Generation Of Ca2 Research Articles

Biosynthesized calcium oxide from calcined conch shells: A potential bactericidal agent against Escherichia coli

The occurrence of ineffective antibiotics and an abundance of waste shell has provided a research opportunity to study calcium compounds through natural recycling, derived from the abundant gleaned species of gastropod conch (Canarium urceus) shells. Particularly, calcium oxide from waste shells were investigated as a value-added application for inhibiting pathogenic Escherichia coli. Hence, this study investigated its bactericidal activity through the Kirby-Bauer disc diffusion method to measure its growth inhibition ability. Waste conch shells were collected from Maitum, Barangay Dahican, City of Mati, Davao Oriental. Conch shell powder was obtained through calcination in a muffle furnace for two hours at 800°C (CS800) and 900°C (CS900), both at 50% and 75% (w/v) concentrations, for comparison. Among the four tested samples with three replicates each, results indicated that CS800 (50%) exhibited the highest inhibitory effects against E. coli. Findings also revealed that samples at 50% concentration demonstrated higher antibacterial activity than those at 75%. This statistically suggests that concentration level significantly influenced the bactericidal activity, whereas the 100°C difference in calcination temperature did not. The antibacterial activity of calcined conch shells can be attributed to CaO through two mechanisms: primarily, the strong alkalinity of the medium, and secondarily, the generation of Ca2+ and reactive oxygen species (ROS) on the surface of CaO, which cause cell wall rupture. Another possible mechanism is also associated with generating superoxides

Dietary Amino Acids Promote Glucagon-like Hormone Release to Generate Novel Calcium Waves in Adipose Tissues.

Nutrient sensing and the subsequent metabolic responses are fundamental functions of animals, closely linked to diseases such as type 2 diabetes and various obesity-related morbidities. Among different metabolic regulatory signals, cytosolic Ca2+ plays pivotal roles in metabolic regulation, including glycolysis, gluconeogenesis, and lipolysis. Recently, intercellular calcium waves (ICWs), the propagation of Ca2+ signaling through tissues, have been found in different systems to coordinate multicellular responses. Nevertheless, our understanding of how ICWs are modulated and operate within living organisms remains limited. In this study, we explore the real-time dynamics, both in organ culture and free-behaving animals, of ICWs in Drosophila larval and adult adipose tissues. We identified Adipokinetic hormone (AKH), the fly functional homolog of mammalian glucagon, as the key factor driving Ca2+ activities in adipose tissue. Interestingly, we found that AKH, which is released in a pulsatile manner into the circulating hemolymph from the AKH-producing neurosecretory cells (APCs) in the brain, stimulates ICWs in the larval fat by a previously unrecognized gap-junction-independent mechanism to promote lipolysis. In the adult fat body, however, gap-junction-dependent random ICWs are triggered by a presumably uniformly diffused AKH. This highlights the stage-specific interplay of hormone secretion, extracellular diffusion, and intercellular communication in the regulation of Ca2+ dynamics. Additionally, we discovered that specific dietary amino acids activate the APCs, leading to increased intracellular Ca2+ and subsequent AKH secretion. Altogether, our findings identify that dietary amino acids regulate the release of AKH peptides from the APCs, which subsequently stimulates novel gap-junction-independent ICWs in adipose tissues, thereby enhancing lipid metabolism.

Signaling switches maintain intercellular communication in the vascular endothelium

