Whole-cell Ca2 Research Articles

Modeling Effects of L-Type Ca2+ Current and Na+-Ca2+ Exchanger on Ca2+ Trigger Flux in Rabbit Myocytes with Realistic T-Tubule Geometries

The transverse tubular system of rabbit ventricular myocytes consists of cell membrane invaginations (t-tubules) that are essential for efficient cardiac excitation-contraction coupling. In this study, we investigate how t-tubule micro-anatomy, L-type Ca2+ channel (LCC) clustering, and allosteric activation of Na+/Ca2+ exchanger by L-type Ca2+ current affects intracellular Ca2+ dynamics. Our model includes a realistic 3D geometry of a single t-tubule and its surrounding half-sarcomeres for rabbit ventricular myocytes. The effects of spatially distributed membrane ion-transporters (LCC, Na+/Ca2+ exchanger, sarcolemmal Ca2+ pump, and sarcolemmal Ca2+ leak), and stationary and mobile Ca2+ buffers (troponin C, ATP, calmodulin, and Fluo-3) are also considered. We used a coupled reaction-diffusion system to describe the spatio-temporal concentration profiles of free and buffered intracellular Ca2+. We obtained parameters from voltage-clamp protocols of L-type Ca2+ current and line-scan recordings of Ca2+ concentration profiles in rabbit cells, in which the sarcoplasmic reticulum is disabled. Our model results agree with experimental measurements of global Ca2+ transient in myocytes loaded with 50 μM Fluo-3. We found that local Ca2+ concentrations within the cytosol and sub-sarcolemma, as well as the local trigger fluxes of Ca2+ crossing the cell membrane, are sensitive to details of t-tubule micro-structure and membrane Ca2+ flux distribution. The model additionally predicts that local Ca2+ trigger fluxes are at least threefold to eightfold higher than the whole-cell Ca2+ trigger flux. We found also that the activation of allosteric Ca2+-binding sites on the Na+/Ca2+ exchanger could provide a mechanism for regulating global and local Ca2+ trigger fluxes in vivo. Our studies indicate that improved structural and functional models could improve our understanding of the contributions of L-type and Na+/Ca2+ exchanger fluxes to intracellular Ca2+ dynamics.

Toward an Integrative Computational Model of the Guinea Pig Cardiac Myocyte

The local control theory of excitation-contraction (EC) coupling asserts that regulation of calcium (Ca2+) release occurs at the nanodomain level, where openings of single L-type Ca2+ channels (LCCs) trigger openings of small clusters of ryanodine receptors (RyRs) co-localized within the dyad. A consequence of local control is that the whole-cell Ca2+ transient is a smooth continuous function of influx of Ca2+ through LCCs. While this so-called graded release property has been known for some time, its functional importance to the integrated behavior of the cardiac ventricular myocyte has not been fully appreciated. We previously formulated a biophysically based model, in which LCCs and RyRs interact via a coarse-grained representation of the dyadic space. The model captures key features of local control using a low-dimensional system of ordinary differential equations. Voltage-dependent gain and graded Ca2+ release are emergent properties of this model by virtue of the fact that model formulation is closely based on the sub-cellular basis of local control. In this current work, we have incorporated this graded release model into a prior model of guinea pig ventricular myocyte electrophysiology, metabolism, and isometric force production. The resulting integrative model predicts the experimentally observed causal relationship between action potential (AP) shape and timing of Ca2+ and force transients, a relationship that is not explained by models lacking the graded release property. Model results suggest that even relatively subtle changes in AP morphology that may result, for example, from remodeling of membrane transporter expression in disease or spatial variation in cell properties, may have major impact on the temporal waveform of Ca2+ transients, thus influencing tissue level electromechanical function.

