Differential Regulation of Ca<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript> Signaling and Membrane Trafficking by Multiple P2 Receptors in Brown Adipocytes

Extracellular ATP triggers changes in intracellular Ca²⁺, ion channel function, and membrane trafficking in adipocytes. The aim of the present study was to determine which P2 receptors might mediate the Ca²⁺ signaling and membrane trafficking responses to ATP in brown fat cells. RT-PCR was used to determine which P2 receptors are expressed in brown fat cells. Responses to nucleotide agonists and antagonists were characterized using fura-2 fluorescence imaging of Ca²⁺ responses, and FM 1-43 fluorescence imaging and membrane capacitance measurements to assess membrane trafficking. The pharmacology of the Ca²⁺ responses fits the properties of the P2Y receptors for which mRNA is expressed, but the agonist and antagonist sensitivity of the membrane-trafficking response was not consistent with any P2 receptor described to date. Brown adipocytes expressed mRNA for P2Y₂, P2Y₆, and P2Y₁₂ metabotropic receptors and P2X₁, P2X₂, P2X₃, P2X₄, P2X₅, and P2X₇ ionotropic receptors. The agonists ATP, ADP, UTP, UDP and 2′, 3′-(benzoylbenzoyl) ATP (BzATP) increased intracellular Ca²⁺, while 100 μM suramin, pyridoxal-phosphate-6-azophenyl-2′ 4′-disulfonic acid (PPADS), or Reactive Blue 2 partially blocked Ca²⁺ responses. ATP, but not ADP, UTP, UDP or BzATP activated membrane trafficking. The membrane response could be blocked completely with 1 μM PPADS but not by the antagonist MRS2179. We conclude that multiple P2 receptors mediate the ATP responses of brown fat cells, and that membrane trafficking is regulated by a P2 receptor showing unusual properties.     

Boric acid inhibits stored Ca<Superscript>2+</Superscript> release in DU-145 prostate cancer cells

Boron (B) is a developmental and reproductive toxin. It is also essential for some organisms. Plants use uptake and efflux transport proteins to maintain homeostasis, and in humans, boron has been reported to reduce prostate cancer. Ca²⁺ signaling is one of the primary mechanisms used by cells to respond to their environment. In this paper, we report that boric acid (BA) inhibits NAD⁺ and NADP⁺ as well as mechanically induced release of stored Ca²⁺ in growing DU-145 prostate cancer cells. Cell proliferation was inhibited by 30% at 100μM, 60% at 250μM, and 97% at 1,000μM BA. NAD⁺-induced Ca²⁺ transients were partly inhibited at 250μM BA and completely at 1,000μM BA, whereas both NADP⁺ and mechanically induced transients were inhibited by 1,000μM BA. Expression of CD38 protein increased in proportion to BA exposure (0–1,000μM). In vitro mass spectrometry analysis showed that BA formed adducts with the CD38 products and Ca²⁺ channel agonists cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). Vesicles positive for the Ca²⁺ fluorophore fluo-3 acetoxymethyl ester accumulated in cells exposed to 250 and 1,000μM BA. The BA analog, methylboronic acid (MBA; 250 and 1,000μM), did not inhibit cell proliferation or NAD⁺, NADP⁺, or mechanically stimulated Ca²⁺ store release. Nor did MBA increase CD38 expression or cause the formation of intracellular vesicles. Thus, mammalian cells can distinguish between BA and its synthetic analog MBA and exhibit graded concentration-dependent responses. Based on these observations, we hypothesize that toxicity of BA stems from the ability of high concentrations to impair Ca²⁺ signaling.     

