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In microglia, changes in intracellular calcium concentration ([Ca2+]i) may regulate process motility, inflammasome activation, and phagocytosis. However, while neurons and astrocytes exhibit frequent spontaneous Ca2+ activity, microglial Ca2+ signals are much rarer and poorly understood. Here, we studied [Ca2+]i changes of microglia in acute brain slices using Fluo-4–loaded cells and mice expressing GCaMP5g in microglia. Spontaneous Ca2+ transients occurred ~ 5 times more frequently in individual microglial processes than in their somata. We assessed whether microglial Ca2+ responses change in Alzheimer's disease (AD) using AppNL−G−F knock-in mice. Proximity to Aβ plaques strongly affected microglial Ca2+ activity. Although spontaneous Ca2+ transients were unaffected in microglial processes, they were fivefold more frequent in microglial somata near Aβ plaques than in wild-type microglia. Microglia away from Aβ plaques in AD mice showed intermediate properties for morphology and Ca2+ responses, partly resembling those of wild-type microglia. By contrast, somatic Ca2+ responses evoked by tissue damage were less intense in microglia near Aβ plaques than in wild-type microglia, suggesting different mechanisms underlying spontaneous vs. damage-evoked Ca2+ signals. Finally, as similar processes occur in neurodegeneration and old age, we studied whether ageing affected microglial [Ca2+]i. Somatic damage-evoked Ca2+ responses were greatly reduced in microglia from old mice, as in the AD mice. In contrast to AD, however, old age did not alter the occurrence of spontaneous Ca2+ signals in microglial somata but reduced the rate of events in processes. Thus, we demonstrate distinct compartmentalised Ca2+ activity in microglia from healthy, aged and AD-like brains. [ABSTRACT FROM AUTHOR]

Intracellular Ca2+ ([Ca2+]i) signaling and catecholamine (CA) exocytosis from adrenal chromaffin cells (CCs) differ between mammalian species. These differences partly result from the different contributions of Ca2+-induced Ca2+-release (CICR) from internal stores, which boosts intracellular Ca2+ signals. Transient inhibition of the sarcoendoplasmic reticulum (SERCA) Ca2+ pump with cyclopiazonic acid (CPA) reduces CICR. Recently, Martínez-Ramírez et al. found that CPA had contrasting effects on catecholamine secretion and intracellular Ca2+ signals in mouse and bovine CCs, where it enhanced and inhibited exocytosis, respectively. After CPA withdrawal, exocytosis diminished in mouse CCs and increased in bovine CCs. These differences can be explained if mouse CCs have weak CICR and strong Ca2+ uptake, and the reverse is true for bovine CCs. Surprisingly, CPA slightly reduced the amplitude of Ca2+ signals in both mouse and bovine CCs. Here we examined the effects of CPA on stimulated CA exocytosis and Ca2+ signaling in rat CCs and investigated if it alters differently the responses of CCs from normotensive (WKY) or hypertensive (SHR) rats, which differ in the gain of CICR. Our results demonstrate that CPA application strongly inhibits voltage-gated exocytosis and Ca2+ transients in rat CCs, regardless of strain (SHR or WKY). Thus, despite the greater phylogenetic distance from the most recent common ancestors, suppression of endoplasmic reticulum (ER) Ca2+ uptake through CPA inhibits the CA secretion in rat CCs more similarly to bovine than mouse CCs, unveiling divergent evolutionary relationships in the mechanism of CA exocytosis of CCs between rodents. Agents that inhibit the SERCA pump, such as CPA, suppress catecholamine secretion equally well in WKY and SHR CCs and are not potential therapeutic agents for hypertension. Rat CCs display Ca2+ signals of varying widths. Some even show early and late Ca2+ components. Narrowing the Ca2+ transients by CPA and ryanodine suggests that the late component is mainly due to CICR. Simultaneous recordings of Ca2+ signaling and amperometry in CCs revealed the existence of a robust and predictable correlation between the kinetics of the whole-cell intracellular Ca2+ signal and the rate of exocytosis at the single-cell level. [ABSTRACT FROM AUTHOR]

Mechanisms of synergistic agonist stimulation and modulation of the electrochemical driving force for anion secretion are still not fully explored in human pancreatic duct epithelial cells. The first objective of this study was therefore to test whether combined agonist stimulation augments anion transport responses in the Capan-1 monolayer model of human pancreatic duct epithelium. The second objective was to test the influence of H+,K+-ATPase inhibition on anion transport in Capan-1 monolayers. The third objective was to analyze the expression and function of K+ channels in Capan-1, which could support anion secretion and cooperate with H+,K+-ATPases in pH and potassium homeostasis. The human pancreatic adenocarcinoma cell line Capan-1 was cultured conventionally or as polarized monolayers that were analyzed by Ussing chamber electrophysiological recordings. Single-cell intracellular calcium was assayed with Fura-2. mRNA isolated from Capan-1 was analyzed by use of the nCounter assay or RT-PCR. Protein expression was assessed by immunofluorescence and western blot analyses. Combined stimulation with different physiological agonists enhanced anion transport responses compared to single agonist stimulation. The responsiveness of Capan-1 cells to histamine was also revealed in these experiments. The H+,K+-ATPase inhibitor omeprazole reduced carbachol- and riluzole-induced anion transport responses. Transcript analyses revealed abundant TASK-2, TWIK-1, TWIK-2, TASK-5, KCa3.1, and KCNQ1 mRNA expression. KCNE1 mRNA and TREK-1, TREK-2, TASK-2, and KCNQ1 protein expression were also shown. This study shows that the Capan-1 model recapitulates key physiological aspects of a bicarbonate-secreting epithelium and constitutes a valuable model for functional studies on human pancreatic duct epithelium. [ABSTRACT FROM AUTHOR]

We studied the efficacy of a near-infrared laser (1475 nm) to activate rat dorsal root ganglion (DRG) neurons with short punctate radiant heat pulses (55 µm diameter) and investigated temporal and spatial summation properties for the transduction process for noxious heat at a subcellular level. Strength-duration curves (10–80 ms range) indicated a minimum power of 30.2mW for the induction of laser-induced calcium transients and a chronaxia of 13.9 ms. However, threshold energy increased with increasing stimulus duration suggesting substantial radial cooling of the laser spot. Increasing stimulus duration demonstrated suprathreshold intensity coding of calcium transients with less than linear gains (Stevens exponents 0.29/35mW, 0.38/60mW, 0.46/70mW). The competitive TRPV1 antagonist capsazepine blocked responses to short near-threshold stimuli and significantly reduced responses to longer duration suprathreshold heat. Heating 1/3 of the soma of a neuron was sufficient to induce calcium transients significantly above baseline (p < 0.05), but maximum amplitude was only achieved by centering the laser over the entire neuron. Heat-induced calcium increase was highest in heated cell parts but rapidly reached unstimulated areas reminiscent of spreading depolarization and opening of voltage-gated calcium channels. Full intracellular equilibrium took about 3 s, consistent with a diffusion process. In summary, we investigated transduction mechanisms for noxious laser heat pulses in native sensory neurons at milliseconds temporal and subcellular spatial resolution and characterized strength duration properties, intensity coding, and spatial summation within single neurons. Thermal excitation of parts of a nociceptor spread via both membrane depolarization and intracellular calcium diffusion. [ABSTRACT FROM AUTHOR]

Since damage induced by ischemia–reperfusion (I/R) involves alterations in Ca2+ homeostasis and is reduced by ischemic postconditioning (IP) and that CoCl2 can trigger changes resembling the response to a hypoxic event in normoxia and its blockade on Ca2+ current in heart muscle, our aim was to evaluate CoCl2 as an IP therapeutic tool. Mechanic and energetic parameters of isolated and arterially perfused male Wistar rat heart ventricles were simultaneously analyzed in a model of I/R in which 0.23 mmol/L CoCl2 was introduced upon reperfusion and kept or withdrawn after 20 min or introduced after 20 min of reperfusion. The presence of CoCl2 did not affect diastolic pressure but increased post-ischemic contractile recovery, which peaked at 20 min and decreased at the end of reperfusion. This decrease was prevented when CoCl2 was removed at 20 min of reperfusion. Total heat release increased throughout reperfusion, while economy increased between 15 and 25 min. No effect was observed when CoCl2 was introduced at 20 min of reperfusion. In addition, both the area under the contracture curve evoked by 10 mmol/L caffeine–36 mmol/L Na+ and the contracture tension relaxation rate were higher with CoCl2. Furthermore, CoCl2 decreased the number of arrhythmias during reperfusion and the ventricular damaged area. The presence of CoCl2 in reperfusion induces cardioprotection consistent with the improvement in cellular calcium handling. The use of CoCl2 constitutes a potential cardioprotective tool of clinical relevance. [ABSTRACT FROM AUTHOR]