Lineage Reprogramming from Cardiomyocytes to Pacemaker Cells via a Single Transcription Factor

The heartbeat originates in a small number of highly-specialized pacemaker cells found in the sinoatrial node (SAN). In an effort to create pacemaker cells from working cardiomyocytes, we expressed Tbx18, a transcription factor indispensible for embryonic development of the SAN. Within days, Tbx18-transduced neonatal rat cardiomyocytes (NRVMs) exhibited rhythmic spontaneous electrical firing. The mechanism of induced pacemaker activity resembled that of SAN cells, with enhanced voltage- and Ca2+-dependent “clock” pathways. The maximum diastolic potential was more positive in Tbx18-NRVMs in comparison to controls (−47±10 mV vs. −73±6 mV) with accompanying reduction in IK1. Line-scan confocal imaging of Tbx18-NRVMs loaded with Rhod-2 resolved localized Ca2+ release events (LCRs) that preceded each whole-cell Ca2+ transient resembling LCRs observed in native SAN cells. LCRs were not detected in control NRVMs. Spontaneous Ca2+-transients in Tbx18-NRVMs were suppressed on superfusion with ryanodine (10 μM) by 47±6% in comparison to 12±2% suppression in controls, indicative of increased relevance of [Ca2+]i stores. A 2.3-fold increase in [Ca2+]i stores was observed in Tbx18-NRVMs which can be attributed to increased sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) activity due to downregulation or phosphorylation of phospholamban (PLN), an inhibitor of SERCA. Tbx18-overexpression led to relative increase in the protein level of phosphorylated PLN (Ser16) similar to higher p-PLN level observed in the adult SAN relative to the left ventricle. In addition, Tbx18-NRVMs exhibited increased levels of cAMP and HCN4, shrank in size with disorganized myofibrils, and underwent epigenetic modifications consistent with durable reprogramming. Focal Tbx18 gene transfer in the guinea-pig ventricle consistently created ectopic pacemaker activity in vivo with a rhythm of 40±20 beats/min (n=5/7). Taken together, the data demonstrate faithful reprogramming of ventricular myocytes to pacemaker cells by a single transcription factor, Tbx18.

Prostaglandin E2 induces spontaneous rhythmic activity in mouse urinary bladder independently of efferent nerves

The acute effects of PGE(2) on bladder smooth muscle and nerves were examined to determine the origin of PGE(2)-induced spontaneous rhythmic contractions. Contraction studies, confocal Ca(2+) imaging and electrophysiological recordings in strips of mouse urinary bladder were used to differentiate the effects of PGE(2) on bladder smooth muscle and efferent nerves. PGE(2) (50 µM) increased the tone and caused phasic contractions of detrusor smooth muscle strips. Confocal Ca(2+) imaging showed that PGE(2) increased the frequency of whole-cell Ca(2+) transients (WCTs) (72 ± 5%) and intracellular recordings showed it increased the frequency of spontaneous depolarizations, from 0.31·s(-1) to 0.90·s(-1). Non-selective inhibition of EP receptors using SC-51322 and AH-6809 (10 µM), or the L-type Ca(2+) channel blocker nifedipine (1 µM), prevented these phasic contractions and WCTs, and reduced the tone (by 45 ± 7% and 59 ± 6%, respectively). Blocking P2X1 receptors with NF449 (10 µM) caused a small but significant reduction in the frequency of PGE(2)-induced phasic contractions (24 ± 9%) and WCTs (28 ± 17%) but had no significant effect on spontaneous depolarizations or tone. Inhibiting muscarinic receptors with cyclopentolate (1 µM) had no significant effect on these measures. Spontaneous WCTs became synchronous in PGE(2), implying enhanced functional coupling between neighbouring cells. However, the electrical input resistance was unchanged. It was concluded that depolarization alone is sufficient to explain a functional increase in intercellular coupling and the ability of PGE(2) to increase detrusor spontaneous rhythmic activity does not require parasympathetic nerves.