Comparative analysis of Ca2+-signaling in brown preadipocytes of the ground squirrel (Spermophillus undulatus) and mouse

We compared the Ca²⁺ response to noradrenalin, tapsigargin, thimerosal, and ionomycin in brown preadipocytes of the ground squirrel (Spermophillus undulatus) and mouse. The ground-squirrel brown preadipocytes did not respond to noradrenalin in concentrations within the physiological range. Stimulation of the plasma membrane Ca²⁺-channels with thimerosal showed a considerable reduction of the calcium entry in cell precursors of the both species. Intracellular calcium stores liberated in the preadipocytes of the both species by tapsigargin and ionomycin in Ca²⁺-free medium were insignificant. Ca²⁺-entry in preadipocytes was not activated by the intracellular Ca²⁺-store depletion. The Ca²⁺ response of the ground squirrel brown preadipocytes was independent of an animal’s physiological state or annual seasons. Brown preadipocytes of ground squirrels (Spermophillus undulatus) may be considered to be high ionomycin-resistant cells with reduced Ca²⁺-signaling systems.     

Convergence of Ca<Superscript>2+</Superscript> signaling pathways in adipocytes. The role of <Emphasis Type="Italic">L</Emphasis>-arginine and protein kinase G in generation of transient and periodic Ca<Superscript>2+</Superscript> signals

Experiments on cultured mouse adipocytes (9 days in vitro) using fluorescent microscopy have shown that activation of α₁- and α₂-adrenoceptors by norepinephrine (NE) or α₂-adrenoreceptors by L-arginine evokes transient Ca²⁺ signals, while activation of m₃-cholinoreceptors by acetylcholine (ACh) or betaine causes sustained or damped Ca²⁺ oscillations. The presence in the incubation medium of L-arginine at a low concentration (100–200 μM) is necessary for a vigorous manifestation of these effects, apparently due to transition of protein kinase G (PKG) and phosphodiesterase V into an active state. In the presence of 1–10 mM L-arginine, the amplitude of the Ca²⁺ transient response to NE increases and signal duration decreases. ACh and NE upon a sequential addition mutually potentiate their effects. Using an inhibitory analysis we show that the observed modes are related to the operation of a signaling pathway with the participation of phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB), endothelial NO synthase (eNOS), cytoplasmic guanylate cyclase (sGC), protein kinase G (PKG), ADP-ribosyl cyclase (CD38), and the ryanodine receptor (RyR). The formation of several loops of positive feedbacks (PF) and negative feedbacks (NF) in the signaling system is possible: (i) short PF loops due to Ca²⁺-induced Ca²⁺ release (CICR) from internal stores through the inositol trisphosphate receptor (IP₃R) and RyR participating in the transient signal formation; (ii) long PF loop Ca²⁺ → eNOS → sGC → PKG → CD38 → RyR → Ca²⁺, which can provide necessary conditions for calcium oscillations arising from short PF loops (CICR); (iii) several NF loops based on PKG-mediated inhibition of IP₃R and activation of Ca²⁺-ATPases of sarco(endo)plasmic reticulum and of the plasma membrane providing a shutdown of signaling by the pathway phospholipase C → IP₃R → Ca²⁺ and limiting Ca²⁺ rise caused by the pathway PI3K → PKB → eNOS → sGC → PKG → CD38 → RyR → Ca²⁺. Convergence of signaling pathways that involve α₁-, α₂-, and m₃-receptors and then Gβγ-subunits of G_q and G_q proteins acting on PI3Kγ can provide activation of cytoplasmic PKG, which plays a key role in producing transient responses, in activation of Ca²⁺ removal and generation of [Ca²⁺]_i oscillations. PKG inhibition (implemented here by KT5823 application) in the presence of any agonist results in rupture of NF loops controlling Ca²⁺ transporting systems activity that leads to uncontrolled [Ca²⁺]_i rise and cell death.     

Change in Ca<Superscript>2+</Superscript>-responses of cultivated brown adipocytes at adrenergic activation

