Local Intracellular Ca Research Articles

A deep learning-based approach for efficient detection and classification of local Ca²⁺ release events in Full-Frame confocal imaging

The release of Ca2+ ions from intracellular stores plays a crucial role in many cellular processes, acting as a secondary messenger in various cell types, including cardiomyocytes, smooth muscle cells, hepatocytes, and many others. Detecting and classifying associated local Ca2+ release events is particularly important, as these events provide insight into the mechanisms, interplay, and interdependencies of local Ca2+release events underlying global intracellular Ca2+signaling. However, time-consuming and labor-intensive procedures often complicate analysis, especially with low signal-to-noise ratio imaging data.Here, we present an innovative deep learning-based approach for automatically detecting and classifying local Ca2+ release events. This approach is exemplified with rapid full-frame confocal imaging data recorded in isolated cardiomyocytes.To demonstrate the robustness and accuracy of our method, we first use conventional evaluation methods by comparing the intersection between manual annotations and the segmentation of Ca2+ release events provided by the deep learning method, as well as the annotated and recognized instances of individual events. In addition to these methods, we compare the performance of the proposed model with the annotation of six experts in the field. Our model can recognize more than 75 % of the annotated Ca2+ release events and correctly classify more than 75 %. A key result was that there were no significant differences between the annotations produced by human experts and the result of the proposed deep learning model.We conclude that the proposed approach is a robust and time-saving alternative to conventional full-frame confocal imaging analysis of local intracellular Ca2+ events.

WNK kinase is a vasoactive chloride sensor in endothelial cells

Endothelial cells (ECs) line the wall of blood vessels and regulate arterial contractility to tune regional organ blood flow and systemic pressure. Chloride (Cl-) is the most abundant anion in ECs and the Cl- sensitive With-No-Lysine (WNK) kinase is expressed in this cell type. Whether intracellular Cl- signaling and WNK kinase regulate EC function to alter arterial contractility is unclear. Here, we tested the hypothesis that intracellular Cl- signaling in ECs regulates arterial contractility and examined the signaling mechanisms involved, including the participation of WNK kinase. Our data obtained using two-photon microscopy and cell-specific inducible knockout mice indicated that acetylcholine, a prototypical vasodilator, stimulated a rapid reduction in intracellular Cl- concentration ([Cl-]i) due to the activation of TMEM16A, a Cl- channel, in ECs of resistance-size arteries. TMEM16A channel-mediated Cl- signaling activated WNK kinase, which phosphorylated its substrate proteins SPAK and OSR1 in ECs. OSR1 potentiated transient receptor potential vanilloid 4 (TRPV4) currents in a kinase-dependent manner and required a conserved binding motif located in the channel C terminus. Intracellular Ca2+ signaling was measured in four dimensions in ECs using a high-speed lightsheet microscope. WNK kinase-dependent activation of TRPV4 channels increased local intracellular Ca2+ signaling in ECs and produced vasodilation. In summary, we show that TMEM16A channel activation reduces [Cl-]i, which activates WNK kinase in ECs. WNK kinase phosphorylates OSR1 which then stimulates TRPV4 channels to produce vasodilation. Thus, TMEM16A channels regulate intracellular Cl- signaling and WNK kinase activity in ECs to control arterial contractility.

Indicator-dependent differences in detection of local intracellular Ca2+ release events evoked by voltage-gated Ca2+ entry in pancreatic β-cells