The endothelium is a sophisticated coordination center that controls a wide range of vascular functions, which include the regulation of blood pressure and blood flow via changes in vascular contractility. To coordinate vascular function, cell communication and interactions are required for tissue level responses to emerge. A significant form of cell-cell communication occurs by the propagation of Ca2+ signals between cells (‘Ca2+ waves’). The objective of our study was to tease apart the mechanisms driving these intercellular Ca2+ waves in the vascular endothelium. We hypothesised that diffusion of free Ca2+ would drive endothelial Ca2+ wave propagation and effect vessel diameter changes. To investigate the mechanisms, intact mesenteric arteries from male Sprague Dawley rats were mounted in an en face preparation, stained with the Ca2+ indicator Cal-520/AM and were imaged on specialised epifluorescence microscopes. Intercellular endothelial signaling was manipulated using caged compounds in concert with precise spatial and temporal control of UV illumination, as well as global or localized drug application. We show that multiple mechanisms maintain communication so that Ca2+ wave propagation occurs irrespective of the status of cell connectivity. Contrary to our hypothesis, regenerative IP3-induced IP3 production transmits Ca2+ signals between adjoining cells and explains the propagated vasodilation that underlies the increased blood flow accompanying tissue activity. By controlling the production of IP3, the inositide is itself suffcient to evoke a regenerative phospholipase C-dependent Ca2+ wave across coupled cells. None of gap junctions, Ca2+ diffusion nor the release of extracellular messengers are required to support this type of intercellular Ca2+ signaling. Surprisingly, when a discontinuity between cells was created using a fire-polished pipette tip, signal was propagated to the other side of the discontinuity and was able to drive propagated vasodilation. Experiments using counter flow and localised perfusion in the absence and presence of P2Y inhibitors revealed that it is ATP released as a diffusible extracellular messenger that transmits Ca2+ signals across endothelial discontinuities. These results show that signaling switches underlie endothelial cell-cell signal transmission, and reveal how communication is maintained in the face of endothelial damage. The findings provide a new framework for understanding Ca2+ wave propagation and cell-cell signaling in the endothelium. This work was funded by the British Heart Foundation (RG/F/20/110007; PG/20/9/34859 ). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

TRP channels: a provocative rationalization for local Ca2+ control in arterial tone development.

Arterial networks are controlled by the consolidated output of stimuli that set "how much" (magnitude) and "where" (distribution) blood flow is delivered. While notable changes in magnitude are tied to network wide responses, altered distribution often arises from focal changes in tone, whose mechanistic foundation remains unclear. We propose herein a framework of focal vasomotor contractility being controlled by pharmacomechanical coupling and the generation of Ca2+ waves via the sarcoplasmic reticulum. We argue the latter is sustained by receptor operated, transient receptor potential (TRP) channels through direct extracellular Ca2+ influx or indirect Na+ influx, reversing the Na+/Ca2+ exchanger. We view this focal regulatory mechanism as complementary, but not redundant with, electromechanical coupling in the precision tuning of blood flow delivery.

Pilot Study of Cytoprotective Mechanisms of Selenium Nanorods (SeNrs) under Ischemia-like Conditions on Cortical Astrocytes.

The cytoprotective properties of the trace element selenium, its nanoparticles, and selenium nanocomplexes with active compounds are shown using a number of models. To date, some molecular mechanisms of the protective effect of spherical selenium nanoparticles under the action of ischemia/reoxygenation on brain cells have been studied. Among other things, the dependence of the effectiveness of the neuroprotective properties of nanoselenium on its diameter, pathways, and efficiency of penetration into astrocytes was established. In general, most research in the field of nanomedicine is focused on the preparation and study of spherical nanoparticles of various origins due to the ease of their preparation; in addition, spherical nanoparticles have a large specific surface area. However, obtaining and studying the mechanisms of action of nanoparticles of a new form are of great interest since nanorods, having all the positive properties of spherical nanoparticles, will also have a number of advantages. Using the laser ablation method, we managed to obtain and characterize selenium nanorods (SeNrs) with a length of 1 μm and a diameter of 100 nm. Using fluorescence microscopy and inhibitory analysis, we were able to show that selenium nanorods cause the generation of Ca2+ signals in cortical astrocytes in an acute experiment through the mobilization of Ca2+ ions from the thapsigargin-sensitive pool of the endoplasmic reticulum. Chronic use of SeNrs leads to a change in the expression pattern of genes encoding proteins that regulate cell fate and protect astrocytes from ischemia-like conditions and reoxygenation through the inhibition of a global increase in the concentration of cytosolic calcium ([Ca2+]i). An important component of the cytoprotective effect of SeNrs during ischemia/reoxygenation is the induction of reactive A2-type astrogliosis in astrocytes, leading to an increase in both baseline and ischemia/reoxygenation-induced phosphoinositide 3-kinase (PI3K) activity and suppression of necrosis and apoptosis. The key components of this cytoprotective action of SeNrs are the actin-dependent process of endocytosis of nanoparticles into cells and activation of the Ca2+ signaling system of astrocytes.