The hypersecretory phenotype of adrenal chromaffin cells (CCs) from early spontaneously hypertensive rats (SHRs) mainly results from enhanced Ca2+-induced Ca2+-release (CICR). A key question is if these abnormalities can be traced to the prehypertensive stage. Spontaneous and stimulus-induced catecholamine exocytosis, intracellular Ca2+ signals, and dense-core granule size and density were examined in CCs from prehypertensive and hypertensive SHRs and compared with age-matched Wistar-Kyoto rats (WKY). During the prehypertensive stage, the depolarization-elicited catecholamine exocytosis was ~ 2.9-fold greater in SHR than in WKY CCs. Interestingly, in half of CCs the exocytosis was indistinguishable from WKY CCs, while it was between 3- and sixfold larger in the other half. Likewise, caffeine-induced exocytosis was ~ twofold larger in prehypertensive SHR. Accordingly, depolarization and caffeine application elicited [Ca2+]i rises ~ 1.5-fold larger in prehypertensive SHR than in WKY CCs. Ryanodine reduced the depolarization-induced secretion in prehypertensive SHR by 57%, compared to 14% in WKY CCs, suggesting a greater contribution of intracellular Ca2+ release to exocytosis. In SHR CCs, the mean spike amplitude and charge per spike were significantly larger than in WKY CCs, regardless of age and stimulus type. This difference in granule content could explain in part the enhanced exocytosis in SHR CCs. However, electron microscopy did not reveal significant differences in granule size between SHRs and WKY rats' adrenal medulla. Nonetheless, preSHR and hypSHR display 63% and 82% more granules than WKY, which could explain in part the enhanced catecholamine secretion. The mechanism responsible for the heterogeneous population of prehypertensive SHR CCs and the bias towards secreting more medium and large granules remains unexplained. [ABSTRACT FROM AUTHOR]

Cardiac alternans closely linked to calcium dysregulation is a crucial risk factor for fatal arrhythmia causing especially sudden death. Calcium overload is well-known to activate Ca2+-dependent protein kinase C (PKC); however, the effects of PKC on arrhythmogenic cardiac alternans have not yet been investigated. This study aimed to determine the contributions of PKC activities in cardiac alternans associated with calcium cycling disturbances. In the present study, action potential duration alternans (APD-ALT) induced by high free intracellular calcium ([Ca2+]i) exerted not only in a calcium concentration–dependent manner but also in a frequency-dependent manner. High [Ca2+]i–induced APD-ALT was suppressed by not only BAPTA-AM but also nifedipine. On the other hand, PKC inhibitors BIM and Gö 6976 eliminated high [Ca2+]i–induced APD-ALT, and PKC activator PMA was found to induce APD-ALT at normal [Ca2+]i condition. Furthermore, BIM effectively prevented calcium transient alternans (CaT-ALT) and even CaT disorders caused by calcium overload. Moreover, BIM not only eliminated electrocardiographic T-wave alternans (TWA) caused by calcium dysregulation, but also lowered the incidence of ventricular arrhythmias in isolated hearts. What's more, BIM prevented the expression of PKC α upregulated by calcium overload in high calcium–perfused hearts. We firstly found that pharmacologically inhibiting Ca2+-dependent PKC over-activation suppressed high [Ca2+]i–induced cardiac alternans. This recognition indicates that inhibition of PKC activities may become a therapeutic target for the prevention of pro-arrhythmogenic cardiac alternans associated with calcium dysregulation. [ABSTRACT FROM AUTHOR]

This document discusses the process of excitation-secretion coupling in chromaffin cells (CCs) of the adrenal gland. It explains that calcium influx through high voltage gated Ca2+ channels (HVCCs) triggers the release of catecholamine (CA)-containing vesicles, which are then released into the circulation. The document also highlights the importance of calcium release from intracellular stores in addition to HVCC Ca2+ influx for CA secretion. The study focuses on cultured CCs from normotensive and hypertensive rats, demonstrating that intracellular Ca2+ release is the major determinant of the excitation-secretion coupling mechanism. The document concludes by emphasizing the importance of CCs research in understanding the physiology and pathophysiology of Ca2+-dependent neurotransmission. [Extracted from the article]

One maladaptive consequence of inflammatory stimulation of the afferent somatosensory system is the manifestation of inflammatory pain. We established and characterized a neuroglial primary culture of the rat superficial dorsal horn (SDH) of the spinal cord to test responses of this structure to neurochemical, somatosensory, or inflammatory stimulation. Primary cultures of the rat SDH consist of neurons (43%), oligodendrocytes (35%), astrocytes (13%), and microglial cells (9%). Neurons of the SDH responded to cooling (7%), heating (18%), glutamate (80%), substance P (43%), prostaglandin E2 (8%), and KCl (100%) with transient increases in the intracellular calcium [Ca2+]i. Short-term stimulation of SDH primary cultures with LPS (10 μg/ml, 2 h) caused increased expression of pro-inflammatory cytokines, inflammatory transcription factors, and inducible enzymes responsible for inflammatory prostaglandin E2 synthesis. At the protein level, increased concentrations of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) were measured in the supernatants of LPS-stimulated SDH cultures and enhanced TNFα and IL-6 immunoreactivity was observed specifically in microglial cells. LPS-exposed microglial cells further showed increased nuclear immunoreactivity for the inflammatory transcription factors NFκB, NF-IL6, and pCREB, indicative of their activation. The short-term exposure to LPS further caused a reduction in the strength of substance P as opposed to glutamate-evoked Ca2+-signals in SDH neurons. However, long-term stimulation with a low dose of LPS (0.01 μg/ml, 24 h) resulted in a significant enhancement of glutamate-induced Ca2+ transients in SDH neurons, while substance P-evoked Ca2+ signals were not influenced. Our data suggest a critical role for microglial cells in the initiation of inflammatory processes within the SDH of the spinal cord, which are accompanied by a modulation of neuronal responses. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H2S) is endogenously produced in pancreatic ß cells and its level is elevated in diabetes. Here, we report that H2S affects insulin secretion via two mechanisms that converge on cytosolic free Ca2+ ([Ca2+]i), a key mediator of insulin exocytosis. Cellular calcium imaging, using Fura-2 or Fluo-4, showed that exposure of INS-1E cells to H2S (30–100 μM) reduced both [Ca2+]i levels (by 21.7 ± 2.3%) and oscillation frequency (p < 0.01, n = 4). Consistent with a role of plasma membrane KATP channels (plasma-KATP), the effects of H2S on [Ca2+]i were blocked by gliclazide (a blocker of plasma-KATP channels), but were mimicked by diazoxide (an activator of plasma-KATP channels). Surprisingly, when Ca2+ entry via plasma membrane was inhibited using Ca2+-free external solutions, H2S increased [Ca2+]i by 39.7 ± 3.6% suggesting Ca2+ release from intracellular stores. H2S-induced [Ca2+]i increases were abolished by either FCCP (which depletes Ca2+ stored in mitochondria) or cyclosporine A (an inhibitor of mitochondrial permeability transition pore, mPTP) suggesting that H2S induces Ca2+ release from mitochondria. Measurement of mitochondrial membrane potential (MMP) suggested that H2S causes MMP depolarization, which was blocked by cyclosporine A. Finally, insulin measurements by ELISA indicated that H2S decreased insulin release from INS-1E cells, but after plasma membrane Ca2+ entry was blocked by nifedipine, H2S-induced mitochondrial Ca2+ release is able to increase insulin release. Together, our results indicate that H2S has dual effects on insulin release suggesting that, with different metabolic conditions, H2S may differentially modulate the insulin release from pancreatic ß cells and play a role in ß cell dysfunction. [ABSTRACT FROM AUTHOR]

Gasotransmitter hydrogen sulphide (H2S) has emerged as a regulator of multiple physiological and pathophysiological processes throughout. Here, we have investigated the effects of NaHS (fast donor of H2S) and GYY4137 (GYY, slow donor of H2S) on the exocytotic release of catecholamines from fast-perifused bovine adrenal chromaffin cells (BCCs) challenged with sequential intermittent pulses of a K+-depolarizing solution. Both donors caused a concentration-dependent facilitation of secretion. This was not due to an augmentation of Ca2+ entry through voltage-activated Ca2+ channels (VACCs) because, in fact, NaHS and GYY caused a mild inhibition of whole-cell Ca2+ currents. Rather, the facilitation of exocytosis seemed to be associated to an augmented basal [Ca2+]c and the K+-elicited [Ca2+]c transients; such effects of H2S donors are aborted by cyclopiazonic acid (CPA), that causes endoplasmic reticulum (ER) Ca2+ depletion through sarcoendoplasmic reticulum Ca2+ ATPase inhibition and by protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), that impedes the ability of mitochondria to sequester cytosolic Ca2+ during cell depolarization. Inasmuch as CPA and FCCP reversed the facilitation of secretion triggered by K+ in the presence of NaHS and GYY, is seems that such facilitation is tightly coupled to Ca2+ handling by the ER and mitochondria. On the basis of these results, we propose that H2S regulates catecholamine secretory responses triggered by K+ in BCCs by (i) mobilisation of ER Ca2+ and (ii) interference with mitochondrial Ca2+ circulation. In so doing, the clearance of the [Ca2+]c transient will be delayed and the Ca2+-dependent trafficking of secretory vesicles will be enhanced to overfill the secretory machinery with new vesicles to enhance exocytosis. [ABSTRACT FROM AUTHOR]