Genetically encoded Ca2+ indicators have become widely used in cell signalling studies as they offer advantages over cell-loaded dye indicators in enabling specific cellular or subcellular targeting. Comparing responses from dye and protein-based indicators may provide information about indicator properties and cell physiology, but side-by-side recordings in cells are scarce. In this study, we compared cytoplasmic Ca2+ concentration ([Ca2+]i) changes in insulin-secreting β-cells recorded with commonly used dyes and indicators based on circularly permuted fluorescent proteins. Total internal reflection fluorescence (TIRF) imaging of K+ depolarization-triggered submembrane [Ca2+]i increases showed that the dyes Fluo-4 and Fluo-5F mainly reported stable [Ca2+]i elevations, whereas the proteins R-GECO1 and GCaMP5G more often reported distinct [Ca2+]i spikes from an elevated level. [Ca2+]i spiking occurred also in glucose-stimulated cells. The spikes reflected Ca2+ release from the endoplasmic reticulum, triggered by autocrine activation of purinergic receptors after exocytotic release of ATP and/or ADP, and the spikes were consequently prevented by SERCA inhibition or P2Y1-receptor antagonism. Widefield imaging, which monitors the entire cytoplasm, increased the spike detection by the Ca2+ dyes. The indicator-dependent response patterns were unrelated to Ca2+ binding affinity, buffering and mobility, and probably reflects the much slower dissociation kinetics of protein compared to dye indicators. Ca2+ dyes thus report signalling within the submembrane space excited by TIRF illumination, whereas the protein indicators also catch Ca2+ events originating outside this volume. The study highlights that voltage-dependent Ca2+ entry in β-cells is tightly linked to local intracellular Ca2+ release mediated via an autocrine route that may be more important than previously reported direct Ca2+ effects on phospholipase C or on intracellular channels mediating calcium-induced calcium release.

Influence of Mpv17 on Hair-Cell Mitochondrial Homeostasis, Synapse Integrity, and Vulnerability to Damage in the Zebrafish Lateral Line.

Noise exposure is particularly stressful to hair-cell mitochondria, which must produce enough energy to meet high metabolic demands as well as regulate local intracellular Ca2+ concentrations. Mitochondrial Inner Membrane Protein 17 (Mpv17) functions as a non-selective cation channel and plays a role in maintaining mitochondrial homeostasis. In zebrafish, hair cells in mpv17a9/a9 mutants displayed elevated levels of reactive oxygen species (ROS), elevated mitochondrial calcium, hyperpolarized transmembrane potential, and greater vulnerability to neomycin, indicating impaired mitochondrial function. Using a strong water current to overstimulate hair cells in the zebrafish lateral line, we observed mpv17a9/a9 mutant hair cells were more vulnerable to morphological disruption than wild type (WT) siblings simultaneously exposed to the same stimulus. To determine the role of mitochondrial homeostasis on hair-cell synapse integrity, we surveyed synapse number in mpv17a9/a9 mutants and WT siblings as well as the sizes of presynaptic dense bodies (ribbons) and postsynaptic densities immediately following stimulus exposure. We observed mechanically injured mpv17a9/a9 neuromasts were not more vulnerable to synapse loss; they lost a similar number of synapses per hair cell relative to WT. Additionally, we quantified the size of hair cell pre- and postsynaptic structures following stimulation and observed significantly enlarged WT postsynaptic densities, yet relatively little change in the size of mpv17a9/a9 postsynaptic densities following stimulation. These results suggest chronically impaired hair-cell mitochondrial activity influences postsynaptic size under homeostatic conditions but does not exacerbate synapse loss following mechanical injury.

Ca2+ Dependence of Volume-Regulated VRAC/LRRC8 and TMEM16A Cl- Channels.

All vertebrate cells activate Cl– currents (ICl,swell) when swollen by hypotonic bath solution. The volume-regulated anion channel VRAC has now been identified as LRRC8/SWELL1. However, apart from VRAC, the Ca2+-activated Cl– channel (CaCC) TMEM16A and the phospholipid scramblase and ion channel TMEM16F were suggested to contribute to cell swelling-activated whole-cell currents. Cell swelling was shown to induce Ca2+ release from the endoplasmic reticulum and to cause subsequent Ca2+ influx. It is suggested that TMEM16A/F support intracellular Ca2+ signaling and thus Ca2+-dependent activation of VRAC. In the present study, we tried to clarify the contribution of TMEM16A to ICl,swell. In HEK293 cells coexpressing LRRC8A and LRRC8C, we found that activation of ICl,swell by hypotonic bath solution (Hypo; 200 mosm/l) was Ca2+ dependent. TMEM16A augmented the activation of LRRC8A/C by enhancing swelling-induced local intracellular Ca2+ concentrations. In HT29 cells, knockdown of endogenous TMEM16A attenuated ICl,swell and changed time-independent swelling-activated currents to VRAC-typical time-dependent currents. Activation of ICl,swell by Hypo was attenuated by blocking receptors for inositol trisphosphate and ryanodine (IP3R; RyR), as well as by inhibiting Ca2+ influx. The data suggest that TMEM16A contributes directly to ICl,swell as it is activated through swelling-induced Ca2+ increase. As activation of VRAC is shown to be Ca2+-dependent, TMEM16A augments VRAC currents by facilitating Hypo-induced Ca2+ increase in submembraneous signaling compartments by means of ER tethering.

