maximal stiffness of ≈1200 μN⋅m−1 occurred when bundles were bathed in solutions containing 250 μM Ca2+, approximately the concentration found in frog endolymph. For cells exposed to solutions with sufficient chelator capacity to reduce the Ca2+ concentration below ≈100 nM, hair-bundle stiffness fell to ≈200 μN⋅m−1 and no longer exhibited Ca2+-dependent changes. Because cells so treated lost mechanoelectrical transduction, we attribute the reduction in bundle stiffness to tip-link disruption. The results indicate that gating springs are not linearly elastic; instead, they stiffen with increased strain, which rises with adaptation-motor activity at the physiological extracellular Ca2+ concentration.

Ca2+/calmodulin-dependent kinase II mediates simultaneous enhancement of gap-junctional conductance and glutamatergic transmission

While chemical synapses are very plastic and modifiable by defined activity patterns, gap junctions, which mediate electrical transmission, have been classically perceived as passive intercellular channels. Excitatory transmission between auditory afferents and the goldfish Mauthner cell is mediated by coexisting gap junctions and glutamatergic synapses. Although an increased intracellular Ca2+ concentration is expected to reduce gap junctional conductance, both components of the synaptic response were instead enhanced by postsynaptic increases in Ca2+ concentration, produced by patterned synaptic activity or intradendritic Ca2+ injections. The synaptically induced potentiations were blocked by intradendritic injection of KN-93, a Ca2+/calmodulin-dependent kinase (CaM-K) inhibitor, or CaM-KIINtide, a potent and specific peptide inhibitor of CaM-KII, whereas the responses were potentiated by injection of an activated form of CaM-KII. The striking similarities of the mechanisms reported here with those proposed for long-term potentiation of mammalian glutamatergic synapses suggest that gap junctions are also similarly regulated and indicate a primary role for CaM-KII in shaping and regulating interneuronal communication, regardless of its modality.