Mechanobiological modulation of blood-brain barrier permeability by laser stimulation of endothelial-targeted nanoparticles.

The blood-brain barrier (BBB) maintains an optimal environment for brain homeostasis but excludes most therapeutics from entering the brain. Strategies that reversibly increase BBB permeability are essential for treating brain diseases and are the focus of significant preclinical and translational interest. Picosecond laser excitation of tight junction-targeted gold nanoparticles (AuNPs) generates a nanoscale mechanical perturbation and induces a graded and reversible increase in BBB permeability (OptoBBB). Here we advanced this technique by showing that targeting endothelial glycoproteins leads to >10-fold higher targeting efficiency than targeting tight junctions both in vitro and in vivo. With both tight-junction and glycoprotein targeting, we demonstrate that OptoBBB is associated with a transient elevation and propagation of Ca2+, actin polymerization, and phosphorylation of ERK1/2 (extracellular signal-regulated protein kinase). These collectively activate the cytoskeleton resulting in increased paracellular permeability. The Ca2+ response involves internal Ca2+ depletion and Ca2+ influx with contributions from mechanosensitive ion channels (TRPV4, Piezo1). We provide insight into how the excitation of tight junction protein (JAM-A)-targeted and endothelial (glycocalyx)-targeted AuNPs leads to similar mechanobiological modulation of BBB permeability while targeting the glycocalyx significantly improves the nanoparticle accumulation in the brain. The results will be critical for guiding the future development of this technology for brain disease treatment.

Comparative Analysis of the Cytotoxic Effect of a Complex of Selenium Nanoparticles Doped with Sorafenib, "Naked" Selenium Nanoparticles, and Sorafenib on Human Hepatocyte Carcinoma HepG2 Cells.

Despite the use of sorafenib as one of the most effective drugs for the treatment of liver cancer, its significant limitations remain—poor solubility, the need to use high doses with the ensuing complications on healthy tissues and organs, and the formation of cell resistance to the drug. At the same time, there is more and more convincing evidence of the anticancer effect of selenium-containing compounds and nanoparticles. The aim of this work was to develop a selenium–sorafenib nanocomplex and study the molecular mechanisms of its anticancer effect on human hepatocyte carcinoma cells, where nanoselenium is not only a sorafenib transporter, but also an active compound. We have created a selenium–sorafenib nanocomplex based on selenium nanoparticles with size 100 nm. Using vitality tests, fluorescence microscopy, and PCR analysis, it was possible to show that selenium nanoparticles, both by themselves and doped with sorafenib, have a pronounced pro-apoptotic effect on HepG2 cells with an efficiency many times greater than that of sorafenib (So). “Naked” selenium nanoparticles (SeNPs) and the selenium–sorafenib nanocomplex (SeSo), already after 24 h of exposure, lead to the induction of the early stages of apoptosis with the transition to the later stages with an increase in the incubation time up to 48 h. At the same time, sorafenib, at the studied concentrations, began to exert a proapoptotic effect only after 48 h. Under the action of SeNPs and SeSo, both classical pathways of apoptosis induction and ER-stress-dependent pathways involving Ca2+ ions are activated. Thus, sorafenib did not cause the generation of Ca2+ signals by HepG2 cells, while SeNPs and SeSo led to the activation of the Ca2+ signaling system of cells. At the same time, the selenium–sorafenib nanocomplex turned out to be more effective in activating the Ca2+ signaling system of cells, inducing apoptosis and ER stress by an average of 20–25% compared to “naked” selenium nanoparticles. Our data on the mechanisms of action and the created nanocomplex are promising as a platform for the creation of highly selective and effective drugs with targeted delivery to tumors.

The Cl--channel TMEM16A is involved in the generation of cochlear Ca2+ waves and promotes the refinement of auditory brainstem networks in mice.