Concomitant genetic ablation of L-type Cav1.3 (\u03b11D) and T-type Cav3.1 (\u03b11G) Ca2+ channels disrupts heart automaticity

Cardiac automaticity is set by pacemaker activity of the sinus node (SAN). In addition to the ubiquitously expressed cardiac voltage-gated L-type Cav1.2 Ca2+ channel isoform, pacemaker cells within the SAN and the atrioventricular node co-express voltage-gated L-type Cav1.3 and T-type Cav3.1 Ca2+ channels (SAN-VGCCs). The role of SAN-VGCCs in automaticity is incompletely understood. We used knockout mice carrying individual genetic ablation of Cav1.3 (Cav1.3−/−) or Cav3.1 (Cav3.1−/−) channels and double mutant Cav1.3−/−/Cav3.1−/− mice expressing only Cav1.2 channels. We show that concomitant loss of SAN-VGCCs prevents physiological SAN automaticity, blocks impulse conduction and compromises ventricular rhythmicity. Coexpression of SAN-VGCCs is necessary for impulse formation in the central SAN. In mice lacking SAN-VGCCs, residual pacemaker activity is predominantly generated in peripheral nodal and extranodal sites by f-channels and TTX-sensitive Na+ channels. In beating SAN cells, ablation of SAN-VGCCs disrupted late diastolic local intracellular Ca2+ release, which demonstrates an important role for these channels in supporting the sarcoplasmic reticulum based “Ca2+clock” mechanism during normal pacemaking. These data implicate an underappreciated role for co-expression of SAN-VGCCs in heart automaticity and define an integral role for these channels in mechanisms that control the heartbeat.

Axial Tubule Junctions Activate Atrial Ca2+ Release Across Species.

Rationale: Recently, abundant axial tubule (AT) membrane structures were identified deep inside atrial myocytes (AMs). Upon excitation, ATs rapidly activate intracellular Ca2+ release and sarcomeric contraction through extensive AT junctions, a cell-specific atrial mechanism. While AT junctions with the sarcoplasmic reticulum contain unusually large clusters of ryanodine receptor 2 (RyR2) Ca2+ release channels in mouse AMs, it remains unclear if similar protein networks and membrane structures exist across species, particularly those relevant for atrial disease modeling.Objective: To examine and quantitatively analyze the architecture of AT membrane structures and associated Ca2+ signaling proteins across species from mouse to human.Methods and Results: We developed superresolution microscopy (nanoscopy) strategies for intact live AMs based on a new custom-made photostable cholesterol dye and immunofluorescence imaging of membraneous structures and membrane proteins in fixed tissue sections from human, porcine, and rodent atria. Consistently, in mouse, rat, and rabbit AMs, intact cell-wide tubule networks continuous with the surface membrane were observed, mainly composed of ATs. Moreover, co-immunofluorescence nanoscopy showed L-type Ca2+ channel clusters adjacent to extensive junctional RyR2 clusters at ATs. However, only junctional RyR2 clusters were highly phosphorylated and may thus prime Ca2+ release at ATs, locally for rapid signal amplification. While the density of the integrated L-type Ca2+ current was similar in human and mouse AMs, the intracellular Ca2+ transient showed quantitative differences. Importantly, local intracellular Ca2+ release from AT junctions occurred through instantaneous action potential propagation via transverse tubules (TTs) from the surface membrane. Hence, sparse TTs were sufficient as electrical conduits for rapid activation of Ca2+ release through ATs. Nanoscopy of atrial tissue sections confirmed abundant ATs as the major network component of AMs, particularly in human atrial tissue sections.Conclusion: AT junctions represent a conserved, cell-specific membrane structure for rapid excitation-contraction coupling throughout a representative spectrum of species including human. Since ATs provide the major excitable membrane network component in AMs, a new model of atrial “super-hub” Ca2+ signaling may apply across biomedically relevant species, opening avenues for future investigations about atrial disease mechanisms and therapeutic targeting.