The thermogenic capability of brown adipose tissue is controlled by noradrenaline. By interacting with α1- and β-adrenoreceptors of adipocytes, noradrenaline (NA) increases the intracellular concentration of Ca²⁺ ([Ca²⁺]_i) and cAMP. The changes in [Ca²⁺]_i under the action of NA and selective agonists of α1- and β-adrenoreceptors, i.e., cirazoline and isoproterenol (IP), are recorded on individual cells of the primary culture of adipocytes during the day in vitro (DIV) 1, DIV 3, and DIV 6. The change in [Ca²⁺]_i under the effect of IP as compared to the response to cirazoline in cells of DIV 1 is characterized by a higher amplitude and shorter duration of impulses in the entire diapason of the used physiological concentrations. After DIV 3, these differences are insignificant and, after DIV 6, the differences in kinetics are nearly absent. For all three agonists, the kinetics of the [Ca²⁺]_i change in the proliferating and differentiated cells is significantly different; i.e., the response amplitude increases with the age of the culture and the duration of transitory response decreases, while sensitivity to agonists of adrenoreceptors increases. It can be seen from the rise in [Ca²⁺]_i with an inhibitor of Ca²⁺-ATPase of the endoplasmic reticulum thapsigargin in calcium-free medium that the source of calcium ions in the endoplasmic reticulum rises with the growth and development of cells in culture, while the rate at which Ca²⁺ is pumped out of cells, which characterizes the activity of Ca²⁺-ATPase of the plasma membrane, increases.     

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.     

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.     

Inhibition of ATP-Induced Ca<Superscript>2+</Superscript> Influx by Corticosterone in Dorsal Root Ganglion Neurons

In addition to the classic genomic effects, it is well known that glucocorticoids also have rapid, nongenomic effects on neurons. In the present study, the effect of corticosterone (CORT) on ATP-induced Ca²⁺ mobilization in cultured dorsal root ganglion (DRG) neurons were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator that could monitor real-time alterations of intracellular calcium concentration ([Ca²⁺]i). ATP, an algesic agent, caused [Ca²⁺]i increase in DRG neurons by activation of P2X receptor. Pretreatment with CORT (1 nM–1 μM for 5 min) inhibited ATP-induced [Ca²⁺]i increase in DRG neurons. The rapid inhibition of ATP-induced Ca²⁺ response by CORT was concentration-dependent, reversible and could be blocked by glucocorticoid receptor antagonist RU38486 (10 μM). Furthermore, the inhibitory effect of CORT was abolished by protein kinase A inhibitor H89 (10 μM), but was not influenced by protein kinase C inhibitor Chelerythrine chloride (10 μM). On the other hand, membrane-impermeable bovine serum albumin-conjugated corticosterone had no effect on ATP-induced [Ca²⁺]i transients. These observations suggest that a nongenomic pathways may be involved in the effect of CORT on ATP-induced [Ca²⁺]i transients in cultured DRG neurons.     

Life after the birth of the mitochondrial Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchanger,NCLX

Powered by the mitochondrial membrane potential, Ca²⁺ permeates the mitochondria via a Ca²⁺ channel termed Ca²⁺ uniporter and is pumped out by a Na⁺/Ca²⁺ exchanger, both of which are located on the inner mitochondrial membrane. Mitochondrial Ca²⁺ transients are critical for metabolic activity and regulating global Ca²⁺ responses. On the other hand, failure to control mitochondrial Ca²⁺ is a hallmark of ischemic and neurodegenerative diseases. Despite their importance, identifying the uniporter and exchanger remains elusive and their inhibitors are non-specific. This review will focus on the mitochondrial exchanger, initially describing how it was molecularly identified and linked to a novel member of the Na⁺/Ca²⁺ exchanger superfamily termed NCLX. Molecular control of NCLX expression provides a selective tool to determine its physiological role in a variety of cell types. In lymphocytes, NCLX is essential for refilling the endoplasmic reticulum Ca²⁺ stores required for antigendependent signaling. Communication of NCLX with the store-operated channel in astroglia controls Ca²⁺ influx and thereby neuro-transmitter release and cell proliferation. The refilling of the Ca²⁺ stores in the sarcoplasmic reticulum, which is controlled by NCLX, determines the frequency of action potential and Ca²⁺ transients in cardiomyocytes. NCLX is emerging as a hub for integrating glucose-dependent Na⁺ and Ca²⁺ signaling in pancreatic β cells, and the specific molecular control of NCLX expression resolved the controversy regarding its role in neurons and β cells. Future studies on an NCLX knockdown mouse model and identification of human NCLX mutations are expected to determine the role of mitochondrial Ca²⁺ efflux in organ activity and whether NCLX inactivation is linked to ischemic and/or neurodegenerative syndromes. Structure-function analysis and protein analysis will identify the NCLX mode of regulation and its partners in the inner membrane of the mitochondria.     