Before hearing onset (postnatal day 12 in mice), inner hair cells (IHCs) spontaneously fire action potentials, thereby driving pre-sensory activity in the ascending auditory pathway. The rate of IHC action potential bursts is modulated by inner supporting cells (ISCs) of Kölliker's organ through the activity of the Ca2+-activated Cl--channel TMEM16A (ANO1). Here, we show that conditional deletion of Ano1 (Tmem16a) in mice disrupts Ca2+ waves within Kölliker's organ, reduces the burst-firing activity and the frequency selectivity of auditory brainstem neurons in the medial nucleus of the trapezoid body (MNTB), and also impairs the functional refinement of MNTB projections to the lateral superior olive. These results reveal the importance of the activity of Kölliker's organ for the refinement of central auditory connectivity. In addition, our study suggests the involvement of TMEM16A in the propagation of Ca2+ waves, which may also apply to other tissues expressing TMEM16A.

The role of action potential changes in depolarization-induced failure of excitation contraction coupling in mouse skeletal muscle.

Excitation-contraction coupling (ECC) is the process by which electrical excitation of muscle is converted into force generation. Depolarization of skeletal muscle resting potential contributes to failure of ECC in diseases such as periodic paralysis, intensive care unit acquired weakness and possibly fatigue of muscle during vigorous exercise. When extracellular K+ is raised to depolarize the resting potential, failure of ECC occurs suddenly, over a narrow range of resting potentials. Simultaneous imaging of Ca2+ transients and recording of action potentials (APs) demonstrated failure to generate Ca2+ transients when APs peaked at potentials more negative than -30mV. An AP property that closely correlated with failure of the Ca2+ transient was the integral of AP voltage with respect to time. Simultaneous recording of Ca2+ transients and APs with electrodes separated by 1.6mm revealed AP conduction fails when APs peak below -21mV. We hypothesize propagation of APs and generation of Ca2+ transients are governed by distinct AP properties: AP conduction is governed by AP peak, whereas Ca2+ release from the sarcoplasmic reticulum is governed by AP integral. The reason distinct AP properties may govern distinct steps of ECC is the kinetics of the ion channels involved. Na channels, which govern propagation, have rapid kinetics and are insensitive to AP width (and thus AP integral) whereas Ca2+ release is governed by gating charge movement of Cav1.1 channels, which have slower kinetics such that Ca2+ release is sensitive to AP integral. The quantitative relationships established between resting potential, AP properties, AP conduction and Ca2+ transients provide the foundation for future studies of failure of ECC induced by depolarization of the resting potential.

Activation of Cx43 Hemichannels Induces the Generation of Ca2+ Oscillations in White Adipocytes and Stimulates Lipolysis.

The aim of the study was to investigate the mechanisms of Ca2+ oscillation generation upon activation of connexin-43 and regulation of the lipolysis/lipogenesis balance in white adipocytes through vesicular ATP release. With fluorescence microscopy it was revealed that a decrease in the concentration of extracellular calcium ([Ca2+]ex) results in two types of Ca2+ responses in white adipocytes: Ca2+ oscillations and transient Ca2+ signals. It was found that activation of the connexin half-channels is involved in the generation of Ca2+ oscillations, since the blockers of the connexin hemichannels—carbenoxolone, octanol, proadifen and Gap26—as well as Cx43 gene knockdown led to complete suppression of these signals. The activation of Cx43 in response to the reduction of [Ca2+]ex was confirmed by TIRF microscopy. It was shown that in response to the activation of Cx43, ATP-containing vesicles were released from the adipocytes. This process was suppressed by knockdown of the Cx43 gene and by bafilomycin A1, an inhibitor of vacuolar ATPase. At the level of intracellular signaling, the generation of Ca2+ oscillations in white adipocytes in response to a decrease in [Ca2+]ex occurred due to the mobilization of the Ca2+ ions from the thapsigargin-sensitive Ca2+ pool of IP3R as a result of activation of the purinergic P2Y1 receptors and phosphoinositide signaling pathway. After activation of Cx43 and generation of the Ca2+ oscillations, changes in the expression levels of key genes and their encoding proteins involved in the regulation of lipolysis were observed in white adipocytes. This effect was accompanied by a decrease in the number of adipocytes containing lipid droplets, while inhibition or knockdown of Cx43 led to inhibition of lipolysis and accumulation of lipid droplets. In this study, we investigated the mechanism of Ca2+ oscillation generation in white adipocytes in response to a decrease in the concentration of Ca2+ ions in the external environment and established an interplay between periodic Ca2+ modes and the regulation of the lipolysis/lipogenesis balance.

