Ryanodine-induced vasoconstriction of the gerbil spiral modiolar artery depends on the Ca<Superscript>2+</Superscript> sensitivity but not on Ca<Superscript>2+</Superscript> sparks or BK channels

Background

In many vascular smooth muscle cells (SMCs), ryanodine receptor-mediated Ca²⁺ sparks activate large-conductance Ca²⁺-activated K⁺ (BK) channels leading to lowered SMC [Ca²⁺]_i and vasodilation. Here we investigated whether Ca²⁺ sparks regulate SMC global [Ca²⁺]_i and diameter in the spiral modiolar artery (SMA) by activating BK channels.

Methods

SMAs were isolated from adult female gerbils, loaded with the Ca²⁺-sensitive flourescent dye fluo-4 and pressurized using a concentric double-pipette system. Ca²⁺ signals and vascular diameter changes were recorded using a laser-scanning confocal imaging system. Effects of various pharmacological agents on Ca²⁺ signals and vascular diameter were analyzed.

Results

Ca²⁺ sparks and waves were observed in pressurized SMAs. Inhibition of Ca²⁺ sparks with ryanodine increased global Ca²⁺ and constricted SMA at 40 cmH₂O but inhibition of Ca²⁺ sparks with tetracaine or inhibition of BK channels with iberiotoxin at 40 cmH₂O did not produce a similar effect. The ryanodine-induced vasoconstriction observed at 40 cmH₂O was abolished at 60 cmH₂O, consistent with a greater Ca²⁺-sensitivity of constriction at 40 cmH₂O than at 60 cmH₂O. When the Ca²⁺-sensitivity of the SMA was increased by prior application of 1 nM endothelin-1, ryanodine induced a robust vasoconstriction at 60 cmH₂O.

Conclusions

The results suggest that Ca²⁺ sparks, while present, do not regulate vascular diameter in the SMA by activating BK channels and that the regulation of vascular diameter in the SMA is determined by the Ca²⁺-sensitivity of constriction.

     

The mechanism of neuroprotection by positive modulation of Ca<Superscript>2+</Superscript>-activated K<Superscript>+</Superscript> channels of cerebellar neurons in primary culture

Here we show that positive modulators (CyPPA and NS309) of Ca²⁺-activated K⁺ channels of small (SK) and intermediate (IK) conductances in cerebellar neurons decrease glutamate-evoked Ca²⁺ entry into neurons independently on the presence of Mg²⁺ in extracellular media. An analysis of neuronal viability after long-term (240 min) glutamate treatments demonstrated neuroprotective action of CyPPA and NS309. Extracellular Mg²⁺ did not protect neurons from apoptosis during prolonged treatment with glutamate. Activation of SK and IK channels results in local membrane hyperpolarization, which enhances Mg²⁺ block of NMDA receptors and reduces activation of voltage-dependent Ca²⁺ channels, which can explain neuroprotection caused by CyPPA or NS309. The obtained results reveal an important role Ca²⁺-activated K⁺ channels of small and intermediate conductance in the regulation of Ca²⁺ entry into cerebellar neurons via NMDA receptors and voltage-gated Ca²⁺ channels.     

Ca<Superscript>2+</Superscript> channels and Ca<Superscript>2+</Superscript> signals involved in abiotic stress responses in plant cells: recent advances

Calcium (Ca²⁺) signals are essential transducers and regulators in many adaptive and developmental processes in plants. Protective responses of plants to a variety of environmental stress factors are mediated by transient changes of Ca²⁺ concentration in plant cells. Ca²⁺ ions are quickly transported by channel proteins present on the plasma membrane. During responses to external stimuli, various signal molecules are transported directly from extracellular to intracellular compartments via Ca²⁺ channel proteins. Three types of Ca²⁺ channels have been identified in plant cell membranes: voltage-dependent Ca²⁺-permeable channels (VDCCs), which is sorted to depolarization-activated Ca²⁺-permeable channels (DACCs) and hyperpolarization-activated Ca²⁺-permeable channels (HACCs), voltage-independent Ca²⁺-permeable channels (VICCs). They make functions in the abiotic stress such as TPCs, CNGCs, MS channels, annexins which distribute in the organelles, plasma membrane, mitochondria, cytosol, intracelluar membrane. This review summarizes recent advances in our knowledge of many types of Ca²⁺ channels and Ca²⁺ signals involved in abiotic stress resistance and responses in plant cells.     