Currently, there are no tailored therapies available for the treatment of right ventricular (RV) hypertrophy, and the cellular mechanisms that underlie the disease are poorly understood. We investigated the cellular changes that occur early in the progression of the disease, when RV hypertrophy is evident, but prior to the onset of heart failure. Intracellular Ca2+ ([Ca2+]i) handling was examined in a rat model of monocrotaline (MCT)-induced pulmonary hypertension and subsequent RV hypertrophy. [Ca2+]i and stress production were measured in isolated RV trabeculae under baseline conditions (1-Hz stimulation, 1.5 mM [Ca2+]o, 37 °C), and in response to inotropic interventions (5-Hz stimulation or 1-μM isoproterenol). Under baseline conditions, MCT trabeculae had impaired Ca2+ release in response to stimulation with a 45% delay in the time-to-peak Ca2+, but there was no difference in the amplitude and decay of the Ca2+ transient, or active stress relative to RV trabeculae from normotensive hearts (CON). Increasing stimulation frequency from 1 to 5 Hz increased stress in CON, but not MCT trabeculae. Similarly, β-adrenergic stimulation with isoproterenol increased Ca2+ transient amplitude and active stress in CON, but not in MCT trabeculae, despite accelerating Ca2+ transient decay in trabeculae from both groups. During isoproterenol treatment, MCT trabeculae showed increased diastolic Ca2+ leak, which may explain the blunted inotropic response to β-adrenergic stimulation. Confocal imaging of trabeculae fixed following functional measurements showed that myocytes were on average wider, and transverse-tubule organisation was disrupted in MCT which provides a mechanism to explain the observed slower release of Ca2+. [ABSTRACT FROM AUTHOR]

The so-called calcium paradoxon of renin describes the phenomenon that exocytosis of renin from juxtaglomerular cells of the kidney is stimulated by lowering of the extracellular calcium concentration. The yet poorly understood effect of extracellular calcium on renin secretion appears to depend on the function of the gap junction protein connexin 40 (Cx40) in renin-producing cells. This study aimed to elucidate the role of Cx40 for the calcium dependency of renin secretion in more detail by investigating if Cx40 function is really essential for the influence of extracellular calcium on renin secretion, if and how Cx40 affects intracellular calcium dynamics in renin-secreting cells and if Cx40-mediated gap junctional coupling of renin-secreting cells with the mesangial cell area is relevant for the influence of extracellular calcium on renin secretion. Renin secretion was studied in isolated perfused mouse kidneys. Calcium measurements were performed in renin-producing cells of microdissected glomeruli. The ultrastructure of renin-secreting cells was examined by electron microscopy. We found that Cx40 was not essential for stimulation of renin secretion by lowering of the extracellular calcium concentration. Instead, Cx40 increased the sensitivity of renin secretion response towards lowering of the extracellular calcium concentration. In line, the sensitivity and dynamics of intracellular calcium in response to lowering of extracellular calcium were dampened when renin-secreting cells lacked Cx40. Disruption of gap junctional coupling of renin-secreting cells by selective deletion of Cx40 from mesangial cells, however, did not change the stimulation of renin secretion by lowering of the extracellular calcium concentration. Deletion of Cx40 from renin cells but not from mesangial cells was associated with a shift of renin expression from perivascular cells of afferent arterioles to extraglomerular mesangial cells. Our findings suggest that Cx40 is not directly involved in the regulation of renin secretion by extracellular calcium. Instead, it appears that in renin-secreting cells of the kidney lacking Cx40, intracellular calcium dynamics and therefore also renin secretion are desensitized towards changes of extracellular calcium. Whether the dampened calcium response of renin-secreting cells lacking Cx40 function results from a direct involvement of Cx40 in intracellular calcium regulation or from the cell type shift of renin expression from perivascular to mesangial cells remains to be clarified. In any case, Cx40-mediated gap junctional coupling between renin and mesangial cells is not relevant for the calcium paradoxon of renin secretion. [ABSTRACT FROM AUTHOR]

Polycystic kidney disease 2-like-1 (PKD2L1), or polycystin-L or TRPP2, formerly TRPP3, is a transient receptor potential (TRP) superfamily member. It is a calcium-permeable non-selective cation channel that regulates intracellular calcium concentration and thereby calcium signaling. PKD2L1 has been reported to take part in hedgehog signaling in renal primary cilia and sour tasting coupling with PKD1L3. In addition to the previous reports, PKD2L1 is recently found to play a crucial role in localization with β2-adrenergic receptor (β2AR) on the neuronal primary cilia. The disruption of PKD2L1 leads to the loss of β2AR on the primary cilia and reduction in intracellular concentration of cyclic adenosine monophosphate (cAMP). Since the role of cAMP and PKA is frequently mentioned in the studies of PKD diseases, we investigated on the mechanism of cAMP regulation in relation to the function of PKD2L1 channel. In this study, we observed the activity of PKD2L1 channel increased by the downstream cascades of β2AR and found the clustered phosphorylation sites, Ser-682, Ser-685, and Ser-686 that are significant in the channel regulation by phosphorylation. [ABSTRACT FROM AUTHOR]

The role of gamma-aminobutyric acid (GABA) in adrenal medulla chromaffin cell (CC) function is just beginning to unfold. GABA is stored in catecholamine (CA)-containing dense core granules and is presumably released together with CA, ATP, and opioids in response to physiological stimuli, playing an autocrine-paracrine role on CCs. The reported paradoxical 'dual action' of GABA-R activation (enhancement of CA secretion and inhibition of synaptically evoked CA release) is only one aspect of GABA's multifaceted actions. In this review, we discuss recent physiological experiments on rat CCs in situ which suggest that GABA regulation of CC function may depend on the physiological context: During non-stressful conditions, GABA-R activation by endogenous GABA tonically inhibits acetylcholine release from splanchnic nerve terminals and decreases spontaneous Ca fluctuations in CCs, preventing unwanted CA secretion. During intense stress, splanchnic nerve terminals release acetylcholine, which depolarizes CCs and allows the Ca influx that triggers the release of CA and GABA. With time, CA secretion declines, due to voltage-independent inhibition of Cachannels and desensitization of cholinergic nicotinic receptors. Nonetheless, acute activation of GABA-R is depolarizing in about 50% of CCs, and thus GABA, acting as an autocrine/paracrine mediator, could help to maintain CA exocytosis under stress. GABA-R activation is not excitatory in about half of CCs' population because it hyperpolarizes them or elicits no response. This percentage possibly varies, depending on functional demands, since GABA-R-mediated actions are determined by the intracellular chloride concentration ([Cl]) and therefore on the activity of cation-chloride co transporters, which is functionally regulated. These findings underscore a potential importance of a novel and complex GABA-mediated regulation of CC function and of CA secretion. [ABSTRACT FROM AUTHOR]

Cardiac neuronal nitric oxide synthase (nNOS) is an important molecule that regulates intracellular Ca homeostasis and contractility of healthy and diseased hearts. Here, we examined the effects of nNOS on fatty acid (FA) regulation of left ventricular (LV) myocyte contraction in sham and angiotensin II (Ang II)-induced hypertensive (HTN) rats. Our results showed that palmitic acid (PA, 100 μM) increased the amplitudes of sarcomere shortening and intracellular ATP in sham but not in HTN despite oxygen consumption rate (OCR) was increased by PA in both groups. Carnitine palmitoyltransferase-1 inhibitor, etomoxir (ETO), reduced OCR and ATP with PA in sham and HTN but prevented PA potentiation of sarcomere shortening only in sham. PA increased nNOS-derived NO only in HTN. Inhibition of nNOS with S-methyl- l-thiocitrulline (SMTC) prevented PA-induced OCR and restored PA potentiation of myocyte contraction in HTN. Mechanistically, PA increased intracellular Ca transient ([Ca]) without changing Ca influx via L-type Ca channel (I-) and reduced myofilament Ca sensitivity in sham. nNOS inhibition increased [Ca], I- and reduced myofilament Ca sensitivity prior to PA supplementation; as such, normalized PA increment of [Ca]. In HTN, PA reduced I- without affecting [Ca] or myofilament Ca sensitivity. However, PA increased I-, [Ca] and reduced myofilament Ca sensitivity following nNOS inhibition. Myocardial FA oxidation (F-fluoro-6-thia-heptadecanoic acid, F-FTHA) was comparable between groups, but nNOS inhibition increased it only in HTN. Collectively, PA increases myocyte contraction through stimulating [Ca] and mitochondrial activity in healthy hearts. PA-dependent cardiac inotropy was limited by nNOS in HTN, predominantly due to its modulatory effect on [Ca] handling. [ABSTRACT FROM AUTHOR]

The aim of the present study was to examine the contribution of intracellular and extracellular calcium sources in contraction caused by noradrenaline (NA) of the human internal thoracic artery (ITA) in vitro. Distal segments of ITA were obtained from 20 patients (aged 38-73, at the time of routine coronary artery surgical revascularization (CABG)). Contractile responses to 10 mol/L NA in the physiological salt solution and in Ca-free solution without and after incubation with 10 mol/L thapsigargin (TSG) were recorded under isometric conditions. Responses of ITA rings to 1 μM NA without incubation with TSG accounted (% of reaction to 80 mM KCl) 224.70 ± 14.06% in PSS solution, 141.30 ± 8.66% in Ca-free solution, and 80.03 ± 1.71% after PSS restoration and were statistically significantly different ( p < 0.0001, one-way ANOVA). Responses of ITA rings to 1 μM NA with 1 μM TSG accounted (% of reaction to 80 mM KCl) 114.50 ± 2.79% in Ca-free solution and 36.70 ± 2.38% after PSS restoration. Responses in Ca-free solution and after PSS restoration without and with TSG were statistically significantly different ( p = 0.0257 and p < 0.0001, respectively- t test). ITA contraction is caused by calcium derived not only from the SR and the extracellular matrix. The delivery of calcium to the space surrounding tissue does not immediately deliver calcium to the myofilaments. [ABSTRACT FROM AUTHOR]