Structure of the human plasma membrane Ca2+-ATPase 1 in complex with its obligatory subunit neuroplastin

Plasma membrane Ca2+-ATPases (PMCAs) are key regulators of global Ca2+ homeostasis and local intracellular Ca2+ dynamics. Recently, Neuroplastin (NPTN) and basigin were identified as previously unrecognized obligatory subunits of PMCAs that dramatically increase the efficiency of PMCA-mediated Ca2+ clearance. Here, we report the cryo-EM structure of human PMCA1 (hPMCA1) in complex with NPTN at a resolution of 4.1 Å for the overall structure and 3.9 Å for the transmembrane domain. The single transmembrane helix of NPTN interacts with the TM8-9-linker and TM10 of hPMCA1. The subunits are required for the hPMCA1 functional activity. The NPTN-bound hPMCA1 closely resembles the E1-Mg2+ structure of endo(sarco)plasmic reticulum Ca2+ ATPase and the Ca2+ site is exposed through a large open cytoplasmic pathway. This structure provides insight into how the subunits bind to the PMCAs and serves as an important basis for understanding the functional mechanisms of this essential calcium pump family.

Abstract P508: Inhibition of Big-conductance Calcium-activated Potassium Channels in Vascular Myocytes is a Key Novel Mechanism for Tacrolimus-induced Hypertension

Background and Purpose: Tacrolimus (TAC, also called FK506), a common immunosuppressive drug, causes hypertension. In this study, we tested a novel hypothesis that TAC inhibits big-conductance Ca 2+ -activated K + (BK) channels in artery smooth muscle cells (SMCs), leading to hypertension. Experimental Approach: Noninvasive and invasive blood pressure measurements were used to determine hypertension; organ bath technique and live videomicroscopy to assess vasoconstriction; fluorescence imaging and confocal microscope to detect whole-cell and local intracellular Ca 2+ concentration; patch-clamp techniques to record channel activity; Western blot analysis and real-time RT-PCR to evaluate channel expression; and gene knockout mice to determine its functions. Key Results: Intraperitoneal injection of TAC once a day for a week causes hypertension with a peak at 4 weeks. Application of norepinephrine (NE) induces a larger vasoconstriction in arteries and increase in [Ca 2+ ] i in artery SMCs from mice treated with TAC for 4 weeks. Whole-cell BK channel activity is significantly decreased in SMCs from TAC-treated mice, whereas Ca 2+ spark activity is increased. The voltage sensitivity, Ca 2+ sensitivity and open time of single BK channels are all decreased in TAC-administrated mouse cells. BK channel β1, but not α, subunit protein and mRNA expression levels are significantly decreased in arteries from TAC-treated mice. Chronic administration of TAC fails to further increase blood pressure in BK channel β1 knockout mice with hypertension. Conclusion and Implications: Our findings provide novel evidence that TAC inhibits BK channel β1 subunit expression and functions in artery SMCs, thereby leading to enhanced vasoconstriction and hypertension.

Local coupling of TRPC6 to ANO1/TMEM16A channels in smooth muscle cells amplifies vasoconstriction in cerebral arteries.

Anoctamin-1 [ANO1, also known as transmembrane protein 16A (TMEM16A)] is a Ca(2+)-activated Cl(-) channel expressed in arterial myocytes that regulates membrane potential and contractility. Signaling mechanisms that control ANO1 activity in arterial myocytes are poorly understood. In cerebral artery myocytes, ANO1 channels are activated by local Ca(2+) signals generated by plasma membrane nonselective cation channels, but the molecular identity of these proteins is unclear. Arterial myocytes express several different nonselective cation channels, including multiple members of the transient receptor potential receptor (TRP) family. The goal of this study was to identify localized ion channels that control ANO1 currents in cerebral artery myocytes. Coimmunoprecipitation and immunofluorescence resonance energy transfer microscopy experiments indicate that ANO1 and canonical TRP 6 (TRPC6) channels are present in the same macromolecular complex and localize in close spatial proximity in the myocyte plasma membrane. In contrast, ANO1 is not near TRPC3, TRP melastatin 4, or inositol trisphosphate receptor 1 channels. Hyp9, a selective TRPC6 channel activator, stimulated Cl(-) currents in myocytes that were blocked by T16Ainh-A01, an ANO1 inhibitor, ANO1 knockdown using siRNA, and equimolar replacement of intracellular EGTA with BAPTA, a fast Ca(2+) chelator that abolishes local Ca(2+) signaling. Hyp9 constricted pressurized cerebral arteries, and this response was attenuated by T16Ainh-A01. In contrast, T16Ainh-A01 did not alter depolarization-induced (60 mM K(+)) vasoconstriction. These data indicate that TRPC6 channels generate a local intracellular Ca(2+) signal that activates nearby ANO1 channels in myocytes to stimulate vasoconstriction.