Physiological roles of NAADP-mediated Ca<Superscript>2+</Superscript> signaling

Nicotinic acid dinucleotide phosphate (NAADP) is unique amongst Ca²⁺ mobilizing messengers in that its principal function is to mobilize Ca²⁺ from acidic organelles. Early studies indicated that it was likely that NAADP activates a novel Ca²⁺ release channel distinct from the well characterized Ca²⁺ release channels on the (sarco)-endoplasmic reticulum (ER), inositol trisphosphate and ryanodine receptors. In this review, we discuss the emergence of a novel family of endolysosomal channels, the two-pore channels (TPCs), as likely targets for NAADP, and how molecular and pharmacological manipulation of these channels is enhancing our understanding of the physiological roles of NAADP as an intracellular Ca²⁺ mobilizing messenger.     

Contrasting Effects of Cd<Superscript>2+</Superscript> and Co<Superscript>2+</Superscript> on the Blocking/Unblocking of Human Ca<Subscript>v</Subscript>3 Channels

Inorganic ions have been used widely to investigate biophysical properties of high voltage-activated calcium channels (HVA: Ca_v1 and Ca_v2 families). In contrast, such information regarding low voltage-activated calcium channels (LVA: Ca_v3 family) is less documented. We have studied the blocking effect of Cd²⁺, Co²⁺ and Ni²⁺ on T-currents expressed by human Ca_v3 channels: Ca_v3.1, Ca_v3.2, and Ca_v3.3. With the use of the whole-cell configuration of the patch-clamp technique, we have recorded Ca²⁺ (2 mM) currents from HEK−293 cells stably expressing recombinant T-type channels. Cd²⁺ and Co²⁺ block was 2- to 3-fold more potent for Ca_v3.2 channels (EC₅₀ = 65 and 122 μM, respectively) than for the other two LVA channel family members. Current-voltage relationships indicate that Co²⁺ and Ni²⁺ shift the voltage dependence of Ca_v3.1 and Ca_v3.3 channels activation to more positive potentials. Interestingly, block of those two Ca_v3 channels by Co²⁺ and Ni²⁺ was drastically increased at extreme negative voltages; in contrast, block due to Cd²⁺ was significantly decreased. This unblocking effect was slightly voltage-dependent. Tail-current analysis reveals a differential effect of Cd²⁺ on Ca_v3.3 channels, which can not close while the pore is occupied with this metal cation. The results suggest that metal cations affect differentially T-type channel activity by a mechanism involving the ionic radii of inorganic ions and structural characteristics of the channels pore.     

Regulation of Ca<Superscript>2+</Superscript> influx in proliferating and differentiating murine myoblasts with participation of L-type Ca<Superscript>2+</Superscript> channels

The basic mechanisms of regulation of Ca²⁺ influx have been studied in murine myoblasts proliferating and differentiating in culture. The presence of L-type Ca²⁺ channels in proliferating myoblasts is shown for the first time. It is also shown that the influx of Ca²⁺ through these channels is regulated by the adrenergic system. The influx of Ca²⁺ after activation of the adrenergic system by addition of adrenaline has been estimated in comparison with the contribution of reticular stocks exhausted by ATP in calcium-free medium. The Ca²⁺ influx in proliferating myoblasts is regulated by β-2 adrenergic receptors whose action is mediated by adenylate cyclase through L-type calcium channels. In differentiating myoblasts, the adrenaline-induced Ca²⁺ influx is substantially lower than in proliferating cells, and maximal influx of Ca²⁺ may be reached only upon exhaustion of reticular stocks.     

The activity of plasma membrane hyperpolarization-activated Ca<Superscript>2+</Superscript> channels during pollen development of <Emphasis Type="Italic">Pyrus pyrifolia</Emphasis>