Synchrony of spontaneous Ca transients among venular mural cells (smooth muscle cells and pericytes) in visceral organs relies on the intercellular spread of L-type voltage-dependent Ca channel (LVDCC)-dependent depolarisations. However, the mechanisms underlying the synchrony of spontaneous Ca transients between arteriolar mural cells are less understood. The spontaneous intracellular Ca dynamics of arteriolar mural cells in the rat rectal submucosa were visualised by Cal-520 Ca imaging to analyse their synchrony. The mural cells in fine arterioles that had a rounded cell body with several extended processes developed spontaneous 'synchronous' Ca transients arising from Ca released from sarcoendoplasmic reticulum Ca stores. Gap junction blockers (3 μM carbenoxolone, 10 μM 18β-glycyrrhetinic acid), a Ca-activated Cl channel (CaCC) blocker (100 μM 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid) or lowering extracellular Cl concentration (from 134.4 to 12.4 mM) disrupted the synchrony of Ca transients between arteriolar mural cells. Blockers of T-type voltage-dependent Ca channels (TVDCCs, 1 μM mibefradil or ML218) or LVDCCs (1 μM nifedipine) reduced the Ca transient frequency or their area under curve (AUC), respectively. However, neither TVDCC nor LVDCC blockers disrupted the synchrony of Ca transients among arteriolar mural cells. This is in contrast with rectal venules in which nifedipine disrupted the synchrony of spontaneous Ca transients. Thus, spontaneous transient depolarisations arising from the opening of CaCCs may effectively spread to neighbouring arteriolar mural cells via gap junctions to maintain the Ca transient synchrony. Activation of TVDCCs appears to accelerate spontaneous Ca transients, while LVDCCs predominantly contribute to the duration of Ca transients. [ABSTRACT FROM AUTHOR]

This study demonstrated that PDE1 (phosphodiesterase 1) existing in the ciliary beat frequency (CBF)-regulating metabolon regulates CBF in procaterol-stimulated lung airway ciliary cells of mouse. Procaterol (an β-agonist) increased the ciliary bend angle (CBA) and CBF via cAMP accumulation in the ciliary cells of mice: interestingly, the time course of CBF increase was slower than that of CBA increase. However, IBMX (3-isobutyl-1-methylxanthine, an inhibitor of PDE) increased CBA and CBF in an identical time course. Lowering an intracellular Ca concentration ([Ca]) caused by switching to an EGTA-containing Ca-free solution from normal one elevated the procaterol-induced increasing rate of CBF. These observations suggest that Ca-dependent PDE1 controls cAMP-stimulated CBF increase. Either application of 8MmIBMX (8-methoxymethyl-IBMX, a selective PDE1 inhibitor), BAPTA-AM (an intracellular Ca chelator), or calmidazolium (an inhibitior of calmodulin) alone increased CBA and CBF in the lung airway ciliary cells and increased cAMP contents in the isolated lung cells, and like IBMX, each application of the compound made the time courses of CBA and CBF increase stimulated by procaterol identical. The immunoelectron microscopic examinations revealed that PDE1A exists in the space between the nine doublet tubules ring and plasma membrane in the lung airway cilium, where the outer dynein arm (a molecular motor regulating CBF) functions. In conclusion, PDE1A is a key factor slowing the time course of the procaterol-induced increase in CBF via degradation of cAMP in the CBF-regulating metabolon of the mouse lung airway cilia. [ABSTRACT FROM AUTHOR]

The Src tyrosine kinase family contributes to the signalling mechanism mediating serotonin (5-hydroxytryptamine (5-HT))-induced vasoconstriction. These kinases were reported to influence the calcium sensitivity of the contractile apparatus. Whether Src kinases affect also the intracellular calcium concentration during constriction of intact arteries is unknown. Thus, we tested the hypothesis that constriction of arteries is associated with a Src kinase-dependent alteration of the intracellular calcium concentration. Contractility of gracilis arteries of Wistar rats was studied using isometric and isobaric myography. The intracellular calcium concentration was measured simultaneously with tension by FURA-2 fluorimetry. Inhibition of Src kinases with 10 μM PP2, 30 μM dasatinib and 100 μM AZM 475271 resulted in a strong attenuation of 5-HT-induced contractions. Vessel incubation with 10 μM PP3, an inactive analogue of PP2, had no effect. Removal of the endothelium did not alter vessel contractile responses to 5-HT nor the action of the Src-kinase inhibitor PP2. The PP2-mediated inhibition of 5-HT-induced contraction was associated with a reduced response of [Ca] to 5-HT. In particular, inhibition of Src kinases attenuates 5-HT-induced calcium influx as well as calcium release from intracellular stores. In contrast, the calcium sensitivity of the contractile apparatus and the filling state of the sarcoplasmic reticulum were not influenced by Src kinases during 5-HT-induced contractions. We conclude that Src kinase activation is a powerful mechanism to produce vasoconstriction of small skeletal muscle arteries of rats. This effect is endothelium-independent. The data further suggest that the action of c-Src kinases is associated with a change in the intracellular calcium concentration that involves Ca entry and Ca release pathways. [ABSTRACT FROM AUTHOR]

It is widely accepted that aldosterone induces atrial fibrillation (AF) by promoting structural changes, but its effects on the function of primary atrial myocytes remain unknown. We have investigated this point in adult rat atrial myocytes, chronically exposed to the hormone. This treatment produced larger amplitude of Ca transients, longer action potential (AP) duration, and higher incidence of unsynchronized Ca oscillations. Moreover, it also gave rise to increases in both cell membrane capacitance (C, 30 %) and activity of L-type Ca channels (LTCCs, 100 %). Concerning K currents, a twofold increase was also observed, but only in a delayed rectifier component (I). Interestingly, the maximal conductance (G) of Na channels was also enhanced, but it occurred in the face of a negative shift in the voltage dependence of inactivation. Thus, at physiological potentials, a decreased fraction of available channels neutralized the effect on G. With regard to the effects on both C and LTCCs, they involved activation of mineralocorticoid receptors (MRs), were dose-dependent (EC ∼20-130 nM), and developed and recovered in days. Neither gating currents nor protein levels of LTCCs were altered. Instead, the effect on LTCCs was mimicked by cAMP, reverted by a PKA inhibitor, and attenuated by a nitric oxide donor (short-term exposures). Both EGTA and the antioxidant NAC prevented the increase in C, without significantly interfering with the upregulation of LTCCs. Overall, these results show that chronic exposures to aldosterone result in dire functional changes at the single myocyte level, which may explain the link between aldosteronism and AF. [ABSTRACT FROM AUTHOR]

Multiphoton imaging has been utilized to analyze in vivo immune cell dynamics over the last 15 years. Particularly, it has deepened the understanding of how immune responses are organized by immune cell migration and interactions. In this review, we first describe the following technical advances in recent imaging studies that contributed to the new findings on the regulation of immune responses and inflammation. Improved multicolor imaging of immune cell behavior has revealed that their interactions are spatiotemporally coordinated to achieve efficient and long-term immunity. The use of photoactivatable and photoconvertible fluorescent proteins has increased duration and volume of cell tracking, even enabling the analysis of inter-organ migration of immune cells. In addition, visualization of immune cell activation using biosensors for intracellular calcium concentration and signaling molecule activities has started to give further mechanistic insights. Then, we also introduce recent imaging analyses of interactions between immune cells and non-immune cells including endothelial, fibroblastic, epithelial, and nerve cells. It is argued that future imaging studies that apply updated technical advances to analyze interactions between immune cells and non-immune cells will be important for thorough physiological understanding of the immune system. [ABSTRACT FROM AUTHOR]

Anions such as Cl and HCO are well known to play an important role in glucose-stimulated insulin secretion (GSIS). In this study, we demonstrate that glucose-induced Cl efflux from β-cells is mediated by the Ca-activated Cl channel anoctamin 1 (Ano1). Ano1 expression in rat β-cells is demonstrated by reverse transcriptase-polymerase chain reaction, western blotting, and immunohistochemistry. Typical Ano1 currents are observed in whole-cell and inside-out patches in the presence of intracellular Ca: at 1 μM, the Cl current is outwardly rectifying, and at 2 μM, it becomes almost linear. The relative permeabilities of monovalent anions are NO (1.83 ± 0.10) > Br (1.42 ± 0.07) > Cl (1.0). A linear single-channel current-voltage relationship shows a conductance of 8.37 pS. These currents are nearly abolished by blocking Ano1 antibodies or by the inhibitors 2-(5-ethyl-4-hydroxy-6-methylpyrimidin-2-ylthio)- N-(4-(4-methoxyphenyl)thiazol-2-yl)acetamide (T-AO1) and tannic acid (TA). These inhibitors induce a strong decrease of 16.7-mM glucose-stimulated action potential rate (at least 87 % on dispersed cells) and a partial membrane repolarization with T-AO1. They abolish or strongly inhibit the GSIS increment at 8.3 mM and at 16.7 mM glucose. Blocking Ano1 antibodies also abolish the 16.7-mM GSIS increment. Combined treatment with bumetanide and acetazolamide in low Cl and HCO media provokes a 65 % reduction in action potential (AP) amplitude and a 15-mV AP peak repolarization. Although the mechanism triggering Ano1 opening remains to be established, the present data demonstrate that Ano1 is required to sustain glucose-stimulated membrane potential oscillations and insulin secretion. [ABSTRACT FROM AUTHOR]