Ca2+ channel currents of cortical neurons from pure and mixed cultures

Voltage-gated Ca(2+) channels (VGCCs) are key regulators of many neuronal functions, and involved in multiple central nervous system diseases. In the last 30years, a large number of injury and disease models have been established based on cultured neurons. Culture with serum develops a mixture of neurons and glial cells, while culture without serum develops pure neurons. Both of these neuronal-culture methods are widely used. However, the properties of Ca(2+) currents in neurons from these two cultures have not been compared. In this study, we cultured rat cortical neurons in serum-containing or -free medium and then recorded the Ca(2+) channel currents using patch-clamp technique. Our results showed that there were significant differences in the amplitude and activation properties of whole-cell Ca(2+) channel currents, and of non-L-type Ca(2+) channel currents between the neurons from these two culture systems. Our data suggested that the difference of whole-cell Ca(2+) currents may result from the differences in non-L-type currents. Understanding of these properties will considerably advance studies of VGCCs in neurons from pure or mixed culture.

Acute Alterations of Somatodendritic Action Potential Dynamics in Hippocampal CA1 Pyramidal Cells after Kainate-Induced Status Epilepticus in Mice

Pathophysiological remodeling processes at an early stage of an acquired epilepsy are critical but not well understood. Therefore, we examined acute changes in action potential (AP) dynamics immediately following status epilepticus (SE) in mice. SE was induced by intraperitoneal (i.p.) injection of kainate, and behavioral manifestation of SE was monitored for 3–4 h. After this time interval CA1 pyramidal cells were studied ex vivo with whole-cell current-clamp and Ca2+ imaging techniques in a hippocampal slice preparation. Following acute SE both resting potential and firing threshold were modestly depolarized (2–5 mV). No changes were seen in input resistance or membrane time constant, but AP latency was prolonged and AP upstroke velocity reduced following acute SE. All cells showed an increase in AP halfwidth and regular (rather than burst) firing, and in a fraction of cells the notch, typically preceding spike afterdepolarization (ADP), was absent following acute SE. Notably, the typical attenuation of backpropagating action potential (b-AP)-induced Ca2+ signals along the apical dendrite was strengthened following acute SE. The effects of acute SE on the retrograde spread of excitation were mimicked by applying the Kv4 current potentiating drug NS5806. Our data unveil a reduced somatodendritic excitability in hippocampal CA1 pyramidal cells immediately after acute SE with a possible involvement of both Na+ and K+ current components.

Phosphodiesterase 4D Regulates Baseline Sarcoplasmic Reticulum Ca 2+ Release and Cardiac Contractility, Independently of L-Type Ca 2+ Current

Baseline contractility of mouse hearts is modulated in a phosphatidylinositol 3-kinase-γ-dependent manner by type 4 phosphodiesterases (PDE4), which regulate cAMP levels within microdomains containing the sarcoplasmic reticulum (SR) calcium ATPase type 2a (SERCA2a). The goal of this study was to determine whether PDE4D regulates basal cardiac contractility. At 10 to 12 weeks of age, baseline cardiac contractility in PDE4D-deficient (PDE4D(-/-)) mice was elevated mice in vivo and in Langendorff perfused hearts, whereas isolated PDE4D(-/-) cardiomyocytes showed increased whole-cell Ca2+ transient amplitudes and SR Ca2+content but unchanged L-type calcium current, compared with littermate controls (WT). The protein kinase A inhibitor R(p)-adenosine-3',5' cyclic monophosphorothioate (R(p)-cAMP) lowered whole-cell Ca2+ transient amplitudes and SR Ca2+ content in PDE4D(-/-) cardiomyocytes to WT levels. The PDE4 inhibitor rolipram had no effect on cardiac contractility, whole-cell Ca2+ transients, or SR Ca2+ content in PDE4D(-/-) preparations but increased these parameters in WT myocardium to levels indistinguishable from those in PDE4D(-/-). The functional changes in PDE4D(-/-) myocardium were associated with increased PLN phosphorylation but not cardiac ryanodine receptor phosphorylation. Rolipram increased PLN phosphorylation in WT cardiomyocytes to levels indistinguishable from those in PDE4D(-/-) cardiomyocytes. In murine and failing human hearts, PDE4D coimmunoprecipitated with SERCA2a but not with cardiac ryanodine receptor. PDE4D regulates basal cAMP levels in SR microdomains containing SERCA2a-PLN, but not L-type Ca2+ channels or ryanodine receptor. Because whole-cell Ca2+ transient amplitudes are reduced in failing human myocardium, these observations may have therapeutic implications for patients with heart failure.