Calcium (Ca²⁺) plays crucial roles in regulation of pollen tube growth. The influx of Ca²⁺ into the pollen tube is mediated by ion channels, and the density and activity of Ca²⁺ channels in pollen plasma membranes critically determines their electrical properties. In this report, using whole-cell and single-channel patch-clamping techniques, we investigated developmental changes of hyperpolarization-activated Ca²⁺ channel activity in pear (Pyrus pyrifolia) pollen and its relationship with pollen viability. For both pollen and pollen tubes, hyperpolarization-activated Ca²⁺ channels had the same conductance and cAMP sensitivity, indicating that they were the same channels. However, the Ca²⁺ current density in pollen tube protoplasts was greater than that in pollen protoplasts. Compared with day-3 flowers’ pollen, hyperpolarization-activated Ca²⁺ current density was significantly lower in day 0 and day 3 flowers’ pollen, which was consistent with the pollen germination and pollen tube growth, indicating that pollen protoplasts’ increased Ca²⁺ current density may have enhanced the pollen viability. During pollen tube elongation, pollen tube plasma membrane Ca²⁺ current density increased with increased length pollen tubes up to 300 μm. All of these results indicated that hyperpolarization-activated Ca²⁺ channel activity was associated with in pear pollen development and may have a causal link between Ca²⁺ channel activity and pollen viability.     

Minocycline chelates Ca<Superscript>2+</Superscript>, binds to membranes,and depolarizes mitochondria by formation of Ca<Superscript>2+</Superscript>-dependent ion channels

Minocycline (an anti-inflammatory drug approved by the FDA) has been reported to be effective in mouse models of amyotrophic lateral sclerosis and Huntington disease. It has been suggested that the beneficial effects of minocycline are related to its ability to influence mitochondrial functioning. We tested the hypothesis that minocycline directly inhibits the Ca²⁺-induced permeability transition in rat liver mitochondria. Our data show that minocycline does not directly inhibit the mitochondrial permeability transition. However, minocycline has multiple effects on mitochondrial functioning. First, this drug chelates Ca²⁺ ions. Secondly, minocycline, in a Ca²⁺-dependent manner, binds to mitochondrial membranes. Thirdly, minocycline decreases the proton-motive force by forming ion channels in the inner mitochondrial membrane. Channel formation was confirmed with two bilayer lipid membrane models. We show that minocycline, in the presence of Ca²⁺, induces selective permeability for small ions. We suggest that the beneficial action of minocycline is related to the Ca²⁺-dependent partial uncoupling of mitochondria, which indirectly prevents induction of the mitochondrial permeability transition.     

Regulation of Insulin Secretion in Islets of Langerhans by Ca<Superscript>2+</Superscript>Channels

Insulin secretion from β-cells of the pancreatic islets of Langerhans is triggered by Ca²⁺ influx through voltage-dependent Ca²⁺ channels. Electrophysiological and molecular studies indicate that β-cells express several subtypes of these channels. This review discusses their roles in regulating insulin secretion, focusing on recent studies using β-cells, exogenous expression systems, and Ca²⁺ channel knockout mice. These investigations reveal that L-type Ca²⁺ channels in the β-cell physically interact with the secretory apparatus by binding to synaptic proteins on the plasma membrane and insulin granule. As a result, Ca²⁺ influx through L-type channels efficiently and rapidly stimulates release of a pool of insulin granules in close contact with the channels. Thus, L-type Ca²⁺ channel activity is preferentially coupled to exocytosis in the β-cell, and plays a critical role in regulating the dynamics of insulin secretion. Non-L-type channels carry a significant portion of the total voltage-dependent Ca²⁺ current in β-cells and cell lines from some species, but nevertheless account for only a small fraction of insulin secretion. These channels may regulate exocytosis indirectly by affecting membrane potential or second messenger signaling pathways. Finally, voltage-independent Ca²⁺ entry pathways and their potential roles in β-cell function are discussed. The emerging picture is that Ca²⁺ channels regulate insulin secretion at multiple sites in the stimulus-secretion coupling pathway, with the specific role of each channel determined by its biophysical and structural properties.This revised version was published online in June 2005 with a corrected cover date.     

Distinctive characteristics and functions of multiple mitochondrial Ca<Superscript>2+</Superscript> influx mechanisms