Ca-activated Cl channels (CaCCs) are a class of Cl channels activated by intracellular Ca that are known to mediate numerous physiological functions. In 2008, the molecular identity of CaCCs was found to be anoctamin 1 (ANO1/TMEM16A). Its roles have been studied in electrophysiological, histological, and genetic aspects. ANO1 is known to mediate Cl secretion in secretory epithelia such as airways, salivary glands, intestines, renal tubules, and sweat glands. ANO1 is a heat sensor activated by noxious heat in somatosensory neurons and mediates acute pain sensation as well as chronic pain. ANO1 is also observed in vascular as well as airway smooth muscles, controlling vascular tone as well as airway hypersensitivity. ANO1 is upregulated in numerous types of cancers and thus thought to be involved in tumorigenesis. ANO1 is also found in proliferating cells. In addition to ANO1, involvement of its paralogs in pathophysiological conditions was also reported. ANO2 is involved in olfaction, whereas ANO6 works as a scramblase whose mutation causes a rare bleeding disorder, the Scott syndrome. ANO5 is associated with muscle and bone diseases. Recently, an X-ray crystal structure of a fungal TMEM16 was reported, which explains a precise molecular gating mechanism as well as ion conduction or phospholipid transport across the plasma membrane. [ABSTRACT FROM AUTHOR]

During cell swelling, Cl channels are activated to lower intracellular Cl concentrations and to reduce cell volume, a process termed regulatory volume decrease (RVD). We show that anoctamin 6 (ANO6; TMEM16F) produces volume-regulated anion currents and controls cell volume in four unrelated cell types. Volume regulation is compromised in freshly isolated intestinal epithelial cells from Ano6−/− mice and also in lymphocytes from a patient lacking expression of ANO6. Ca influx is activated and thus ANO6 is stimulated during cell swelling by local Ca increase probably in functional nanodomains near the plasma membrane. This leads to stimulation of phospholipase A (PLA) and generation of plasma membrane lysophospholipids, which activates ANO6. Direct application of lysophospholipids also activates an anion current that is inhibited by typical ANO6 blocker. An increase in intracellular Ca supports activation of ANO6, but is not required when PLA is fully activated, while re-addition of arachidonic acid completely blocked ANO6. Moreover, ANO6 is activated by low intracellular Cl concentrations and may therefore operate as a cellular osmosensor. High intracellular Cl concentration inhibits ANO6 and activation by PLA. Taken together, ANO6 supports volume regulation and volume activation of anion currents by action as a Cl channel or by scrambling membrane phospholipids. Thereby, it may support the function of LRRC8 proteins. [ABSTRACT FROM AUTHOR]

Using fluorescence [Ca] imaging in rat adrenal slices, we characterized the effects of agonists and antagonists of the GABA receptor (GABA-R) on resting intracellular Ca ([Ca]) and spontaneous [Ca] fluctuations (SCFs) in hundreds of individual chromaffin cells (CCs) recorded simultaneously in situ. Muscimol, a GABA-R agonist (20 μM; 25 s), induced an increase of resting [Ca] in 43 ± 3 % of CCs, a decrease in 26 ± 2 %, and no response in 30 ± 5 %. In Ca-free external medium, SCFs ceased completely and muscimol failed to elicit [Ca] rises. All muscimol-induced [Ca] changes were blocked by the GABA-R antagonist bicuculline, suggesting that they result from changes in membrane potential depending on the cell's Cl equilibrium potential. Unexpectedly, bicuculline increased the amplitude and frequency of SCFs in 54 % of CCs, revealing a tonic inhibition of SCFs by ambient GABA acting through GABA-R. Mecamylamine (a specific nicotinic cholinergic blocker) decreased basal SCF activity in 18 % of CCs and inhibited bicuculline-induced SCF intensification, suggesting that spontaneous acetylcholine (ACh) release from nerve endings contributes to SCF generation in CCs in situ and that blockade of presynaptic GABA-Rs intensifies SCFs in part through the disinhibition of spontaneous cholinergic transmission. Electrophysiological experiments confirmed that spontaneous excitatory postsynaptic currents recorded from CCs in situ were enhanced by bicuculline. To our knowledge, this is the first description of a regulatory effect of endogenous GABA on synaptic currents and SCFs of adrenal CCs. These findings denote a novel GABA-mediated presynaptic and postsynaptic regulatory mechanism of CC activity which may participate in the control of catecholamine secretion. [ABSTRACT FROM AUTHOR]

Postcapillary venules (PCVs) play a critical role in regulating capillary hydrostatic pressure, but their contractile mechanisms are not well understood. We examined the properties of spontaneous vasomotion and corresponding Ca transients in gastric PCV. In the rat gastric submucosa, changes in PCV diameter and intracellular Ca dynamics were visualised by video tracking system and fluorescent Ca imaging, respectively, while PCV morphology was examined by immunohistochemistry. Stellate-shaped PCV mural cells expressing α-smooth muscle actin exhibited synchronised spontaneous Ca transients to develop vasomotion which was abolished by nifedipine (1 μM), cyclopiazonic acid (10 μM), or Ca-activated Cl channel inhibitors (100 μM niflumic acid, 1 μM T16Ainh-A01). A gap junction blocker (3 μM carbenoxolone) disrupted the synchrony of spontaneous Ca transients amongst PCV mural cells and attenuated spontaneous vasomotion. Low chloride solution ([Cl] = 12.4 mM) also disrupted the synchrony of spontaneous Ca transients and abolished vasomotion. Na-K-Cl co-transporter inhibitors (10 μM bumetanide, 30 μM furosemide) suppressed spontaneous Ca transients and vasoconstrictions. A phosphodiesterase type 5 (PDE5) inhibitor (1 μM tadalafil) disrupted the spontaneous Ca transient synchrony and abolished vasomotion in a nitric oxide (NO)-dependent manner. Thus, gastric PCVs exhibit spontaneous vasomotion, resulting from synchronised spontaneous Ca transients within a network of stellate-shaped PCV mural cells. An active Cl accumulation partly via Na-K-Cl co-transport appears to be fundamental in maintaining depolarisation upon the opening of Ca-activated Cl channels that triggers Ca influx via voltage-dependent L-type Ca channels. Basal PDE5 activity may continuously counteract vaso-relaxing effects of endothelial NO to maintain spontaneous vasomotion. [ABSTRACT FROM AUTHOR]

Low-voltage-activated Ca3 channels are distinguished among other voltage-activated calcium channels by the most negative voltage activation threshold. The voltage dependence of current activation is virtually identical in all three Ca3 channels while the current kinetics of the Ca3.3 current is one order slower than that of the Ca3.1 and Ca3.2 channels. We have analyzed the voltage dependence and kinetics of charge (Q) movement in human recombinant Ca3.3 and Ca3.1 channels. The voltage dependence of voltage sensor activation (Q-V) of the Ca3.3 channel was significantly shifted with respect to that of the Ca3.1 channel by +18.6 mV and the kinetic of Q activation in the Ca3.3 channel was significantly slower than that of the Ca3.1 channel. Removal of the gating brake in the intracellular loop connecting repeats I and II in the Ca3.3 channel in the ID12 mutant channel shifted the Q-V relation to a value even more negative than that for the Ca3.1 channel. The kinetic of Q activation was not significantly different between ID12 and Ca3.1 channels. Deletion of the gating brake in the Ca3.1 channel resulted in a GD12 channel with the voltage dependence of the gating current activation significantly shifted toward more negative potentials. The Q kinetic was not significantly altered. ID12 and GD12 mutants did not differ significantly in voltage dependence nor in the kinetic of voltage sensor activation. In conclusion, the putative gating brake in the intracellular loop connecting repeats I and II controls the gating current of the Ca3 channels. We suggest that activation of the voltage sensor in domain I is limiting both the voltage dependence and the kinetics of Ca3 channel activation. [ABSTRACT FROM AUTHOR]

Intestinal epithelial electrolyte secretion is activated by increase in intracellular cAMP or Ca and opening of apical Cl channels. In infants and young animals, but not in adults, Ca-activated chloride channels may cause secretory diarrhea during rotavirus infection. While detailed knowledge exists concerning the contribution of cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) channels, analysis of the role of Ca-dependent Cl channels became possible through identification of the anoctamin (TMEM16) family of proteins. We demonstrate expression of several anoctamin paralogues in mouse small and large intestines. Using intestinal-specific mouse knockout models for anoctamin 1 (Ano1) and anoctamin 10 (Ano10) and a conventional knockout model for anoctamin 6 (Ano6), we demonstrate the role of anoctamins for Ca-dependent Cl secretion induced by the muscarinic agonist carbachol (CCH). Ano1 is preferentially expressed in the ileum and large intestine, where it supports Ca-activated Cl secretion. In contrast, Ano10 is essential for Ca-dependent Cl secretion in jejunum, where expression of Ano1 was not detected. Although broadly expressed, Ano6 has no role in intestinal cholinergic Cl secretion. Ano1 is located in a basolateral compartment/membrane rather than in the apical membrane, where it supports CCH-induced Ca increase, while the essential and possibly only apical Cl channel is CFTR. These results define a new role of Ano1 for intestinal Ca-dependent Cl secretion and demonstrate for the first time a contribution of Ano10 to intestinal transport. [ABSTRACT FROM AUTHOR]