Multi-target novel neuroprotective compound ITH33/IQM9.21 inhibits calcium entry, calcium signals and exocytosis

Compound ITH33/IQM9.21 (ITH/IQM) belongs to a new family of l-glutamic acid derivatives with antioxidant and neuroprotective properties on in vitro and in vivo models of stroke. Because neuronal damage after brain ischemia is tightly linked to excess Ca2+ entry and neuronal Ca2+ overload, we have investigated whether compound ITH/IQM antagonises the elevations of the cytosolic Ca2+ concentrations ([Ca2+]c) and the ensuing exocytotic responses triggered by depolarisation of bovine chromaffin cells. In fluo-4-loaded cell populations, ITH/IQM reduced the K+-evoked [Ca2+]c transients with an IC50 of 5.31μM. At 10μM, the compound decreased the amplitude and area of the Ca2+ transient elicited by challenging single fura-2-loaded cells with high K+, by 40% and 80%, respectively. This concentration also caused a blockade of K+-induced catecholamine release at the single-cell level (78%) and cell populations (55%). These effects are likely due to blockade of the whole-cell inward Ca2+ currents (IC50=6.52μM). At 10μM, ITH/IQM also inhibited the Ca2+-dependent outward K+ current, leaving untouched the voltage-dependent component of IK. The inward Na+ current was unaffected. Inhibition of depolarisation-elicited Ca2+ entry, [Ca2+]c elevation and exocytosis could contribute to the neuroprotective effects of ITH/IQM in vulnerable neurons undergoing depolarisation during brain ischemia.

Mechanism underlying the block of human Cav3.2 T-type Ca2+ channels by benidipine, a dihydropyridine Ca2+ channel blocker

AimsBenidipine, a dihydropyridine Ca2+ channel blocker, has been reported to block T-type Ca2+ channels; however, the mechanism underlying this effect was unclear. In this study, we characterized the mechanism responsible for this blocking activity. Furthermore, the blocking activity was compared between two enantiomers of benidipine, (S, S)- and (R, R)-benidipine. Main methodsHuman Cav3.2 (hCav3.2) T-type Ca2+ channels stably expressed in the human embryonic kidney cell line, HEK-293, were studied in whole-cell patch-clamp recordings and Ca2+ mobilization assay. Key findingsIn whole-cell patch-clamp recordings, benidipine blocked hCav3.2 T-type Ca2+ currents elicited by depolarization to a comparable extent as efonidipine. The block was dependent on stimulation frequency and holding potential, but not test potential. Benidipine significantly shifted the steady-state inactivation curve to the hyperpolarizing direction, but had no effect on the activation curve. Benidipine prolonged the recovery from inactivation of hCav3.2 T-type Ca2+ channels without any effect on the kinetics of activation, inactivation, or deactivation. In the Ca2+ mobilization assay, benidipine was more potent than efonidipine in blocking Ca2+ influx through hCav3.2 T-type Ca2+ channels. (S, S)-Benidipine was more potent than (R, R)-benidipine in blocking hCav3.2 T-type Ca2+ currents, but there was no difference in blocking the Ca2+ influx. SignificanceWe have characterized the blocking activity of benidipine against hCav3.2 Ca2+ channels and revealed the difference between the two enantiomers of benidipine. The blocking action of benidipine could be mediated by stabilizing hCav3.2 Ca2+ channels in an inactivated state.