Intracellular Ca²⁺ is vital for cell physiology. Disruption of Ca²⁺ homeostasis contributes to human diseases such as heart failure, neuron-degeneration, and diabetes. To ensure an effective intracellular Ca²⁺ dynamics, various Ca²⁺ transport proteins localized in different cellular regions have to work in coordination. The central role of mitochondrial Ca²⁺ transport mechanisms in responding to physiological Ca²⁺ pulses in cytosol is to take up Ca²⁺ for regulating energy production and shaping the amplitude and duration of Ca²⁺ transients in various micro-domains. Since the discovery that isolated mitochondria can take up large quantities of Ca²⁺ approximately 5 decades ago, extensive studies have been focused on the functional characterization and implication of ion channels that dictate Ca²⁺ transport across the inner mitochondrial membrane. The mitochondrial Ca²⁺ uptake sensitive to non-specific inhibitors ruthenium red and Ru360 has long been considered as the activity of mitochondrial Ca²⁺ uniporter (MCU). The general consensus is that MCU is dominantly or exclusively responsible for the mitochondrial Ca²⁺ influx. Since multiple Ca²⁺ influx mechanisms (e.g. L-, T-, and N-type Ca²⁺ channel) have their unique functions in the plasma membrane, it is plausible that mitochondrial inner membrane has more than just MCU to decode complex intracellular Ca²⁺ signaling in various cell types. During the last decade, four molecular identities related to mitochondrial Ca²⁺ influx mechanisms have been identified. These are mitochondrial ryanodine receptor, mitochondrial uncoupling proteins, LETM1 (Ca²⁺/H⁺ exchanger), and MCU and its Ca²⁺ sensing regulatory subunit MICU1. Here, we briefly review recent progress in these and other reported mitochondrial Ca²⁺ influx pathways and their differences in kinetics, Ca²⁺ dependence, and pharmacological characteristics. Their potential physiological and pathological implications are also discussed.     

Regulation of the RyR channel gating by Ca<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript>

Ryanodine receptors (RyRs) are the Ca²⁺ release channels in the sarcoplasmic reticulum in striated muscle which play an important role in excitation-contraction coupling and cardiac pacemaking. Single channel recordings have revealed a wealth of information about ligand regulation of RyRs from mammalian skeletal and cardiac muscle (RyR1 and RyR2, respectively). RyR subunit has a Ca²⁺ activation site located in the luminal and cytoplasmic domains of the RyR. These sites synergistically feed into a common gating mechanism for channel activation by luminal and cytoplasmic Ca²⁺. RyRs also possess two inhibitory sites in their cytoplasmic domains with Ca²⁺ affinities of the order of 1 μM and 1 mM. Magnesium competes with Ca²⁺ at these sites to inhibit RyRs and this plays an important role in modulating their Ca²⁺-dependent activity in muscle. This review focuses on how these sites lead to RyR modulation by Ca²⁺ and Mg²⁺ and how these mechanisms control Ca²⁺ release in excitation-contraction coupling and cardiac pacemaking.     

Damage-induced cell–cell communication in different cochlear cell types via two distinct ATP-dependent Ca<Superscript>2+</Superscript> waves

Intercellular Ca²⁺ waves can coordinate the action of large numbers of cells over significant distances. Recent work in many different systems has indicated that the release of ATP is fundamental for the propagation of most Ca²⁺ waves. In the organ of hearing, the cochlea, ATP release is involved in critical signalling events during tissue maturation. ATP-dependent signalling is also implicated in the normal hearing process and in sensing cochlear damage. Here, we show that two distinct Ca²⁺ waves are triggered during damage to cochlear explants. Both Ca²⁺ waves are elicited by extracellular ATP acting on P2 receptors, but they differ in their source of Ca²⁺, their velocity, their extent of spread and the cell type through which they propagate. A slower Ca²⁺ wave (14 μm/s) communicates between Deiters’ cells and is mediated by P2Y receptors and Ca²⁺ release from IP₃-sensitive stores. In contrast, a faster Ca²⁺ wave (41 μm/s) propagates through sensory hair cells and is mediated by Ca²⁺ influx from the external environment. Using inhibitors and selective agonists of P2 receptors, we suggest that the faster Ca²⁺ wave is mediated by P2X₄ receptors. Thus, in complex tissues, the expression of different receptors determines the propagation of distinct intercellular communication signals.     