Structural Mechanisms of Store-Operated and Mitochondrial Calcium Regulation: Initiation Points for Drug Discovery.

Calcium (Ca2+) is a universal signaling ion that is essential for the life and death processes of all eukaryotes. In humans, numerous cell stimulation pathways lead to the mobilization of sarco/endoplasmic reticulum (S/ER) stored Ca2+, resulting in the propagation of Ca2+ signals through the activation of processes, such as store-operated Ca2+ entry (SOCE). SOCE provides a sustained Ca2+ entry into the cytosol; moreover, the uptake of SOCE-mediated Ca2+ by mitochondria can shape cytosolic Ca2+ signals, function as a feedback signal for the SOCE molecular machinery, and drive numerous mitochondrial processes, including adenosine triphosphate (ATP) production and distinct cell death pathways. In recent years, tremendous progress has been made in identifying the proteins mediating these signaling pathways and elucidating molecular structures, invaluable for understanding the underlying mechanisms of function. Nevertheless, there remains a disconnect between using this accumulating protein structural knowledge and the design of new research tools and therapies. In this review, we provide an overview of the Ca2+ signaling pathways that are involved in mediating S/ER stored Ca2+ release, SOCE, and mitochondrial Ca2+ uptake, as well as pinpoint multiple levels of crosstalk between these pathways. Further, we highlight the significant protein structures elucidated in recent years controlling these Ca2+ signaling pathways. Finally, we describe a simple strategy that aimed at applying the protein structural data to initiating drug design.

Metabolic Cost of Dendritic Ca2+ Action Potentials in Layer 5 Pyramidal Neurons.

Pyramidal neurons consume most signaling-related energy to generate action potentials (APs) and perform synaptic integration. Dendritic Ca2+ spike is an important integration mechanism for coupling inputs from different cortical layers. Our objective was to quantify the metabolic energy associated with the generation of Ca2+ APs in the dendrites. We used morphology-based computational models to simulate the dendritic Ca2+ spikes in layer 5 pyramidal neurons. We calculated the energy cost by converting Ca2+ influx into the number of ATP required to restore and maintain the homeostasis of intracellular Ca2+ concentrations. We quantified the effects of synaptic inputs, dendritic voltage, back-propagating Na+ spikes, and Ca2+ inactivation on Ca2+ spike cost. We showed that much more ATP molecules were required for reversing Ca2+ influx in the dendrites than for Na+ ion pumping in the soma during a Ca2+ AP. Increasing synaptic input increased the rate of dendritic depolarization and underlying Ca2+ influx, resulting in higher ATP consumption. Depolarizing dendritic voltage resulted in the inactivation of Ca2+ channels and reduced the ATP cost, while dendritic hyperpolarization increased the spike cost by de-inactivating Ca2+ channels. A back-propagating Na+ AP initiated in the soma increased Ca2+ spike cost in the apical dendrite when it coincided with a synaptic input within a time window of several milliseconds. Increasing Ca2+ inactivation rate reduced Ca2+ spike cost, while slowing Ca2+ inactivation increased the spike cost. The results revealed that the energy demand of a Ca2+ AP was dynamically dependent on the state of dendritic activity. These findings were important for predicting the energy budget for signaling in pyramidal cells, interpreting functional imaging data, and designing energy-efficient neuromorphic devices.

Expression of the Inositol 1,4,5-Trisphosphate Receptor and the Ryanodine Receptor Ca2+-Release Channels in the Beta-Cells and Alpha-Cells of the Human Islets of Langerhans.

Calcium signaling regulates secretion of hormones and many other cellular processes in the islets of Langerhans. The three subtypes of the inositol 1,4,5-trisphosphate receptors (IP3Rs), inositol 1,4,5-trisphosphate receptor type 1 (IP3R1), 1,4,5-trisphosphate receptor type 2 (IP3R2), 1,4,5-trisphosphate receptor type 3 (IP3R3), and the three subtypes of the ryanodine receptors (RyRs), ryanodine receptor 1 (RyR1), ryanodine receptor 2 (RyR2) and ryanodine receptor 3 (RyR3) are the main intracellular Ca2+-release channels. The identity and the relative levels of expression of these channels in the alpha-cells, and the beta-cells of the human islets of Langerhans are unknown. We have analyzed the RNA sequencing data obtained from highly purified human alpha-cells and beta-cells for quantitatively identifying the mRNA of the intracellular Ca2+-release channels in these cells. We found that among the three IP3Rs the IP3R3 is the most abundantly expressed one in the beta-cells, whereas IP3R1 is the most abundantly expressed one in the alpha-cells. In addition to the IP3R3, beta-cells also expressed the IP3R2, at a lower level. Among the RyRs, the RyR2 was the most abundantly expressed one in the beta-cells, whereas the RyR1 was the most abundantly expressed one in the alpha-cells. Information on the relative abundance of the different intracellular Ca2+-release channels in the human alpha-cells and the beta-cells may help the understanding of their roles in the generation of Ca2+ signals and many other related cellular processes in these cells.