Bile acids play important physiological role in the solubilisation and absorption of dietary lipids. However, under pathophysiological conditions, such as short bowel syndrome, they can reach the colon in high concentrations inducing diarrhoea. In this study, our aim was to characterise the cellular pathomechanism of bile-induced diarrhoea using human samples. Colonic crypts were isolated from biopsies of patients (controls with negative colonoscopic findings) and of cholecystectomised/ileum-resected patients with or without diarrhoea. In vitro measurement of the transporter activities revealed impaired Na/H exchanger (NHE) and Cl/HCO exchanger (CBE) activities in cholecystectomised/ileum-resected patients suffering from diarrhoea, compared to control patients. Acute treatment of colonic crypts with 0.3 mM chenodeoxycholate caused dose-dependent intracellular acidosis; moreover, the activities of acid/base transporters (NHE and CBE) were strongly impaired. This concentration of chenodeoxycholate did not cause morphological changes in colonic epithelial cells, although significantly reduced the intracellular ATP level, decreased mitochondrial transmembrane potential and caused sustained intracellular Ca elevation. We also showed that chenodeoxycholate induced Ca release from the endoplasmic reticulum and extracellular Ca influx contributing to the Ca elevation. Importantly, our results suggest that the chenodeoxycholate-induced inhibition of NHE activities was ATP-dependent, whereas the inhibition of CBE activity was mediated by the sustained Ca elevation. We suggest that bile acids inhibit the function of ion transporters via cellular energy breakdown and Ca overload in human colonic epithelial cells, which can reduce fluid and electrolyte absorption in the colon and promote the development of diarrhoea. [ABSTRACT FROM AUTHOR]

We have recently identified a class of dihydropyridine (DHP) analogues with 30-fold selectivity for T-type over L-type calcium channels that could be attributed to a modification of a key ester moiety. Based on these results, we examined a second series of compounds with similar attributes to determine if they had enhanced affinity for T-type channels. Whole-cell patch clamp experiments in transfected tsA-201 cells were used to screen these DHP derivatives for high affinity and selectivity for Ca3.2 over Ca1.2 L-type channels. The effects of the two lead compounds, termed N10 and N12, on Ca3.2 channel activity and gating were characterized in detail. When delivered intrathecally or intraperitoneally, these compounds mediated analgesia in a mouse model of acute inflammatory pain. The best compound from the initial screening, N12, was also able to reverse mechanical hyperalgesia produced by nerve injury. The compounds were ineffective in Ca3.2 null mice. Altogether, our data reveal a novel class of T-type channel blocking DHPs for potential pain therapies. [ABSTRACT FROM AUTHOR]

The functional presence of type-2 cannabinoid receptors (CBRs) in layer II/III pyramidal neurons of the rat medial prefrontal cortex (mPFC) was recently demonstrated. In the present study, we show that the application of the endocannabinoids (eCBs) 2-arachidonoylglycerol (2-AG) and methanandamide [a stable analog of the eCB anandamide (AEA)] can activate CBRs of mPFC layer II/III pyramidal neurons, which subsequently induces a Cl current. In addition, we show that action potential (AP) firing evoked by 20-Hz current injections results in an eCB-mediated opening of Cl channels via CBR activation. This AP-evoked synthesis of eCBs is dependent on the Ca influx through N-type voltage-gated calcium channels. Our results indicate that 2-AG is the main eCB involved in this process. Finally, we demonstrate that under physiologically relevant intracellular Cl conditions, 20-Hz AP firing leads to a CBR-dependent reduction in neuronal excitability. Altogether, our data indicate that eCBs released upon action potential firing can modulate, through CBR activation, neuronal activity in the mPFC. We discuss how this may be a mechanism to prevent excessive neuronal firing. [ABSTRACT FROM AUTHOR]

In addition to the TSH-cyclic AMP signalling pathway, calcium signalling is of crucial importance in thyroid cells. Although the importance of calcium signalling has been thoroughly investigated for several decades, the nature of the calcium channels involved in signalling is unknown. In a recent series of investigations using the well-studied rat thyroid FRTL-5 cell line, we showed that these cells exclusively express the transient receptor potential canonical 2 (TRPC2) channel. Our results suggested that the TRPC2 channel is of significant importance in regulating thyroid cell function. These investigations were the first to show that thyroid cells express a member of the TRPC family of ion channels. In this review, we will describe the importance of the TRPC2 channel in regulating TSH receptor expression, thyroglobulin maturation, intracellular calcium and iodide homeostasis and that the channel also regulates thyroid cell proliferation. [ABSTRACT FROM AUTHOR]

The presence of Ca-activated Cl channels (CaCCs) in vascular smooth muscle cells (SMCs) is well established. Their molecular identity is, however, elusive. Two distinct Ca-activated Cl currents ( I) were previously characterized in SMCs. We have shown that the cGMP-dependent I depends on bestrophin expression, while the 'classical' I is not. Downregulation of bestrophins did not affect arterial contraction but inhibited the rhythmic contractions, vasomotion. In this study, we have used in vivo siRNA transfection of rat mesenteric small arteries to investigate the role of a putative CaCC, TMEM16A. Isometric force, [Ca], and SMC membrane potential were measured in isolated arterial segments. I and GTPγS-induced nonselective cation current were measured in isolated SMCs. Downregulation of TMEM16A resulted in inhibition of both the cGMP-dependent I and the 'classical' I in SMCs. TMEM16A downregulation also reduced expression of bestrophins. TMEM16A downregulation suppressed vasomotion both in vivo and in vitro. Downregulation of TMEM16A reduced agonist (noradrenaline and vasopressin) and K-induced contractions. In accordance with the depolarizing role of CaCCs, TMEM16A downregulation suppressed agonist-induced depolarization and elevation in [Ca]. Surprisingly, K-induced depolarization was unchanged but Ca entry was reduced. We suggested that this is due to reduced expression of the L-type Ca channels, as observed at the mRNA level. Thus, the importance of TMEM16A for contraction is, at least in part, independent from membrane potential. This study demonstrates the significance of TMEM16A for two SMCs I and vascular function and suggests an interaction between TMEM16A and L-type Ca channels. [ABSTRACT FROM AUTHOR]

In this article, we review recent advances in our understanding of arrhythmia mechanisms in the failing heart. We focus on changes in repolarization, conduction, and intracellular calcium cycling because of their importance to the vast majority of clinical arrhythmias in heart failure. We highlight recent efforts to combat arrhythmias using gene-based approaches that target ion channel, gap junction, and calcium cycling proteins. We further discuss the advantages and limitations associated with individual approaches. [ABSTRACT FROM AUTHOR]

Skeletal muscle excitation-contraction (E-C) coupling is altered in several models of phosphatidylinositol phosphate (PtdIns P) phosphatase deficiency and ryanodine receptor activity measured in vitro was reported to be affected by certain PtdIns Ps, thus prompting investigation of the physiological role of PtdIns Ps in E-C coupling. We measured intracellular Ca transients in voltage-clamped mouse muscle fibres microinjected with a solution containing a PtdIns P substrate (PtdIns(3,5) P or PtdIns(3) P) or product (PtdIns(5) P or PtdIns) of the myotubularin phosphatase MTM1. No significant change was observed in the presence of either PtdIns(5) P or PtdIns but peak SR Ca release was depressed by ~30% and 50% in fibres injected with PtdIns(3,5) P and PtdIns(3) P, respectively, with no concurrent alteration in the membrane current signals associated with the DHPR function as well as in the voltage dependence of Ca release inactivation. In permeabilized muscle fibres, the frequency of spontaneous Ca release events was depressed in the presence of the three tested phosphorylated forms of PtdIns P with PtdIns(3,5) P being the most effective, leading to an almost complete disappearance of Ca release events. Results support the possibility that pathological accumulation of MTM1 substrates may acutely depress ryanodine receptor-mediated Ca release. Overexpression of a mCherry-tagged form of MTM1 in muscle fibres revealed a striated pattern consistent with the triadic area. Ca release remained although unaffected by MTM1 overexpression and was also unaffected by the PtdIns-3-kinase inhibitor LY2940002, suggesting that the 3-phosphorylated PtdIns lipids active on voltage-activated Ca release are inherently maintained at a low level, inefficient on Ca release in normal conditions. [ABSTRACT FROM AUTHOR]

The large-conductance Ca- and voltage-activated K (MaxiK, BK, BK, Slo1, K1.1) channel role in cell signalling is becoming apparent as we learn how the channel interacts with a multiplicity of proteins not only at the plasma membrane but also in intracellular organelles including the endoplasmic reticulum, nucleus, and mitochondria. In this review, we focus on the interactions of MaxiK channels with seven-transmembrane G protein-coupled receptors and discuss information suggesting that, the channel big C-terminus may act as the nucleus of signalling molecules including kinases relevant for cell death and survival. Increasing evidence indicates that the channel is able to associate with a variety of receptors including β-adrenergic receptors, G protein-coupled estrogen receptors, acetylcholine receptors, thromboxane A2 receptors, and angiotensin II receptors, which highlights the varied functions that the channel has (or may have) not only in regulating contraction/relaxation of muscle cells or neurotransmission in the brain but also in cell metabolism, proliferation, migration, and gene expression. In line with this view, MaxiK channels have been implicated in obesity and in brain, prostate, and mammary cancers. A better understanding on the molecular mechanisms underlying or triggered by MaxiK channel abnormalities like overexpression in certain cancers may lead to new therapeutics to prevent devastating diseases. [ABSTRACT FROM AUTHOR]