Cav1.3 L-Type Calcium Channels-Mediated Ryanodine Receptor Dependent Calcium Release Controls Heart Rate

Pacemaker activity of the sino-atrial node (SAN) controls heart rate. However, the mechanism underling SAN pacemaker activity is not completely understood and in particular, the respective physiological importance of ion channels and ryanodine receptor (RyR)-dependent Ca2+ release in pacemaking is hotly debated. We have investigated Ca2+ handling in SAN pacemaker cells of wild-type (WT) and mice lacking L-type Cav1.3 (Cav1.3-/-) channels. In isolated Cav1.3-/- SAN cells the frequency of Ca2+ transients was reduced by 45% compared to WT pacemaker cells. Loss of Cav1.3 channels also blunted by about 47% the positive chronotropic effect induced by 0.01 μM isoproterenol (ISO). Furthermore, in Cav1.3-/- pacemaker cells, local Ca2+ release (LCR) occurring during the diastolic phase was reduced by 71%. In SAN cells from mice in which L-type Cav1.2 channels have been rendered insensitive to dihydropyridines (Cav1.2DHP-/-), application of 0.3 μM isradipine decreased diastolic LCR by 78 %, thus showing that Cav1.3 channels are major regulators of RyR-dependent LCR during the diastolic phase. In individual cells of isolated intact SAN, pacemaking of Cav1.3-/- cells was characterized by reduced (37%) frequency of Ca2+ transients and an increase in Ca2+ waves. Normal pacemaking in Cav1.3-/- isolated SAN cells and intact tissue could be observed only after direct activation of RyR-dependent Ca2+ release by low doses of caffeine (200 μM). Experiments with high doses of caffeine (10 mM) in Cav1.3-/- cells, showed that the reduction in diastolic LCR and in the frequency Ca2+ transients could not be ascribed to a decrease in sarcoplasmic reticulum (SR) Ca2+ content. Our results show that in SAN pacemaker cell, LCR Ca2+ release is tightly controlled by Cav1.3 channels and that such a control is critical for promoting the formation of whole-cell Ca2+ transients.Support: FWF (P20670, P22528), ANR-06-PHYSIO-004-01.

Optically Probing Heterogeneous Synaptic Activities in Hair Cells

At the first auditory synapse, the ribbon synapse of inner hair cell, neurotransmitter release is triggered by Ca2+ influx through clusters of Cav1.3 channels. Previous work in our lab has shown marked heterogeneity in the size of Ca2+-channel cluster (Meyer et al., 2009), as well as the amplitude and voltage dependence of microdomain Ca2+ (Frank et al. 2009) at active zones within single inner hair cells. Such heterogeneity of presynaptic Ca2+ signaling may contribute to the divergent sound coding properties of the postsynaptic spiral ganglion neurons that connect to the same hair cell. While potentially relevant for explaining large dynamic range sound encoding, its underlying mechanisms and the link to transmitter release are not well understood. Here we aimed at estimating the number of functional Ca2+ channels at the single synapses by means of an optical fluctuation analysis approach. The fluctuation analysis approach is compared to whole-cell Ca2+ current measurements in which single-synapse resolution is achieved by local pharmacological manipulation. In order to more systematically address synaptic heterogeneity we combined hair cell patch-clamp with spinning-disk confocal microscopy for simultaneous fast confocal Ca2+ imaging at multiple active zones. This method also allows us to analyze microdomain properties as a function of location in 3D-reconstructed hair cells. In addition, measurement of exocytosis at individual synapse is essential in linking Ca2+ signal amplitude to transmitter release. We are currently working on using optical reporters to monitor vesicle fusion at confocal resolution. Preliminary work demonstrates localized changes in VGLUT1-pHluorin fluorescence in response to voltage-gated Ca2+ influx.

Self-Organization of Pacemaking Sites for Calcium Waves and Oscillations in Cardiac Myocytes