Cochlea-Specific Deletion of Cav1.3 Calcium Channels Arrests Inner Hair Cell Differentiation and Unravels Pitfalls of Conditional Mouse Models.

Inner hair cell (IHC) Cav1.3 Ca2+ channels are multifunctional channels mediating Ca2+ influx for exocytosis at ribbon synapses, the generation of Ca2+ action potentials in pre-hearing IHCs and gene expression. IHCs of deaf systemic Cav1.3-deficient (Cav1.3-/-) mice stay immature because they fail to up-regulate voltage- and Ca2+-activated K+ (BK) channels but persistently express small conductance Ca2+-activated K+ (SK2) channels. In pre-hearing wildtype mice, cholinergic neurons from the superior olivary complex (SOC) exert efferent inhibition onto spontaneously active immature IHCs by activating their SK2 channels. Because Cav1.3 plays an important role for survival, health and function of SOC neurons, SK2 channel persistence and lack of BK channels in systemic Cav1.3-/- IHCs may result from malfunctioning neurons of the SOC. Here we analyze cochlea-specific Cav1.3 knockout mice with green fluorescent protein (GFP) switch reporter function, Pax2::cre;Cacna1d-eGFPflex/flex and Pax2::cre;Cacna1d-eGFPflex/-. Profound hearing loss, lack of BK channels and persistence of SK2 channels in Pax2::cre;Cacna1d-eGFPflex/- mice recapitulated the phenotype of systemic Cav1.3-/- mice, indicating that in wildtype mice, regulation of SK2 and BK channel expression is independent of Cav1.3 expression in SOC neurons. In addition, we noticed dose-dependent GFP toxicity leading to death of basal coil IHCs of Pax2::cre;Cacna1d-eGFPflex/flex mice, likely because of high GFP concentration and small repair capacity. This and the slower time course of Pax2-driven Cre recombinase in switching two rather than one Cacna1d-eGFPflex allele lead us to study Pax2::cre;Cacna1d-eGFPflex/- mice. Notably, control Cacna1d-eGFPflex/- IHCs showed a significant reduction in Cav1.3 channel cluster sizes and currents, suggesting that the intronic construct interfered with gene translation or splicing. These pitfalls are likely to be a frequent problem of many genetically modified mice with complex or multiple gene-targeting constructs or fluorescent proteins. Great caution and appropriate controls are therefore required.

Effect of CAX1a TILLING mutations and calcium concentration on some primary metabolism processes in Brassica rapa plants

Cation/H+ exchanger transporters (CAXs) are crucial in Ca2+ homeostasis and in the generation of Ca2+ profiles involved in signalling processes. Given the crucial role of CAX1 in Ca2+ homeostasis, CAX1 modifications could have effects on plant metabolism. Three Brassica rapa mutants for CAX1 were obtained through TILLING. The aim of this work is to assess the effect of the different mutations and different Ca2+ doses on plant metabolism. For this, the mutants and the parental line were grown under low, control and high Ca2+ doses and parameters related to nitrogen (N) and tricarboxylic acid (TCA) metabolisms, and amino acid (AAs) and phytohormone profiles were measured. The results show that BraA.cax1a mutations affect metabolism especially under high Ca2+ dose. Thus, BraA.cax1a-7 inhibited some N metabolism enzymes and activated photorespiration activity. On the opposite side, BraA.cax1a-12 mutation provides a better tolerance to high Ca2+ dose. This tolerance could be provided by an improved N and TCA metabolisms enzymes, and a higher glutamate, malate, indole-3-acetic acid and abscisic acid concentrations. Therefore, BraA.cax1a-12 mutation could be used for B. rapa improving; the metabolomics changes observed in this mutant could be responsible for a better tolerance to high Ca2+.