The powerful plant-derived irritant allyl isothiocyanate (AITC, aka mustard oil) induces hyperalgesia to heat in rodents and humans through mechanisms that are not yet fully understood. It is generally believed that AITC activates the broadly tuned chemosensory cation channel transient receptor potential cation channel subfamily A member 1 (TRPA1), triggering an inflammatory response that sensitizes the heat sensor transient receptor potential cation channel subfamily V member 1 (TRPV1). In the view of recent data demonstrating that AITC can directly activate TRPV1, we here explored the possibility that this compound sensitizes TRPV1 to heat stimulation in a TRPA1-independent manner. Patch-clamp recordings and intracellular Ca imaging experiments in HEK293T cells over-expressing mouse TRPV1 revealed that the increase in channel activation induced by heating is larger in the presence of AITC than in control conditions. The analysis of the effects of AITC and heat on the current-voltage relationship of TRPV1 indicates that the mechanism of sensitization is based on additive shifts of the voltage dependence of activation towards negative voltages. Finally, intracellular Ca imaging experiments in mouse sensory neurons isolated from Trpa1 KO mice yielded that AITC enhances the response to heat, specifically in the subpopulation expressing TRPV1. Furthermore, this effect was strongly reduced by the TRPV1 inhibitor capsazepine and virtually absent in neurons isolated from double Trpa1/ Trpv1 KO mice. Taken together, these findings demonstrate that TRPV1 is a locus for cross sensitization between AITC and heat in sensory neurons and may help explaining, at least in part, the role of this channel in AITC-induced hyperalgesia to heat. [ABSTRACT FROM AUTHOR]

TASK-2 (K 2P5.1) is a background K + channel opened by extra- or intracellular alkalinisation that plays a role in renal bicarbonate handling, central chemoreception and cell volume regulation. Here, we present results that suggest that TASK-2 is also modulated by Gβγ subunits of heterotrimeric G protein. TASK-2 was strongly inhibited when GTP-γ-S was used as a replacement for intracellular GTP. No inhibition was present using GDP-β-S instead. Purified Gβγ introduced intracellularly also inhibited TASK-2 independently of whether GTP or GDP-β-S was present. The effects of GTP-γ-S and Gβγ subunits were abolished by neutralisation of TASK-2 C terminus double lysine residues K257–K258 or K296–K297. Use of membrane yeast two hybrid (MYTH) experiments and immunoprecipitation assays using tagged proteins gave evidence for a physical interaction between Gβ1 and Gβ2 subunits and TASK-2, in agreement with expression of these subunits in proximal tubule cells. Co-immunoprecipitation was impeded by mutating C terminus K257–K258 (but not K296–K297) to alanines. Gating by extra- or intracellular pH was unaltered in GTP-γ-S-insensitive TASK-2-K257A-K258A mutant. Shrinking TASK-2-expressing cells in hypertonic solution decreased the current to 36 % of its initial value. The same manoeuvre had a significantly diminished effect on TASK-2-K257A-K258A- or TASK-2-K296-K297-expressing cells, or in cells containing intracellular GDP-β-S. Our data are compatible with the concept that TASK-2 channels are modulated by Gβγ subunits of heterotrimeric G protein. We propose that this modulation is a novel way in which TASK-2 can be tuned to its physiological functions. [ABSTRACT FROM AUTHOR]

Cardiac atrophy as a consequence of mechanical unloading develops following exposure to microgravity or prolonged bed rest. It also plays a central role in the reverse remodelling induced by left ventricular unloading in patients with heart failure. Surprisingly, the intracellular Ca 2+ transients which are pivotal to electromechanical coupling and to cardiac plasticity were repeatedly found to remain unaffected in early cardiac atrophy. To elucidate the mechanisms underlying the preservation of the Ca 2+ transients, we investigated Ca 2+ cycling in cardiomyocytes from mechanically unloaded (heterotopic abdominal heart transplantation) and control (orthotopic) hearts in syngeneic Lewis rats. Following 2 weeks of unloading, sarcoplasmic reticulum (SR) Ca 2+ content was reduced by ~55 %. Atrophic cardiac myocytes also showed a much lower frequency of spontaneous diastolic Ca 2+ sparks and a diminished systolic Ca 2+ release, even though the expression of ryanodine receptors was increased by ~30 %. In contrast, current clamp recordings revealed prolonged action potentials in endocardial as well as epicardial myocytes which were associated with a two to fourfold higher sarcolemmal Ca 2+ influx under action potential clamp. In addition, Cav1.2 subunits which form the pore of L-type Ca 2+ channels (LTCC) were upregulated in atrophic myocardium. These data suggest that in early cardiac atrophy induced by mechanical unloading, an augmented sarcolemmal Ca 2+ influx through LTCC fully compensates for a reduced systolic SR Ca 2+ release to preserve the Ca 2+ transient. This interplay involves an electrophysiological remodelling as well as changes in the expression of cardiac ion channels. [ABSTRACT FROM AUTHOR]

Phosphatidylserine (PS) exposure in red blood cells (RBCs) from sickle cell disease (SCD) patients is increased compared to levels in normal individuals and may participate in the anaemic and ischaemic complications of SCD. Exposure is increased by deoxygenation and occurs with elevation of intracellular Ca 2+ to low micromolar levels. The Ca 2+ entry step has not been defined but a role for the deoxygenation-induced pathway, P sickle, is postulated. Partial P sickle inhibitors 4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonic acid (SITS), 4,4′-dithiocyano-2,2′-stilbene-disulphonic acid (DIDS) and dipyridamole inhibited deoxygenation-induced PS exposure (DIDS IC 50, 118 nM). Inhibitors and activators of other pathways (including these stimulated by depolarisation, benzodiazepines, glutamate and stretch) were without effect. Zn 2+ and Gd 3+ stimulated PS exposure to high levels. In the case of Zn 2+, this effect was independent of oxygen (and hence HbS polymerisation and RBC sickling) but required extracellular Ca 2+. The effect was completely abolished when Zn 2+ (100 μM) was added to RBCs suspended in autologous plasma, implying a requirement of high levels of free Zn 2+. [ABSTRACT FROM AUTHOR]

The inward rectifier K + current (I K1) plays an important role in terminal repolarization and stabilization of the resting potential in cardiac cells. Although I K1 was shown to be sensitive to changes in intracellular Ca 2+ concentration ([Ca 2+] i), the nature of this Ca 2+ sensitivity—in spite of its deep influence on action potential morphology—is controversial. Therefore, we aimed to investigate the effects of a nonadrenergic rise in [Ca 2+] i on the amplitude of I K1 in canine and human ventricular myocardium and its consequences on cardiac repolarization. I K1, defined as the current inhibited by 10 μM Ba 2+, was significantly increased in isolated canine myocytes following a steady rise in [Ca 2+] i. Enhanced I K1 was also observed when [Ca 2+] i was not buffered by ethylene glycol tetraacetic acid, and [Ca 2+] I transients were generated. This [Ca 2+] i-dependent augmentation of I K1 was largely attenuated after inhibition of CaMKII by 1 μM KN-93. Elevation of [Ca 2+] o in multicellular canine and human ventricular preparations resulted in shortening of action potentials and acceleration of terminal repolarization. High [Ca 2+] o enhanced the action potential lengthening effect of the Ba 2+-induced I K1 blockade and attenuated the prolongation of action potentials following a 0.3-μM dofetilide-induced I Kr blockade. Blockade of I Ks by 0.5 μM HMR-1556 had no significant effect on APD 90 in either 2 mM or 4 mM [Ca 2+] o. It is concluded that high [Ca 2+] i leads to augmentation of the Ba 2+-sensitive current in dogs and humans, regardless of the mechanism of the increase. This effect seems to be at least partially mediated by a CaMKII-dependent pathway and may provide an effective endogenous defense against cardiac arrhythmias induced by Ca 2+ overload. [ABSTRACT FROM AUTHOR]

Trimeric intracellular cation-selective (TRIC) channel subtypes, namely TRIC-A and TRIC-B, are derived from distinct genes and distributed throughout the sarco/endoplasmic reticulum (SR/ER) and nuclear membranes. TRIC-A is preferentially expressed at high levels in excitable tissues, while TRIC-B is ubiquitously detected at relatively low levels in various tissues. TRIC channels are composed of ~300 amino acid residues and contain three putative membrane-spanning segments to form a bullet-shaped homo-trimeric assembly. Both native and purified recombinant TRIC subtypes form functional monovalent cation-selective channels in a lipid bilayer reconstitution system. The electrophysiological data indicate that TRIC channels behave as K channels under intracellular conditions, although the detailed channel characteristics remain to be investigated. The pathophysiological defects detected in knockout mice suggest that TRIC channels support SR/ER Ca release mediated by ryanodine (RyR) and inositol trisphosphate receptor (IPR) channels. For example, Tric-a-knockout mice develop hypertension resulting from vascular hypertonicity, and the mutant vascular smooth muscle cells exhibit insufficient RyR-mediated Ca release for inducing hyperpolarization. Tric-b-knockout mice show respiratory failure at birth, and IPR-mediated Ca release essential for surfactant handling is impaired in the mutant alveolar epithelial cells. Moreover, double-knockout mice lacking both TRIC subtypes show embryonic heart failure, and SR Ca handling is deranged in the mutant cardiomyocytes. Current evidence strongly suggests that TRIC channels mediate counter-K movements, in part, to facilitate physiological Ca release from intracellular stores. [ABSTRACT FROM AUTHOR]