Short-term exposure to somatostatin or muscarinic agonists reduce acetylcholine-induced <Superscript>3</Superscript>H-MPP<Superscript>+</Superscript> release from bovine adrenal medullary cells

Summary The aim of this work was to investigate the effect of a short-term exposure to somatostatin (SS), its receptors (SSTR) selective agonists as well as muscarinic receptors agonists upon acetylcholine-induced release of ³H-MPP⁺ from bovine adrenal medullary cells. Acetylcholine (ACH, 100, 500 μM) was found to increase the release of ³H-MPP⁺ by these cells (to 175 and 171% of basal release, respectively). ACH-elicited ³H-MPP⁺ release was significantly reduced by hexamethonium (100 μM) and atropine (100 μM), selective nicotinic and muscarinic antagonists, respectively. Previous exposure to any of two muscarinic agonists, oxotremorine or pilocarpine, led to a significant reduction of ³H-MPP⁺ release in response to 100 μM ACH, to about a maximum of 51% and 78% of control, respectively. Somatostatin (SS, 0.01–0.1 μM), previously applied to the preparation, depressed ACH-elicited ³H-MPP⁺ release by 25–27%, but only when a 500 μM ACH concentration was used. The inhibition exerted by SS upon ACH-evoked ³H-MPP⁺ release appeared to be mediated by its SSTR: (1) SSTR2, 3 and 4 subtype agonists mimicked the effects seen with SS, and (2) the SSTR non-selective antagonist, cyclo-SS, counteracted the SS inhibitory effect. When SS was tested in the presence of any of the muscarinic agonists, oxotremorine or pilocarpine, its inhibitory effect on 500 μM ACH-induced ³H-MPP⁺ release was no longer detectable. These results, showing a somewhat similar effect of short-term exposure to SS and muscarinic agonists over ACH-induced release of ³H-MPP⁺, as well as the loss of effect of SS by the presence of the muscarinic agonists, suggest that these compounds may share signalling pathways.     

Signaling Pathways in the Biphasic Effect of ANG II on Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Exchanger in T84 Cells

The effect of ANG II on pH_i, [Ca²⁺]_i and cell volume was investigated in T84 cells, a cell line originated from colon epithelium, using the probes BCECF-AM, Fluo 4-AM and acridine orange, respectively. The recovery rate of pH_i via the Na⁺/H⁺ exchanger was examined in the first 2 min following the acidification of pH_i with a NH₄Cl pulse. In the control situation, the pH_i recovery rate was 0.118 ± 0.001 (n = 52) pH units/min and ANG II (10⁻¹² M or 10⁻⁹ M) increased this value (by 106% or 32%, respectively) but ANG II (10⁻⁷ M) decreased it to 47%. The control [Ca²⁺]_i was 99 ± 4 (n = 45) nM and ANG II increased this value in a dose-dependent manner. The ANG II effects on cell volume were minor and late and should not interfere in the measurements of pH_i recovery and [Ca²⁺]_i. To document the signaling pathways in the hormonal effects we used: Staurosporine (a PKC inhibitor), W13 (a calcium-dependent calmodulin antagonist), H89 (a PKA inhibitor) or Econazole (an inhibitor of cytochrome P450 epoxygenase). Our results indicate that the biphasic effect of ANG II on Na⁺/H⁺ exchanger is a cAMP-independent mechanism and is the result of: 1) stimulation of the exchanger by PKC signaling pathway activation (at 10⁻¹² – 10⁻⁷ M ANG II) and by increases of [Ca²⁺]_i in the lower range (at 10⁻¹² M ANG II) and 2) inhibition of the exchanger at high [Ca²⁺]_i levels (at 10⁻⁹ – 10⁻⁷ M ANG II) through cytochrome P450 epoxygenase-dependent metabolites of the arachidonic acid signaling pathway.     