Calcium (Ca) sparks are elementary events of intracellular Ca signaling, which tend to occur randomly. Ca waves and whole-cell Ca oscillations occur under Ca overload and disease conditions. How Ca waves emerge from Ca sparks is not completely understood. We developed a three-dimensional model for Ca cycling which contains 100x20x10=20,000 identical Ca release units (CRUs), simulating the CRU network corresponding to a complete cardiac myocyte with dimensions of 100x20x10 micrometers. Using this model, we can generate the well known Ca signaling hierarchy: Ca quarks, Ca sparks, macro-sparks, abortive waves, and full Ca waves. We can also induce spiral waves within the cell, a wave phenomenon widely observed in myocyte experiments. Besides the well known experimental observation that increasing Ca loading promotes these wave dynamics, we also make the following observations: 1) The diffusion rate of Ca is a key parameter. Spontaneous Ca waves occur only when the diffusion rate is above a critical value. 2) When the model is homogeneous, Ca waves originate from different locations via a self-organizing process. This self-organizing process is influenced by, but does not require, heterogeneity. 3) When the model contains heterogeneities, such as heterogeneous Ca release channel distribution, Ca waves can originate from different locations or occur repeatedly from the same location. In real cardiac rabbit ventricular myocytes loaded with Fluo-4 AM to image intracellular Ca, Ca waves typically originate from different locations after successive rapid pacing episodes. In conclusion, our results indicate that Ca waves in cardiac myocytes originate predominantly as a result of self-organizing processes rather than pre-existing heterogeneities.

The IQ Motif is Crucial for Cav1.1 Function

Ca2+-dependent modulation via calmodulin, with consensus CaM-binding IQ motif playing a key role, has been documented for most high-voltage-activated Ca2+ channels. The skeletal muscle Cav1.1 also exhibits Ca2+-/CaM-dependent modulation. Here, whole-cell Ca2+ current, Ca2+ transient, and maximal, immobilization-resistant charge movement (Qmax) recordings were obtained from cultured mouse myotubes, to test a role of IQ motif in function of Cav1.1. The effect of introducing mutation (IQ to AA) of IQ motif into Cav1.1 was examined. In dysgenic myotubes expressing YFP-Cav1.1AA, neither Ca2+ currents nor evoked Ca2+ transients were detectable. The loss of Ca2+ current and excitation-contraction coupling did not appear to be a consequence of defective trafficking to the sarcolemma. The Qmax in dysgenic myotubes expressing YFP-Cav1.1AA was similar to that of normal myotubes. These findings suggest that the IQ motif of the Cav1.1 may be an unrecognized site of structural and functional coupling between DHPR and RyR.

Repeated cocaine exposure decreases dopamine D2‐like receptor modulation of Ca2+ homeostasis in rat nucleus accumbens neurons

The nucleus accumbens (NAc) is a limbic structure in the forebrain that plays a critical role in cognitive function and addiction. Dopamine modulates activity of medium spiny neurons (MSNs) in the NAc. Both dopamine D₁-like and D₂-like receptors (including D1R or D(1,5)R and D2R or D(2,3,4)R, respectively) are thought to play critical roles in cocaine addiction. Our previous studies demonstrated that repeated cocaine exposure (which alters dopamine transmission) decreases excitability of NAc MSNs in cocaine-sensitized, withdrawn rats. This decrease is characterized by a reduction in voltage-sensitive Na(+) currents and high voltage-activated Ca(2+) currents, along with increased voltage-gated K(+) currents. These changes are associated with enhanced activity in the D1R/cAMP/PKA/protein phosphatase 1 pathway and diminished calcineurin function. Although D1R-mediated signaling is enhanced by repeated cocaine exposure, little is known whether and how the D2R is implicated in the cocaine-induced NAc dysfunction. Here, we performed a combined electrophysiological, biochemical, and neuroimaging study that reveals the cocaine-induced dysregulation of Ca(2+) homeostasis with involvement of D2R. Our novel findings reveal that D2R stimulation reduced Ca(2+) influx preferentially via the L-type Ca(2+) channels and evoked intracellular Ca(2+) release, likely via inhibiting the cAMP/PKA cascade, in the NAc MSNs of drug-free rats. However, repeated cocaine exposure abolished the D₂R effects on modulating Ca(2+) homeostasis with enhanced PKA activity and led to a decrease in whole-cell Ca(2+) influx. These adaptations, which persisted for 21 days during cocaine abstinence, may contribute to the mechanism of cocaine withdrawal.