Angiotensin II Induces Automatic Activity of the Isolated Guinea Pig Pulmonary Vein Myocardium through Activation of the IP₃ Receptor and the Na⁺-Ca2+ Exchanger.

The automaticity of the pulmonary vein myocardium is known to be the major cause of atrial fibrillation. We examined the involvement of angiotensin II in the automatic activity of isolated guinea pig pulmonary vein preparations. In tissue preparations, application of angiotensin II induced an automatic contractile activity; this effect was mimicked by angiotensin I and blocked by losartan, but not by PD123,319 or carvedilol. In cardiomyocytes, application of angiotensin II induced an increase in the frequency of spontaneous Ca2+ sparks and the generation of Ca2+ transients; these effects were inhibited by losartan or xestospongin C. In tissue preparations, angiotensin II caused membrane potential oscillations, which lead to repetitive generation of action potentials. Angiotensin II increased the diastolic depolarization slope of the spontaneous or evoked action potentials. These effects of angiotensin II were inhibited by SEA0400. In tissue preparations showing spontaneous firing of action potentials, losartan, xestospongin C or SEA0400 decreased the slope of the diastolic depolarization and inhibited the firing of action potentials. In conclusion, in the guinea pig pulmonary vein myocardium, angiotensin II induces the generation of automatic activity through activation of the IP3 receptor and the Na+-Ca2+ exchanger.

Panglial gap junctions between astrocytes and olfactory ensheathing cells mediate transmission of Ca2+ transients and neurovascular coupling.

Astrocytes are arranged in highly organized gap junction-coupled networks, communicating via the propagation of Ca2+ waves. Astrocytes are gap junction-coupled not only to neighboring astrocytes, but also to oligodendrocytes, forming so-called panglial syncytia. It is not known, however, whether glial cells in panglial syncytia transmit information using Ca2+ signaling. We used confocal Ca2+ imaging to study intercellular communication between astrocytes and olfactory ensheathing glial cells (OECs) in in-toto preparations of the mouse olfactory bulb. Our results demonstrate that Ca2+ transients in juxtaglomerular astrocytes, evoked by local photolysis of "caged" ATP and "caged" tACPD, led to subsequent Ca2+ responses in OECs. This transmission of Ca2+ responses from astrocytes to OECs persisted in the presence of neuronal inhibition, but was absent when gap junctional coupling was suppressed with carbenoxolone. When Ca2+ transients were directly evoked in OECs by puff application of DHPG, they resulted in delayed Ca2+ responses in juxtaglomerular astrocytes, indicating that panglial transmission of Ca2+ signals occurred in a bidirectional manner. In addition, panglial transmission of Ca2+ signals from astrocytes to OECs resulted in vasoconstriction of OEC-associated blood vessels in the olfactory nerve layer. Our results demonstrate functional transmission of Ca2+ signals between different classes of glial cells within gap junction-coupled panglial networks and the resulting regulation of blood vessel diameter in the olfactory bulb.

Cultivation of the causative agent of human neoehrlichiosis from clinical isolates identifies vascular endothelium as a target of infection

ABSTRACT Candidatus (Ca.) Neoehrlichia mikurensis is the cause of neoehrlichiosis, an emerging tick-borne infectious disease characterized by fever and vascular events. The bacterium belongs to the Anaplasmataceae, a family of obligate intracellular pathogens, but has not previously been cultivated, and it is uncertain which cell types it infects. The goals of this study were to cultivate Ca. N. mikurensis in cell lines and to identify possible target cells for human infection. Blood components derived from infected patients were inoculated into cell lines of both tick and human origin. Bacterial growth in the cell cultures was monitored by real-time PCR and imaging flow cytometry. Ca. N. mikurensis was successfully propagated from the blood of immunocompromised neoehrlichiosis patients in two Ixodes spp. tick cell lines following incubation periods of 7–20 weeks. Human primary endothelial cells derived from skin microvasculature as well as pulmonary artery were also susceptible to infection with tick cell-derived bacteria. Finally, Ca. N. mikurensis was visualized within circulating endothelial cells of two neoehrlichiosis patients. To conclude, we report the first successful isolation and propagation of Ca. N. mikurensis from clinical isolates and identify human vascular endothelial cells as a target of infection.