Effects of Levetiracetam,Carbamazepine, Phenytoin,Valproate, Lamotrigine,Oxcarbazepine, Topiramate,Vinpocetine and Sertraline on Presynaptic Hippocampal Na<Superscript>+</Superscript> and Ca<Superscript>2+</Superscript> Channels Permeability

Ion channels are targets of various antiepileptic drugs. In cerebral presynaptic nerve endings Na⁺ and Ca²⁺ channels are particularly abundant, as they control neurotransmitter release, including the release of glutamate (Glu), the most concentrated excitatory amino acid neurotransmitter in the brain. Several pre-synaptic channels are implicated in the mechanism of action of the pro-convulsive agent, 4-aminopyridine (4-AP). In the present study the effects of levetiracetam and other established and newer (vinpocetine) anti-epileptic drugs, as well as of the anti-depressant, sertraline on the increase in Ca²⁺ induced by 4-AP in hippocampal isolated nerve endings were investigated. Also the effects of some of the anti-seizure drugs on the selective increase in Ca²⁺ induced by high K⁺, or on the selective increase in Na⁺ induced by veratridine were tested. Sertraline and vinpocetine effectively inhibited the rise in Ca²⁺ induced by 4-AP, which was dependent on the out-in Na⁺ gradient and tetrodotoxin sensitive. Carbamazepine, phenytoin, lamotrigine and oxcarbazepine inhibited the rise in Ca²⁺ induced by 4-AP too, but at higher concentrations than sertraline and vinpocetine, whereas levetiracetam, valproic acid and topiramate did not. The three latter antiepileptic drugs also failed in modifying other responses mediated by the activation of brain presynaptic Na⁺ or Ca²⁺ channels, including Glu release. This indicates that levetiracetam, valproic acid and topiramate mechanisms of action are unrelated with a decrease in presynaptic Na⁺ or Ca²⁺ channels permeability. It is concluded that depolarized cerebral isolated nerve endings represent a useful tool to unmask potential antiepileptic drugs targeting presynaptic Na⁺ and/or Ca²⁺ channels in the brain; such as vinpocetine or the anti-depressant sertraline, which high effectiveness to control seizures in the animal in vivo has been demonstrated.     

Effects of Copper on T-Type Ca<Superscript>2+</Superscript> Channels in Mouse Spermatogenic Cells

Low voltage-activated, rapidly inactivating T-type Ca²⁺ channels are found in a variety of cells, where they regulate electrical activity and Ca²⁺ entry. In whole-cell patch-clamp recordings from mouse spermatogenic cells, trace element copper (Cu²⁺) inhibited T-type Ca²⁺ current (I _T-Ca) with IC₅₀ of 12.06 μM. Inhibition of I _T-Ca by Cu²⁺ was concentration-dependent and mildly voltage-dependent. When voltage stepped to −20 mV, Cu²⁺ (10 μM) inhibited I _T-Ca by 49.6 ± 4.1%. Inhibition of I _T-Ca by Cu²⁺ was accompanied by a shift of −2.23 mV in the voltage dependence of steady-state inactivation. Cu²⁺ upshifted the current–voltage (I-V) curve. To know the change of the gating kinetics of T-type Ca²⁺ channels, we analyzed the effect of Cu²⁺ on activation, inactivation, deactivation and reactivation of T-type Ca²⁺ channels. Since T-type Ca²⁺ channels are a key component in capacitation and the acrosome reaction, our data suggest that Cu²⁺ can affect male reproductive function through T-type Ca²⁺ channels as a preconception contraceptive material.     

Muscarinic acetylcholine receptor compounds alter net Ca<Superscript>2+</Superscript> flux and contractility in an invertebrate smooth muscle

Responses of a holothurian smooth muscle to a range of muscarinic (M₁ to M₅) acetylcholine receptor (mAChR) agonists and antagonists were surveyed using calcium (Ca²⁺)-selective electrodes and a mechanical recording technique. Most of the mAChR agonists and antagonists tested increased both contractility and net Ca²⁺ efflux, with M₁-specific agents like oxotremorine M being the most potent in their action. To investigate the possible sources of Ca²⁺ used during mAChR activation, agents that disrupt intracellular Ca²⁺ ion sequestration [cyclopiazonic acid (CPA), caffeine, ryanodine], the phosphoinositide signaling pathway [lithium chloride (LiCl)], and L-type Ca²⁺ channels (diltiazem and verapamil) were used to challenge contractions induced by oxotremorine M. These contractions were blocked by treatment with CPA, caffeine, LiCl, and by channel blockers, diltiazem and verapamil, but were unaltered by ryanodine. Our data suggest that this smooth muscle had an M_1,3,5-like receptor that was associated with the phosphoinositide signaling pathway that relied on intracellular Ca²⁺ stores, but secondarily used extracellular Ca²⁺ via the opening of L-type channels.     