Chronic Nicotine Alters Nicotinic Receptor-induced Presynaptic Ca<Superscript>2+</Superscript> Responses in Isolated Nerve Terminals

Brain nicotinic receptors display pronounced permeability for Ca²⁺ and localize to presynaptic nerve terminals, in addition to postsynaptic sites. Chronic exposure to nicotine has been shown to alter brain nicotinic receptor expression, but the functional consequences for presynaptic Ca²⁺ have not been directly examined. Here, we used confocal imaging to assess Ca²⁺ responses in individual nerve terminals from cortices of mice treated up to 14 days with nicotine as compared to vehicle-treated controls. Chronic nicotine treatment led to substantially enhanced amplitudes of presynaptic Ca²⁺ responses to acute application of nicotine at concentrations of 50 nM (2-fold) and 500 nM (1.7-fold), but not 50 μM. In addition, increased expression of high-affinity nicotinic receptors on isolated terminals was observed following chronic treatment, as determined immunocytochemically and pharmacologically. These findings suggest that chronic exposure to nicotine may lead to enhanced sensitivity to nicotine at select presynaptic sites in brain via up-regulation of high-affinity nicotinic receptors.     

Ca<Superscript>2+</Superscript> and CaM are Involved in NO- and H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Adventitious Root Development in Marigold

Our previous results have demonstrated that both nitric oxide (NO) and hydrogen peroxide (H₂O₂) are involved in the promotion of adventitious root development in marigold (Tagetes erecta L.). However, not much is known about the intricate molecular network of adventitious root development triggered by NO and H₂O₂. In this study, the involvement of calcium (Ca²⁺) and calmodulin (CaM) in NO- and H₂O₂-induced adventitious rooting in marigold was investigated. Exogenous Ca²⁺ was capable of promoting adventitious rooting, with a maximal biological response at 50 μM CaCl₂. Ca²⁺ chelators and CaM antagonists prevented NO- and H₂O₂-induced adventitious rooting, indicating that both endogenous Ca²⁺ and CaM may play crucial roles in the adventitious rooting induced by NO and H₂O₂. NO and H₂O₂ treatments increased the endogenous content of Ca²⁺ and CaM, suggesting that NO and H₂O₂ enhanced adventitious rooting by stimulating the endogenous Ca²⁺ and CaM levels. Moreover, treatment with Ca²⁺ enhanced the endogenous levels of NO and H₂O₂. Additionally, Ca²⁺ might be involved as an upstream signaling molecule for CaM during NO- and H₂O₂-induced rooting. Altogether, the results suggest that both Ca²⁺ and CaM are two downstream signaling molecules in adventitious rooting induced by NO and H₂O₂.     

Enhanced Glutathione Efflux from Astrocytes in Culture by Low Extracellular Ca<Superscript>2+</Superscript> and Curcumin

Efflux of glutathione (GSH) from astrocytes has been suggested as a key factor for neuroprotection by astrocytes. Here we evaluated if the Nrf2 activator curcumin affects basal and stimulated (Ca²⁺ omission) GSH efflux from cultures of astroglial cells. Stimulated efflux of GSH was observed at medium concentration of 0, 0.1 mM Ca²⁺, but not at 0.2 or 0.3 mM Ca²⁺. Astroglia treated with 30 μM curcumin increased the cellular content of GSH in parallel with elevated basal and stimulated efflux. Conversely treatment with buthionine sulfoximine lowered efflux of GSH. The efflux stimulated by Ca²⁺- omission was not affected by the P2X7-receptor antagonist Blue Brilliant G (100 nM) or the pannexin mimetic/blocking peptide ¹⁰Panx1 but inhibited by the gap junction blocker carbenoxolone (100 μM) and a hemichannel blocker Gap26 (300 μM). RNAi directed against Nrf2 partly inhibited the effect of curcumin. The results show that elevated cellular GSH by curcumin treatment enhance efflux from astroglial cells, a process which appear to be a prerequisite for astroglial mediated neuroprotection.     