Changes in plasma, extracellular, and intracellular calcium can affect renin secretion from the renal juxtaglomerular (JG) cells. Elevated intracellular calcium directly inhibits renin release from JG cells by decreasing the dominant second messenger intracellular cyclic adenosine monophosphate (cAMP) via actions on calcium-inhibitable adenylyl cyclases and calcium-activated phosphodiesterases. Increased extracellular calcium also directly inhibits renin release by stimulating the calcium-sensing receptor (CaSR) on JG cells, resulting in parallel changes in the intracellular environment and decreasing intracellular cAMP. In vivo, acutely elevated plasma calcium inhibits plasma renin activity (PRA) via parathyroid hormone-mediated elevations in renal cortical interstitial calcium that stimulate the JG cell CaSR. However, chronically elevated plasma calcium or CaSR activation may actually stimulate PRA. This elevation in PRA may be a compensatory mechanism resulting from calcium-mediated polyuria. Thus, changing the extracellular calcium in vitro or in vivo results in inversely related acute changes in cAMP, and therefore renin release, but chronic changes in calcium may result in more complex interactions dependent upon the duration of changes and the integration of the body's response to these changes. [ABSTRACT FROM AUTHOR]

Airway ciliary beat activity (CBA) plays a pivotal role in protecting the body by removing mucus and pathogens from the respiratory tract. Since CBA is complicated and cannot be characterized by merely frequency, we recorded CBA using laser confocal line scanning and defined six parameters for describing CBA. The values of these parameters were all above 0 when measured in beating ciliated cells from mouse tracheae. We subsequently used 10 μM adenosine-5′-triphosphate (ATP) to stimulate ciliated cells and simultaneously recorded intracellular Ca levels and CBA. We found that intracellular Ca levels first increased, followed by an increase in CBA. Among the six parameters, frequency, amplitude, and integrated area significantly increased, whereas rise time, decay time, and full duration at half maximum markedly decreased. The results suggest that these six parameters are appropriate for assessing CBA and that increased intracellular Ca levels might enhance CBA. We next used our established methods to observe changes in mechanically stimulated cilia tips. We found that mechanical stimulation-induced changes in both intracellular Ca levels and CBA were not only similar to those induced by ATP, but were also blocked by treatment with a Ca chelator, BAPTA-AM, (10 μM) for 10 min. Moreover, while the same blockage was observed under Ca-free conditions, addition of 2 mM Ca into the chamber restored increases in both intracellular Ca levels and CBA. Taken together, we have provided a novel method for real-time measurement and complete analysis of CBA as well as demonstrated that mechanical stimulation of cilia tips resulted in Ca influx that led to increased intracellular Ca levels, which in turn triggered CBA enhancement. [ABSTRACT FROM AUTHOR]

Connexin45 (Cx45) hemichannels (HCs) open in the absence of Ca and close in its presence. To elucidate the underlying mechanisms, we examined the role of extra- and intracellular Ca on the electrical properties of HCs. Experiments were performed on HeLa cells expressing Cx45 using electrical (voltage clamp) and optical (Ca imaging) methods. HCs exhibit a time- and voltage-dependent current ( I), activating with depolarization and inactivating with hyperpolarization. Elevation of [Ca] from 20 nM to 2 μM reversibly decreases I, decelerates its rate of activation, and accelerates its deactivation. Our data suggest that [Ca] modifies the channel properties by adhering to anionic sites in the channel lumen and/or its outer vestibule. In this way, it blocks the channel pore and reversibly lowers I and modifies its kinetics. Rapid lowering of [Ca] from 2 mM to 20 nM, achieved early during a depolarizing pulse, led to an outward I that developed with virtually no delay and grew exponentially in time paralleled by unaffected [Ca]. A step increase of [Ca] evoked by photorelease of Ca early during a depolarizing pulse led to a transient decrease of I superimposed on a growing outward I; a step decrease of [Ca] elicited by photoactivation of a Ca scavenger provoked a transient increase in I. Hence, it is tempting to assume that Ca exerts a direct effect on Cx45 hemichannels. [ABSTRACT FROM AUTHOR]

The chemoreception of dietary fat in the oral cavity has largely been attributed to activation of the somatosensory system that conveys the textural properties of fat. However, the ability of fatty acids, which are believed to represent the proximate stimulus for fat taste, to stimulate rat trigeminal neurons has remained unexplored. Here, we found that several free fatty acids are capable of activating trigeminal neurons with different kinetics. Further, a polyunsaturated fatty acid, linoleic acid (LA), activates trigeminal neurons by increasing intracellular calcium concentration and generating depolarizing receptor potentials. Ion substitution and pharmacological approaches reveal that intracellular calcium store depletion is crucial for LA-induced signaling in a subset of trigeminal neurons. Using pseudorabies virus (PrV) as a live cell tracer, we identified a subset of lingual nerve-innervated trigeminal neurons that respond to different subsets of fatty acids. Quantitative real-time PCR of several transient receptor potential channel markers in individual neurons validated that PrV labeled a subset but not the entire population of lingual-innervated trigeminal neurons. We further confirmed that the LA-induced intracellular calcium rise is exclusively coming from the release of calcium stores from the endoplasmic reticulum in this subset of lingual nerve-innervated trigeminal neurons. [ABSTRACT FROM AUTHOR]

Understanding the mechanisms of neuronal dysfunction and death represents a major frontier in contemporary medicine, involving the acute cell death in stroke, and the attrition of the major neurodegenerative diseases, including Parkinson's, Alzheimer's, Huntington's and Motoneuron diseases. A growing body of evidence implicates mitochondrial dysfunction as a key step in the pathogenesis of all these diseases, with the promise that mitochondrial processes represent valuable potential therapeutic targets. Each disease is characterised by the loss of a specific vulnerable population of cells-dopaminergic neurons in Parkinson's disease, spinal motoneurons in Motoneuron disease, for example. We discuss the possible roles of cell type-specific calcium signalling mechanisms in defining the pathological phenotype of each of these major diseases and review central mechanisms of calcium-dependent mitochondrial-mediated cell death. [ABSTRACT FROM AUTHOR]

Mitochondria convert the energy stored in carbohydrate and fat into ATP molecules that power enzymatic reactions within cells, and this process influences cellular calcium signals in several ways. By providing ATP to calcium pumps at the plasma and intracellular membranes, mitochondria power the calcium gradients that drive the release of Ca from stores and the entry of Ca across plasma membrane channels. By taking up and subsequently releasing calcium ions, mitochondria determine the spatiotemporal profile of cellular Ca signals and the activity of Ca-regulated proteins, including Ca entry channels that are themselves part of the Ca circuitry. Ca elevations in the mitochondrial matrix, in turn, activate Ca-dependent enzymes that boost the respiratory chain, increasing the ability of mitochondria to buffer calcium ions. Mitochondria are able to encode and decode Ca signals because the respiratory chain generates an electrochemical gradient for protons across the inner mitochondrial membrane. This proton motive force (Δ p) drives the activity of the ATP synthase and has both an electrical component, the mitochondrial membrane potential (ΔΨ), and a chemical component, the mitochondrial proton gradient (ΔpH). ΔΨ contributes about 190 mV to Δ p and drives the entry of Ca across a recently identified Ca-selective channel known as the mitochondrial Ca uniporter. ΔpH contributes ~30 mV to Δ p and is usually ignored or considered a minor component of mitochondria respiratory state. However, the mitochondrial proton gradient is an essential component of the chemiosmotic theory formulated by Peter Mitchell in 1961 as ΔpH sustains the entry of substrates and metabolites required for the activity of the respiratory chain and drives the activity of electroneutral ion exchangers that allow mitochondria to maintain their osmolarity and volume. In this review, we summarize the mechanisms that regulate the mitochondrial proton gradient and discuss how thermodynamic concepts derived from measurements in purified mitochondria can be reconciled with our recent findings that mitochondria have high proton permeability in situ and that ΔpH decreases during mitochondrial Ca elevations. [ABSTRACT FROM AUTHOR]

The transient receptor potential (TRP) family of ion channels comprises receptors that are activated by a vast variety of physical as well as chemical stimuli. TRP channels interact in a complex manner with several intracellular signaling cascades, both up- and downstream of receptor activation. Investigating cascades stimulated downstream of the cold and menthol receptor TRPM8, we found evidence for both, functional and structural interaction of TRPM8 with Gαq. We demonstrated menthol-evoked increase in intracellular Ca under extracellular Ca-free conditions, which was blocked by the PLC inhibitors U73122 or edelfosine. This metabotropic Ca signal could be observed also in cells expressing a channel-dead (i.e. non-conducting) or a chloride-conducting TRPM8 pore mutant. However, this intracellular metabotropic Ca signal could not be detected in Gαq deficient cells or in the presence of dominant-negative GαqX. Evidence for a close spatial proximity necessary for physical interaction of TRPM8 and Gαq was provided by acceptor bleaching experiments demonstrating FRET between TRPM8-CFP and Gαq-YFP. A Gαq-YFP mobility assay (FRAP) revealed a restricted diffusion of Gαq-YFP under conditions when TRPM8 is immobilized in the plasma membrane. Moreover, a menthol-induced and TRPM8-mediated G protein activation could be demonstrated by FRET experiments monitoring the dissociation of Gαq-YFP from a Gβ/Gγ-CFP complex, and by the exchange of radioactive [S]GTPγS for GDP. Our observations lead to a view that extends the operational range of the TRPM8 receptor from its function as a pure ion channel to a molecular switch with additional metabotropic capacity. [ABSTRACT FROM AUTHOR]