Involvement of calcium-independent phospholipase A<Subscript>2</Subscript> in regulation of Ca<Superscript>2+</Superscript> signals induced by a calmodulin inhibitor in rat thymocytes

Previous studies have shown that micromolar concentrations of calmodulin inhibitor calmidazolium induce fast activation of nonselective Ca²⁺ channels in plasma membranes of Ehrlich ascites carcinoma cells (Zinchenko, V.P., Kasymov, V.A., Li, V.V., and Kaimachnikov, N.P., Biofizika (Rus.), 2005, vol. 50 (6), pp. 1055–1069). In order to detect this type of Ca²⁺ channels in other cells and to establish common regulatory mechanisms, we studied calmidazolium effects on rat thymocytes. It was found that calmidazolium induces biphasic increases in Ca²⁺ content in cytosol of rat thymocytes due to Ca²⁺ entry from external medium and reflects the activity of nonselective Ca²⁺ channels permeable for Mn²⁺ and Ni²⁺ ions. The rate and the amplitude of the fast phase are decreased, while those of the slow phase are increased in the presence of specific inhibitors of Ca²⁺-independent phospholipase A₂ (bromoenol lactone and palmitoyl trifluoromethyl ketone). The rate and the amplitude of the fast phase are also inhibited by arachidonic acid and the lipoxygenase inhibitor nordihydroguaiaretic acid, while the Ca²⁺-dependent phospholipase A₂ inhibitor bromophenacyl bromide, the cyclooxygenase inhibitor indomethacin, the specific store-operated Ca²⁺ channel inhibitor gadolinium and the phospholipase C inhibitor U73122 have no such effect. The rate of the fast phase only slightly depends on temperature, while that of the slow phase shows a strong temperature dependence and increases with a rise in temperatures (Q ₁₀ = 2). The amplitude of the fast phase of the Ca²⁺ signal increases with a decrease of temperatures due to prolongation of the maximum activity of the Ca²⁺ channel. The data obtained suggest that iPLA₂ is an intermediate link in the activation of calmidazolium-induced nonselective Ca²⁺ channels. The iPLA₂ products lysophospholipids and arachidonic acid activate and inhibit Ca²⁺ channels, respectively. The fact that these compounds manifest different affinities for Ca²⁺ channels shed additional light on the mechanisms of biphasic Ca²⁺ elevation in thymus cell cytosol and prolongation of the active state of Ca²⁺ channels at low temperatures.     

Ca<Superscript>2+</Superscript>-Dependent Phosphorylation of RyR2 Can Uncouple Channel Gating from Direct Cytosolic Ca<Superscript>2+</Superscript> Regulation

Phosphorylation of the cardiac ryanodine receptor (RyR2) is thought to be important not only for normal cardiac excitation-contraction coupling but also in exacerbating abnormalities in Ca²⁺ homeostasis in heart failure. Linking phosphorylation to specific changes in the single-channel function of RyR2 has proved very difficult, yielding much controversy within the field. We therefore investigated the mechanistic changes that take place at the single-channel level after phosphorylating RyR2 and, in particular, the idea that PKA-dependent phosphorylation increases RyR2 sensitivity to cytosolic Ca²⁺. We show that hyperphosphorylation by exogenous PKA increases open probability (P _o) but, crucially, RyR2 becomes uncoupled from the influence of cytosolic Ca²⁺; lowering [Ca²⁺] to subactivating levels no longer closes the channels. Phosphatase (PP1) treatment reverses these gating changes, returning the channels to a Ca²⁺-sensitive mode of gating. We additionally found that cytosolic incubation with Mg²⁺/ATP in the absence of exogenously added kinase could phosphorylate RyR2 in approximately 50% of channels, thereby indicating that an endogenous kinase incorporates into the bilayer together with RyR2. Channels activated by the endogenous kinase exhibited identical changes in gating behavior to those activated by exogenous PKA, including uncoupling from the influence of cytosolic Ca²⁺. We show that the endogenous kinase is both Ca²⁺-dependent and sensitive to inhibitors of PKC. Moreover, the Ca²⁺-dependent, endogenous kinase–induced changes in RyR2 gating do not appear to be related to phosphorylation of serine-2809. Further work is required to investigate the identity and physiological role of this Ca²⁺-dependent endogenous kinase that can uncouple RyR2 gating from direct cytosolic Ca²⁺ regulation.     