Towards extracellular Ca<Superscript>2+</Superscript> sensing by MRI: synthesis and calcium-dependent <Superscript>1</Superscript>H and <Superscript>17</Superscript>O relaxation studies of two novel bismacrocyclic Gd<Superscript>3+</Superscript> complexes

Two new bismacrocyclic Gd³⁺ chelates containing a specific Ca²⁺ binding site were synthesized as potential MRI contrast agents for the detection of Ca²⁺ concentration changes at the millimolar level in the extracellular space. In the ligands, the Ca²⁺-sensitive BAPTA-bisamide central part is separated from the DO3A macrocycles either by an ethylene (L¹) or by a propylene (L²) unit [H₄BAPTA is 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; H₃DO₃A is 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid]. The sensitivity of the Gd³⁺ complexes towards Ca²⁺ and Mg²⁺ was studied by ¹H relaxometric titrations. A maximum relaxivity increase of 15 and 10% was observed upon Ca²⁺ binding to Gd₂L¹ and Gd₂L², respectively, with a distinct selectivity of Gd₂L¹ towards Ca²⁺ compared with Mg²⁺. For Ca²⁺ binding, association constants of log K = 1.9 (Gd₂L¹) and log K = 2.7 (Gd₂L²) were determined by relaxometry. Luminescence lifetime measurements and UV–vis spectrophotometry on the corresponding Eu³⁺ analogues proved that the complexes exist in the form of monohydrated and nonhydrated species; Ca²⁺ binding in the central part of the ligand induces the formation of the monohydrated state. The increasing hydration number accounts for the relaxivity increase observed on Ca²⁺ addition. A ¹H nuclear magnetic relaxation dispersion and ¹⁷O NMR study on Gd₂L¹ in the absence and in the presence of Ca²⁺ was performed to assess the microscopic parameters influencing relaxivity. On Ca²⁺ binding, the water exchange is slightly accelerated, which is likely related to the increased steric demand of the central part leading to a destabilization of the Ln–water binding interaction. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Differential Contribution of L-, N-, and P/Q-type Calcium Channels to [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> Changes Evoked by Kainate in Hippocampal Neurons

We investigated the contribution of L-, N- and P/Q-type Ca²⁺ channels to the [Ca²⁺]_i changes, evoked by kainate, in the cell bodies of hippocampal neurons, using a pharmacological approach and Ca²⁺ imaging. Selective Ca²⁺ channel blockers, namely nitrendipine, ω-Conotoxin GVIA (ω-GVIA) and ω-Agatoxin IVA (ω-AgaIVA) were used. The [Ca²⁺]_i changes evoked by kainate presented a high variability, and were abolished by NBQX, a AMPA/kainate receptor antagonist, but the N-methyl-d-aspartate (NMDA) receptor antagonist, D-AP5, was without effect. Each Ca²⁺ channel blocker caused differential inhibitory effects on [Ca²⁺]_i responses evoked by kainate. We grouped the neurons for each blocker in three subpopulations: (1) neurons with responses below 60% of the control; (2) neurons with responses between 60% and 90% of the control, and (3) neurons with responses above 90% of the control. The inhibition caused by nitrendipine was higher than the inhibition caused by ω-GVIA or ω-AgaIVA. Thus, in the presence of nitrendipine, the percentage of cells with responses below 60% of the control was 41%, whereas in the case of ω-GVIA or ω-AgaIVA the values were 9 or 17%, respectively. The results indicate that hippocampal neurons differ in what concerns their L-, N- and P/Q- type Ca²⁺ channels activated by stimulation of the AMPA/kainate receptors. Special issue article in honor of Dr. Ricardo Tapia.     

Swelling-Induced Ca<Superscript>2+</Superscript> Influx and K<Superscript>+</Superscript> Efflux in American Alligator Erythrocytes