Cardiodepressive effect elicited by the essential oil of Alpinia speciosa is related to L-type Ca2+ current blockade

This study was undertaken to elucidate the effect of the essential oil from Alpinia speciosa (EOAs) on cardiac contractility and the underlying mechanisms. The essential oil was obtained from Alpinia speciosa leaves and flowers and the oil was analyzed by GC-MS method. Chemical analysis revealed the presence of at least 18 components. Terpinen-4-ol and 1,8-cineole corresponded to 38% and 18% of the crude oil, respectively. The experiments were conducted on spontaneously-beating right atria and on electrically stimulated left atria isolated from adult rats. The effect of EOAs on the isometric contractions and cardiac frequency in vitro was examined. EOAs decreased rat left atrial force of contraction with an EC₅₀ of 292.2±75.7 μg/ml. Nifedipine, a well known L-type Ca²+ blocker, inhibited in a concentration-dependent manner left atrial force of contraction with an EC₅₀ of 12.1±3.5 μg/ml. Sinus rhythm was diminished by EOAs with an EC₅₀ of 595.4±56.2 μg/ml. Whole-cell L-type Ca²+ currents were recorded by using the patch-clamp technique. EOAs at 25 μg/ml decreased I(Ca,L) by 32.6±9.2% and at 250 μg/ml it decreased by 89.3±7.4%. Thus, inhibition of L-type Ca²+ channels is involved in the cardiodepressive effect elicited by the essential oil of Alpinia speciosa in rat heart.

Pulicatins A−E, Neuroactive Thiazoline Metabolites from Cone Snail-Associated Bacteria

The cone snail Conus pulicarius from the Philippines provides a specific habitat for actinomycetes and other bacteria. A phenotypic screen using primary cultures of mouse dorsal root ganglion neurons revealed that one C. pulicarius associate, Streptomyces sp. CP32, produces a series of natural products that enhance or diminish whole-cell Ca(2+) flux. These compounds include known thiazoline compounds and a series of new derivatives, pulicatins A-E (6-10). Individual compounds were shown to bind to a series of human receptors, with selective binding to the human serotonin 5-HT(2B) receptor. Here, we report the structure elucidation of the new compounds and results of the neurological assays.

The Extracellular Matrix Molecule Hyaluronic Acid Regulates Hippocampal Synaptic Plasticity by Modulating Postsynaptic L-Type Ca 2+ Channels

Although the extracellular matrix plays an important role in regulating use-dependent synaptic plasticity, the underlying molecular mechanisms are poorly understood. Here we examined the synaptic function of hyaluronic acid (HA), a major component of the extracellular matrix. Enzymatic removal of HA with hyaluronidase reduced nifedipine-sensitive whole-cell Ca(2+) currents, decreased Ca(2+) transients mediated by L-type voltage-dependent Ca(2+) channels (L-VDCCs) in postsynaptic dendritic shafts and spines, and abolished an L-VDCC-dependent component of long-term potentiation (LTP) at the CA3-CA1 synapses in the hippocampus. Adding exogenous HA, either by bath perfusion or via local delivery near recorded synapses, completely rescued this LTP component. In a heterologous expression system, exogenous HA rapidly increased currents mediated by Ca(v)1.2, but not Ca(v)1.3, subunit-containing L-VDCCs, whereas intrahippocampal injection of hyaluronidase impaired contextual fear conditioning. Our observations unveil a previously unrecognized mechanism by which the perisynaptic extracellular matrix influences use-dependent synaptic plasticity through regulation of dendritic Ca(2+) channels.

Spark-Induced Sparks As a Mechanism of Intracellular Calcium Alternans in Cardiac Myocytes

Intracellular calcium (Ca) alternans has been widely studied in cardiac myocytes and tissue, yet the underlying mechanism remains controversial. In this study, we used computational modeling and simulation to study how randomly occurring Ca sparks interact collectively to result in whole-cell Ca alternans. We developed a spatially distributed intracellular Ca cycling model in which Ca release units (CRUs) are locally coupled by Ca diffusion throughout the myoplasm and sarcoplasmic reticulum (SR) network. Ca sparks occur randomly in the CRU network when periodically paced with a clamped voltage waveform, but Ca alternans develops as the pacing speeds up. Combining computational simulation with theoretical analysis, we show that Ca alternans emerges as a collective behavior of Ca sparks, determined by 3 critical properties of the CRU network from which Ca sparks arise: "randomness" (of Ca spark activation), "refractoriness" (of a CRU after a Ca spark), and "recruitment" (Ca sparks inducing Ca sparks in adjacent CRUs). We also show that the steep nonlinear relationship between fractional SR Ca release and SR Ca load arises naturally as a collective behavior of Ca sparks, and Ca alternans can occur even when SR Ca is held constant. We present a general theory for the mechanisms of intracellular Ca alternans, which mechanistically links Ca sparks to whole-cell Ca alternans, and is applicable to Ca alternans in both physiological and pathophysiological conditions.