Junctate boosts phagocytosis by recruiting endoplasmic reticulum Ca2+ stores near phagosomes.

Local intracellular Ca(2+) elevations increase the efficiency of phagocytosis, a process that is essential for innate and adaptive immunity. These local Ca(2+) elevations are generated in part by the store-operated Ca(2+) entry (SOCE) sensor STIM1, which recruits endoplasmic reticulum (ER) cisternae to phagosomes and opens phagosomal Ca(2+) channels at ER-phagosome junctions. However, residual ER-phagosome contacts and periphagosomal Ca(2+) hotspots remain in Stim1(-/-) cells. Here, we tested whether junctate (also called ASPH isoform 8), a molecule that targets STIM1 to ER-plasma-membrane contacts upon Ca(2+)-store depletion, cooperates with STIM1 at phagosome junctions. Junctate expression in Stim1(-/-) and Stim1(-/-); Stim2(-/-) phagocytic fibroblasts increased phagocytosis and periphagosomal Ca(2+) elevations, yet with only a minimal impact on global SOCE. These Ca(2+) hotspots were only marginally reduced by the SOCE channel blocker lanthanum chloride (La(3+)) but were abrogated by inositol trisphosphate receptor inhibitors 2-APB and xestospongin-C, revealing that unlike STIM1-mediated hotspots, junctate-mediated Ca(2+) originates predominantly from periphagosomal Ca(2+) stores. Accordingly, junctate accumulates near phagosomes and elongates ER-phagosome junctions in Stim1(-/-) cells. Thus, junctate mediates an alternative mechanism for generating localized Ca(2+) elevations within cells, promoting Ca(2+) release from internal stores recruited to phagosomes, thereby boosting phagocytosis.

Simultaneous Imaging of Local Calcium and Single Sarcomere Length in Rat Neonatal Cardiomyocytes via Expression of Cameleon-Nano in Z-Discs

In cardiac muscle, contraction is regulated by micromolar concentrations of Ca2+ released from the sarcoplasmic reticulum (SR) (i.e., Ca2+-induced Ca2+ release), resulting in the binding of Ca2+ to troponin C and subsequent formation of cross-bridges. In order to enhance our understanding of cardiac excitation-contraction coupling, we in the present study developed a novel experimental system for simultaneous measurement of local intracellular Ca2+ ([Ca2+]i) and single sarcomere length via expression of yellow cameleon-Nano (i.e., FRET-based ultrasensitive Ca2+ indicator) fused to the C-terminus of α-actinin in Z-discs in primary-cultured rat neonatal cardiomyocytes. In a given sarcomere, the fluorescence emission ratio (YFP/CFP) of the expressed fusion protein varied in response to a change in local [Ca2+]i. Under dual-view microscopy, we simultaneously measured local [Ca2+]i and sarcomere length (the latter defined as the distance between the YFP fluorescence profiles), and found that sarcomere length varied in response to a change in YFP/CFP in various local regions of the myocyte. These results demonstrate that the present experimental system is useful for elucidating cardiac excitation-contraction coupling at the single sarcomere level.

Abstract 152: Unleashed intracellular Ca2+ Releases From Ryanodine Receptor-2 Plays An Important Role In Diabetic Cerebral Vasoconstriction