Images of Ca flux in astrocytes: evidence for spatially distinct sites of Ca release and uptake

In this study, we have developed a mathematical method to derive the Ca²⁺ fluxes underlying agonist-evoked Ca²⁺ waves in cultured rat cortical astrocytes. Astrocytes were stimulated with norepinephrine (100 nM) to evoke Ca²⁺ waves, which were recorded by measuring FIuo-3 fluorescence changes with high spatial and temporal resolution. Normalized fluorescence (ΔF/F) was analyzed in discrete cellular spaces in a series of successive slices along the length of the cell. From these data, Ca²⁺ flux was then calculated using a one dimensional reaction-diffusion equation which utilizes the temporal and spatial derivatives of the fluorescence data and the diffusion coefficient of Ca²⁺ in the cytosol. This method identified distinct sites of positive flux (Ca²⁺ release into the cytosol) and of negative flux (Ca²⁺ removal from cytosol) and showed that in astrocytes, sites of Ca²⁺ release from stores regularly alternate with sites of Ca²⁺ removal from the cytosol. Cross correlation analysis of the two distribution patterns gave positive correlation at 2 μm out of phase and a negative correlation in phase. Thapsigargin-induced Ca²⁺ waves were analyzed to determine if the negative flux was due to Ca²⁺ uptake via thapsigargin-sensitive Ca²⁺ pumps. Negative flux sites were still found under these conditions, suggesting that multiple mechanisms of Ca²⁺ removal from the cytosol may contribute to negative flux sites. This method of calculation of flux may serve as a means to describe the distribution of functional ion channels and pumps participating in cellular Ca²⁺ signalling.     

TRPC3 ion channel subunit immunolocalization in the cochlea

Canonical transient receptor potential (TRPC) subunits assemble as tetramers to form ion channels with high calcium (Ca²⁺) permeability. Here, we investigated the possibility that TRPC3 ion channels are broadly expressed in the adult guinea pig and mouse cochleae. Using immunofluorescence, pronounced labeling occurred in the spiral ganglion (SG) neurons, inner hair cells (IHC), outer hair cells (OHC) and epithelial cells lining scala media. TRPC3 expression was homogeneous in the SG throughout the cochlea. In contrast, there was marked spatial variation in the immunolabeling in the cochlear hair cells with respect to location. This likely relates to the tonotopy of these cells. TRPC3 immunolabeling was more pronounced in the IHC than OHC. Both basal region IHC and OHC had higher TRPC3 expression levels than the corresponding cells from the apical region of the cochlea. These data suggest that TRPC3 ion channels contribute to Ca²⁺ homeostasis associated with the hair cells, with higher ion fluxes in more basal regions of the cochlea, and may also be a significant pathway for Ca²⁺ entry associated with auditory neurotransmission via the SG neurons. TRPC3 expression was also identified within the spiral limbus region, inner and outer sulcus, but without evidence for spatial variation in expression level. Expression in these gap junction-coupled epithelial cells lining scala media is indicative of a contribution of TRPC3 channels to cochlear electrochemical homeostasis.     

Analysis of the Ca<Superscript>2+</Superscript> response of mycelial fungi to external effects by the recombinant aequorin method

Using the mutant strain Aspergillus awamori 66A, producing the recombinant Ca²⁺-dependent photosensitive protein aequorin, the dynamics of Ca²⁺ was studied for the first time in the cytosol of micromycetes exposed to stressful factors, such as an increase in extracellular Ca²⁺ to 50 mM, hypoosmotic shock, and mechanical shock. The cell response to stress proved to involve an increase in the Ca²⁺ concentration in the cytosol, which was determined from the amplitude of aequorin luminescence and the time of the amplitude enhancement and relaxation. The level of the Ca²⁺ response depended on the physiological stimulus. Inhibitory analysis with various agents that block Ca²⁺ channels and with agonists that specifically enhance the activity of the channels suggested that (1) the level of Ca²⁺ in the cytosol of micromycetes increases in response to stress because of the ion influx from both the growth medium and intracellular reservoirs and (2) potential-dependent transport systems play the major role in the Ca²⁺ influx into the cytosol of the micromycete cells.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 734–740.Original Russian Text Copyright © 2004 by Kozlova, Egorov, Kupriyanova-Ashina, Rid, El-Registan.