The American alligator can hibernate during winter, which may lead to osmotic imbalance because of reduced kidney function and lack of food consumption during this period. Accordingly, we hypothesized that their red blood cells would have a well-developed regulatory volume decrease (RVD) to cope with the homeostatic challenges associated with torpor. Osmotic fragility was determined optically, mean cell volume was measured by electronic sizing, and changes in intracellular Ca²⁺ concentration were visualized using fluorescence microscopy and fluo-4-AM. Osmotic fragility increased and the ability to regulate volume was inhibited when extracellular Na⁺ was replaced with K⁺, or when cells were exposed to the K⁺ channel inhibitor quinine, indicating a requirement of K⁺ efflux for RVD. Addition of the ionophore gramicidin to the extracellular medium decreased osmotic fragility and also potentiated volume recovery, even in the presence of quinine. In addition, hypotonic shock (0.5× Ringer) caused an increase in cytosolic Ca²⁺, which resulted from Ca²⁺ influx because it was not observed when extracellular Ca²⁺ was chelated with EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid). Furthermore, cells loaded with BAPTA-AM (1,2-bis(2-aminophenoxymethyl)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl) ester) or exposed to a low Ca²⁺-EGTA hypotonic Ringer had a greater osmotic fragility and also failed to recover from cell swelling, indicating that extracellular Ca²⁺ was needed for RVD. Gramicidin reversed the inhibitory effect of low extracellular Ca²⁺. Finally, and surprisingly, the Ca²⁺ ionophore A23187 increased osmotic fragility and inhibited volume recovery. Taken together, our results show that cell swelling activated a K⁺ permeable pathway via a Ca²⁺-dependent mechanism, and this process mediated K⁺ loss during RVD.     

Intracellular Mg<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript> Influences Both Open and Closed Times of a Native Ca<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript>-activated BK Channel in Cultured Human Renal Proximal Tubule Cells

Effects of intracellular Mg²⁺ on a native Ca²⁺-and voltage-sensitive large-conductance K⁺ channel in cultured human renal proximal tubule cells were examined with the patch-clamp technique in the inside-out mode. At an intracellular concentration of Ca²⁺ ([Ca²⁺]_i) of 10⁻⁵–10⁻⁴ M, addition of 1–10 mM Mg²⁺ increased the open probability (P_o) of the channel, which shifted the P_o –membrane potential (V_m) relationship to the negative voltage direction without causing an appreciable change in the gating charge (Boltzmann constant). However, the Mg²⁺-induced increase in P_o was suppressed at a relatively low [Ca²⁺]_i (10^−5.5–10⁻⁶ M). Dwell-time histograms have revealed that addition of Mg²⁺ mainly increased P_o by extending open times at 10⁻⁵ M Ca²⁺ and extending both open and closed times simultaneously at 10^−5.5 M Ca²⁺. Since our data showed that raising the [Ca²⁺]_i from 10⁻⁵ to 10⁻⁴ M increased P_o mainly by shortening the closed time, extension of the closed time at 10^−5.5 M Ca²⁺ would result from the Mg²⁺-inhibited Ca²⁺-dependent activation. At a constant V_m, adding Mg²⁺ enhanced the sigmoidicity of the P_o–[Ca²⁺]_i relationship with an increase in the Hill coefficient. These results suggest that the major action of Mg²⁺ on this channel is to elevate P_o by lengthening the open time, while extension of the closed time at a relatively low [Ca²⁺]_i results from a lowering of the sensitivity to Ca²⁺ of the channel by Mg²⁺, which causes the increase in the Hill coefficient. M. Kubokawa and Y. Sohma contributed equally to this work.     

Hypoxic Modulation of Ca<Superscript>2+</Superscript> Signaling in Human Venous and Arterial Endothelial Cells

Our understanding of vascular endothelial cell physiology is based on studies of endothelial cells cultured from various vascular beds of different species for varying periods of time. Systematic analysis of the properties of endothelial cells from different parts of the vasculature is lacking. Here, we compare Ca²⁺ homeostasis in primary cultures of endothelial cells from human internal mammary artery and saphenous vein and how this is modified by hypoxia, an inevitable consequence of bypass grafting (2.5% O₂, 24 h). Basal [Ca²⁺] _i and store depletion-mediated Ca²⁺ entry were significantly different between the two cell types, yet agonist (ATP)–mediated mobilization from endoplasmic reticulum stores was similar. Hypoxia potentiated agonist-evoked responses in arterial, but not venous, cells but augmented store depletion-mediated Ca²⁺ entry only in venous cells. Clearly, Ca²⁺ signaling and its remodeling by hypoxia are strikingly different in arterial vs. venous endothelial cells. Our data have important implications for the interpretation of data obtained from endothelial cells of varying sources.