Many devastating cerebral vascular diseases occur due to abnormally increased [Ca 2+ ] i and associated contraction in cerebral artery smooth muscle cells (CASMCs); however, the underlying molecular mechanisms are largely unknown. In this study, we assessed the novel hypothesis that FK506 binding protein 12.6 (FKBP12.6) is dissociated from ryanodine receptor-2 (RyR2), leading to unleashed intracellular Ca 2+ release in diabetic CASMCs and thus cerebral vasoconstriction. Using Förster resonance energy transfer and other biochemical assays, we have found that FKBP12.6 was tightly associated with RyR2 in mouse CASMCs. The association of these two Ca 2+ signaling molecules was disrupted in diabetic cells. Investigations using a laser scanning confocal microscope have revealed that targeted gene knockout (KO) of FKBP12.6 significantly increased local intracellular Ca 2+ release (Ca 2+ spark). A similar change in local intracellular Ca 2+ release was observed in diabetic cells. FKBP12.6 KO and diabetes did not produce additive effects. Global (whole-cell) intracellular Ca 2+ release following activation of RyRs with caffeine was increased to a similar extent in both FKBP12.6 KO and diabetic CASMCs. The increased caffeine-induced intracellular Ca 2+ release was not further enhanced in diabetic/FKBP12.6 KO cells. Stimulation of -adrenergic receptors with norepinephrine could result in a large increase in local and global intracellular Ca 2+ release with the same degree in FKBP12.6 KO, diabetic and diabetic/FKBP12.6 KO. FKBP12.6 KO, diabetic and diabetic/FKBP12.6 KO mice were all hypertensive, and their hypertensive levels were similar. In conclusion, we have, for the first time, provided compelling evidence that FKBP12.6 is dissociated from RyR2, which leads to unleashed intracellular Ca 2+ release through RyR2 in CASMCs and associated cerebral vasoconstriction, thereby playing an essential role in the development of diabetic and other cerebral vascular diseases.

Localized mobilization of intracellular calcium promotes epithelial repair in vivo

We report that localized intracellular Ca2+ mobilization occurs at the site of gastric surface epithelial damage in vivo, and investigate the role of intracellular and extracellular Ca2+ in stimulating the epithelial cell migration that mediates tissue repair. Experiments used anesthetized yellow cameleon 3.0 (intracellular Ca2+ sensor) transgenic mice, with the surgically exposed gastric mucosa imaged with confocal/two‐photon microscopy. CFP was excited at 840 nm and Ca2+ dynamics measured as FRET/CFP ratio. Photodamage (high intensity 840 nm light) was imposed on 3–5 gastric epithelial surface cells, leading to a microscopic lesion that healed within 15 min. During repair, intracellular Ca2+ selectively increased in restituting epithelial cells directly adjacent to the damaged area. Pretreatment with either chelerythrine (PKC inhibitor), U‐73122 (phospholipase C [PLC] inhibitor), or verapamil (voltage‐activated Ca2+ channel blocker) inhibited repair of damage, and these agents also inhibited the intracellular Ca2+increase. Addition of 10 mM luminal Ca2+ rescued the slowed repair of damage caused by verapamil or indomethacin (COX inhibitor), and partially rescued the increase of intracellular Ca2. Intracellular Ca2+ mobilization in restituting epithelial cells plays an important role in gastric repair in vivo, and is regulated by eicosanoids, PKC, PLC and voltage operated Ca2+ channels. NIDDK RO1‐DK‐54940 and University of Cincinnati Research Council Funding.

Endothelin‐1 increases the frequency of smooth muscle calcium waves in vena cava but not aorta

Vascular contraction is associated with a global increase in smooth muscle Ca2+. The frequency and amplitude of localized intracellular Ca2+ release events, such as IP3 receptor‐mediated Ca2+ waves, can also regulate excitation‐contraction coupling in smooth muscle. While the vasoconstrictor ET‐1 does increase IP3 formation, it is unclear if IP3 receptor‐mediated Ca2+ waves are a regulator of ET‐1‐induced contraction in arteries and veins. We hypothesized that ET‐1 increases the frequency and amplitude of Ca2+ waves in rat aorta (RA) and vena cava (RVC). RA and RVC were mounted en face in an imaging superfusion chamber and incubated with the intensiometric fluorescent Ca2+ indicator Fluo 4‐AM (10 μM). Using spinning‐disc confocal microscopy, image sequences were first recorded with buffer alone, and then maximal contractile concentrations of norepinephrine (NE, 10 μM) (RVC) or phenylephrine (PE, 10 μM) (RA), and finally ET‐1 (100 nM). ET‐1 (100 nM) increased Ca2+ wave frequency significantly in RVC versus control (0.72±0.16 Hz vs. 0.45±0.13 Hz), but not in RA. Neither NE (10 μM) nor PE (10 μM) altered Ca2+ wave frequency or amplitude in RA and RVC. These data indicate that intracellular Ca2+ mobilization by ET‐1 is different in arteries and veins. These data also provide indirect evidence that ET receptors are coupled to IP3‐mediated Ca2+ release mechanisms in the vena cava but not aorta. Supported by NIH P01HL70687.