A Mathematical Analysis of Agonist- and KCl-Induced Ca2+ Oscillations in Mouse Airway Smooth Muscle Cells

Airway hyperresponsiveness is a major characteristic of asthma and is generally ascribed to excessive airway narrowing associated with the contraction of airway smooth muscle cells (ASMCs). ASMC contraction is initiated by a rise in intracellular calcium concentration ([Ca2+]i), observed as oscillatory Ca2+ waves that can be induced by either agonist or high extracellular K+ (KCl). In this work, we present a model of oscillatory Ca2+ waves based on experimental data that incorporate both the inositol trisphosphate receptor and the ryanodine receptor. We then combined this Ca2+ model and our modified actin-myosin cross-bridge model to investigate the role and contribution of oscillatory Ca2+ waves to contractile force generation in mouse ASMCs. The model predicts that: 1), the difference in behavior of agonist- and KCl-induced Ca2+ waves results principally from the fact that the sarcoplasmic reticulum is depleted during agonist-induced oscillations, but is overfilled during KCl-induced oscillations; 2), regardless of the order in which agonist and KCl are added into the cell, the resulting [Ca2+]i oscillations will always be the short-period, agonist-induced-like oscillations; and 3), both the inositol trisphosphate receptor and the ryanodine receptor densities are higher toward one end of the cell. In addition, our results indicate that oscillatory Ca2+ waves generate less contraction than whole-cell Ca2+ oscillations induced by the same agonist concentration. This is due to the spatial inhomogeneity of the receptor distributions.

Intracellular Ca 2+ Silences L-Type Ca 2+ Channels in Mesenteric Veins

Rationale : Calcium channel blockers (CCBs) exert their antihypertensive effect by reducing cardiac afterload but not preload, suggesting that Ca 2+ influx through L-type Ca 2+ channels (LTCC) mediates arterial but not venous tone. Objective : The object of this study was to resolve the mechanism of venous resistance to CCBs. Methods and Results : We compared the sensitivity of depolarization (KCl)-induced constriction of rat small mesenteric arteries (MAs) and veins (MVs) to the dilator effect of CCBs. Initial findings confirmed that nifedipine progressively dilated depolarization-induced constrictions in MAs but not MVs. However, Western blots showed a similar expression of the α 1C pore-forming subunit of the LTCC in both vessels. Patch-clamp studies revealed a similar density of whole-cell Ca 2+ channel current between single smooth muscle cells (SMCs) of MAs and MVs. Based on these findings, we hypothesized that LTCCs are expressed but “silenced” by intracellular Ca 2+ in venous SMCs. After depletion of intracellular Ca 2+ stores by the SERCA pump inhibitor thapsigargin, depolarization-induced constrictions in MVs were blocked 80% by nifedipine suggesting restoration of Ca 2+ influx through LTCCs. Similarly, KCl-induced constrictions were sensitive to block by nifedipine after depletion of intracellular Ca 2+ stores by caffeine, ryanodine, or 2-aminoethoxydiphenyl borate. Cell-attached patch recordings of unitary LTCC currents confirmed rare channel openings during depolarization of venous compared to arterial SMCs, but chelating intracellular Ca 2+ significantly increased the open-state probability of venous LTCCs. Conclusions : We report that intracellular Ca 2+ inactivates LTCCs in venous SMCs to confer venous resistance to CCB-induced dilation, a fundamental drug property that was previously unexplained.