The Subcellular Distribution of Ryanodine Receptors and L-Type Ca2+ Channels Modulates Ca2+-Transient Properties and Spontaneous Ca2+-Release Events in Atrial Cardiomyocytes.

Spontaneous Ca2+-release events (SCaEs) from the sarcoplasmic reticulum play crucial roles in the initiation of cardiac arrhythmias by promoting triggered activity. However, the subcellular determinants of these SCaEs remain incompletely understood. Structural differences between atrial and ventricular cardiomyocytes, e.g., regarding the density of T-tubular membrane invaginations, may influence cardiomyocyte Ca2+-handling and the distribution of cardiac ryanodine receptors (RyR2) has recently been shown to undergo remodeling in atrial fibrillation. These data suggest that the subcellular distribution of Ca2+-handling proteins influences proarrhythmic Ca2+-handling abnormalities. Here, we employ computational modeling to provide an in-depth analysis of the impact of variations in subcellular RyR2 and L-type Ca2+-channel distributions on Ca2+-transient properties and SCaEs in a human atrial cardiomyocyte model. We incorporate experimentally observed RyR2 expression patterns and various configurations of axial tubules in a previously published model of the human atrial cardiomyocyte. We identify an increased SCaE incidence for larger heterogeneity in RyR2 expression, in which SCaEs preferentially arise from regions of high local RyR2 expression. Furthermore, we show that the propagation of Ca2+ waves is modulated by the distance between RyR2 bands, as well as the presence of experimentally observed RyR2 clusters between bands near the lateral membranes. We also show that incorporation of axial tubules in various amounts and locations reduces Ca2+-transient time to peak. Furthermore, selective hyperphosphorylation of RyR2 around axial tubules increases the number of spontaneous waves. Finally, we present a novel model of the human atrial cardiomyocyte with physiological RyR2 and L-type Ca2+-channel distributions that reproduces experimentally observed Ca2+-handling properties. Taken together, these results significantly enhance our understanding of the structure-function relationship in cardiomyocytes, identifying that RyR2 and L-type Ca2+-channel distributions have a major impact on systolic Ca2+ transients and SCaEs.

Intraluminal pressure triggers myogenic response via activation of calcium spark and calcium-activated chloride channel in rat renal afferent arteriole.

Myogenic contraction of renal arterioles is an important regulatory mechanism for renal blood flow autoregulation. We have previously demonstrated that integrin-mediated mechanical force increases the occurrence of Ca2+ sparks in freshly isolated renal vascular smooth muscle cells (VSMCs). To further test whether the generation of Ca2+ sparks is a downstream signal of mechanotransduction in pressure-induced myogenic constriction, the relationship between Ca2+ sparks and transmural perfusion pressure was investigated in intact VSMCs of pressurized rat afferent arterioles. Spontaneous Ca2+ sparks were found in VSMCs when afferent arterioles were perfused at 80 mmHg. The spark frequency was significantly increased when perfusion pressure was increased to 120 mmHg. A similar increase of spark frequency was also observed in arterioles stimulated with β1-integrin-activating antibody. Moreover, spark frequency was significantly higher in arterioles of spontaneous hypertensive rats at 80 and 120 mmHg. Spontaneous membrane current recorded using whole cell perforated patch in renal VSMCs showed predominant activity of spontaneous transient inward currents instead of spontaneous transient outward currents when holding potential was set close to physiological resting membrane potential. Real-time PCR and immunohistochemistry confirmed the expression of Ca2+-activated Cl- channel (ClCa) TMEM16A in renal VSMCs. Inhibition of TMEM16A with T16Ainh-A01 impaired the pressure-induced myogenic contraction in perfused afferent arterioles. Our study, for the first time to our knowledge, detected Ca2+ sparks in VSMCs of intact afferent arterioles, and their frequencies were positively modulated by the perfusion pressure. Our results suggest that Ca2+ sparks may couple to ClCa channels and trigger pressure-induced myogenic constriction via membrane depolarization.