β2-Adrenergic Receptor (β2-AR) Stimulation of Cytosolic Phospholipase A2 (cPLA2) Is Dependent on PKC and IP3-Mediated Ca2+ Signaling in Atrial Myocytes

We reported that inhibition of adenylate cyclase (AC)/cAMP by cell attachment to laminin (+LMN) or pharmacological (KT5720) inhibition of cAMP-dependent protein kinase (PKA) in cells not attached to LMN (-LMN-PKA), activates β2-AR stimulation of ICa,L via cPLA2 signaling. The present study determined the role of PKC and IP3-mediated Ca2+ release in β2-AR/cPLA2 signaling. As previously reported, 0.1 μM zinterol (β2-AR agonist) stimulation of L-type Ca2+ current (ICa,L) was unaffected by 10 μM AACOCF3 (cPLA2 inhibitor) in cells not attached to LMN (-LMN) but was significantly inhibited in +LMN and -LMN-PKA myocytes. Zinterol stimulation of ICa,L in -LMN-PKA myocytes was blocked by 5 μM U73122 (PLC inhibitor) and significantly inhibited by 4 μM chelerythrine (PKC inhibitor). Zinterol stimulation of ICa,L in -LMN myocytes was unaffected by inhibition of IP3-receptors (IP3Rs) by 2 μM 2-APB, but was significantly inhibited in +LMN and -LMN-PKA myocytes. Cells were cultured on LMN (24 hrs) with an adenovirus IP3 affinity trap to inhibit IP3-dependent Ca2+ signaling. Compared to control cells (β-gal), zinterol stimulation of ICa,L was significantly inhibited in cells infected with IP3 trap. Laser scanning confocal microscopy (fluo-4) revealed that zinterol stimulation of +LMN myocytes elicited local intracellular Ca2+ release events in 1 mM tetracaine (blocks RyR Ca2+ release), that were blocked by 2-APB. We conclude that inhibition of cAMP/PKA activates β2-AR stimulation of ICa,L via cPLA2 which is dependent on PKC and IP3-mediated Ca2+ signaling. These findings may be relevant to the remodeling of β-AR signaling in diseased (fibrotic) and/or aging atria, both of which exhibit decreases in AC activity.

Studies of RyR function in situ

The ryanodine receptors (RyRs) are intracellular Ca2+ release channels of the sarcoplasmic reticulum (SR) involved in many cellular responses, including muscle excitation–contraction coupling. Multiple biochemical and biophysical methods are available to study RyR functions. However, most of them are somewhat limited because they can only be used to examine channels which are purified from the SR and no longer in their natural environment. In this review we discuss optical methods for studying RyR functions in situ. We describe several techniques for the investigation of local (microscopic) intracellular Ca2+ signals (a.k.a Ca2+ sparks) by means of confocal microscopy and flash photolysis of caged compounds. We discuss how these studies can and will continue to contribute to our understanding of RyR function in physiological and pathological conditions.

Calcium microdomains at presynaptic active zones of vertebrate hair cells unmasked by stochastic deconvolution

Signal transduction by auditory and vestibular hair cells involves an impressive ensemble of finely tuned control mechanisms, strictly dependent on the local intracellular Ca(2+) concentration ([Ca(2+)](i)). The study of Ca(2+) dynamics in hair cells typically combines Ca(2+)-sensitive fluorescent indicators (dyes), patch clamp and optical microscopy to produce images of the patterns of fluorescence of a Ca(2+) indicator following various stimulation protocols. Here we describe a novel method that combines electrophysiological recordings, fluorescence imaging and numerical simulations to effectively deconvolve Ca(2+) signals within cytoplasmic microdomains that would otherwise remain inaccessible to direct observation. The method relies on the comparison of experimental data with virtual signals derived from a Monte Carlo reaction-diffusion model based on a realistic reconstruction of the relevant cell boundaries in three dimensions. The model comprises Ca(2+) entry at individual presynaptic active zones followed by diffusion, buffering, extrusion and release of Ca(2+). Our results indicate that changes of the hair cell [Ca(2+)](i) during synaptic transmission are primarily controlled by the Ca(2+) endogenous buffers both at short (<1mu) and at long (tens of microns) distances from the active zones. We provide quantitative estimates of concentration and kinetics of the hair cell endogenous Ca(2+) buffers and Ca(2+)-ATPases. We finally show that experimental fluorescence data collected during Ca(2+) influx are not interpreted correctly if the [Ca(2+)](i) is estimated by assuming that Ca(2+) equilibrates instantly with its reactants. In our opinion, this approach is of potentially general interest as it can be easily adapted to the study of Ca(2+) dynamics in diverse biological systems.

Hypoxia reduces KCa channel activity by inducing Ca2+ spark uncoupling in cerebral artery smooth muscle cells

Arterial smooth muscle cell large-conductance Ca(2+)-activated potassium (K(Ca)) channels have been implicated in modulating hypoxic dilation of systemic arteries, although this is controversial. K(Ca) channel activity in arterial smooth muscle cells is controlled by localized intracellular Ca(2+) transients, termed Ca(2+) sparks, but hypoxic regulation of Ca(2+) sparks and K(Ca) channel activation by Ca(2+) sparks has not been investigated. We report here that in voltage-clamped (-40 mV) cerebral artery smooth muscle cells, a reduction in dissolved O(2) partial pressure from 150 to 15 mmHg reversibly decreased Ca(2+) spark-induced transient K(Ca) current frequency and amplitude to 61% and 76% of control, respectively. In contrast, hypoxia did not alter Ca(2+) spark frequency, amplitude, global intracellular Ca(2+) concentration, or sarcoplasmic reticulum Ca(2+) load. Hypoxia reduced transient K(Ca) current frequency by decreasing the percentage of Ca(2+) sparks that activated a transient K(Ca) current from 89% to 63%. Hypoxia reduced transient K(Ca) current amplitude by attenuating the amplitude relationship between Ca(2+) sparks that remained coupled and the evoked transient K(Ca) currents. Consistent with these data, in inside-out patches at -40 mV hypoxia reduced K(Ca) channel apparent Ca(2+) sensitivity and increased the K(d) for Ca(2+) from approximately 17 to 32 microM, but did not alter single-channel amplitude. In summary, data indicate that hypoxia reduces K(Ca) channel apparent Ca(2+) sensitivity via a mechanism that is independent of cytosolic signaling messengers, and this leads to uncoupling of K(Ca) channels from Ca(2+) sparks. Transient K(Ca) current inhibition due to uncoupling would oppose hypoxic cerebrovascular dilation.

Neuromodulation of short-term synaptic dynamics examined in a mechanistic model based on kinetics of calcium currents

Network plasticity arises in large part due to the effects of exogenous neuromodulators. We investigate the neuromodulatory effects on short-term synaptic dynamics. The synapse from the lateral pyloric (LP) to the pyloric dilator (PD) neuron in the pyloric network of the crab C. borealis has both spike-mediated and non-spike-mediated (graded) components. Previous studies have shown that the graded component of this synapse exhibits short-term depression. Recent results from our lab indicate that in the presence of the neuromodulatory peptide proctolin, low-amplitude presynaptic stimuli switch the short-term dynamics of this graded component from depression to facilitation. In this study, we show that this facilitation is correlated with the activation of a presynaptic inward current that is blocked by Mn 2+, suggesting that it is a slowly accumulating Ca 2+ current. We modify a mechanistic model of synaptic release by assuming that the low-voltage-activating Ca 2+ current in our system is composed of two currents with fast ( I CaF) and slow ( I CaS) kinetics. We show that if proctolin adjusts the activation rate of I CaS, this leads to accumulation of local intracellular Ca 2+ in response to multiple presynaptic voltage stimuli, which, in turn, results in synaptic facilitation. Additionally, we assume that proctolin increases the maximal conductances of Ca 2+ currents in the model, consistent with the increased synaptic release found in the experiments. We find that these two presynaptic actions of proctolin in the model are sufficient to describe its actions on the short-term dynamics of the LP to PD synapse.