Nanosecond Electric Pulses: A Novel Stimulus for Triggering Ca<Superscript>2+</Superscript> Influx into Chromaffin Cells Via Voltage-Gated Ca<Superscript>2+</Superscript> Channels

Exposing bovine chromaffin cells to a single 5 ns, high-voltage (5 MV/m) electric pulse stimulates Ca²⁺ entry into the cells via L-type voltage-gated Ca²⁺ channels (VGCC), resulting in the release of catecholamine. In this study, fluorescence imaging was used to monitor nanosecond pulse-induced effects on intracellular Ca²⁺ level ([Ca²⁺]_i) to investigate the contribution of other types of VGCCs expressed in these cells in mediating Ca²⁺ entry. ω-Conotoxin GVIA and ω-agatoxin IVA, antagonists of N-type and P/Q-type VGCCs, respectively, reduced the magnitude of the rise in [Ca²⁺]_i elicited by a 5 ns pulse. ω-conotoxin MVIIC, which blocks N- and P/Q-type VGCCs, had a similar effect. Blocking L-, N-, and P\Q-type channels simultaneously with a cocktail of VGCC inhibitors abolished the pulse-induced [Ca²⁺]_i response of the cells, suggesting Ca²⁺ influx occurs only via VGCCs. Lowering extracellular K⁺ concentration from 5 to 2 mM or pulsing cells in Na⁺-free medium suppressed the pulse-induced rise in [Ca²⁺]_i in the majority of cells. Thus, both membrane potential and Na⁺ entry appear to play a role in the mechanism by which nanoelectropulses evoke Ca²⁺ influx. However, activation of voltage-gated Na⁺ channels (VGSC) is not involved since tetrodotoxin (TTX) failed to block the pulse-induced rise in [Ca²⁺]_i. These findings demonstrate that a single electric pulse of only 5 ns duration serves as a novel stimulus to open multiple types of VGCCs in chromaffin cells in a manner involving Na⁺ transport across the plasma membrane. Whether Na⁺ transport occurs via non-selective cation channels and/or through lipid nanopores remains to be determined.     

Neuronal Calcium Sensor-1 Regulation of Calcium Channels,Secretion, and Neuronal Outgrowth

Calcium (Ca²⁺) is an important intracellular messenger underlying cell physiology. Ca²⁺ channels are the main entry route for Ca²⁺ into excitable cells, and regulate processes such as neurotransmitter release and neuronal outgrowth. Neuronal Calcium Sensor-1 (NCS-1) is a member of the Calmodulin superfamily of EF-hand Ca²⁺ sensing proteins residing in the subfamily of NCS proteins. NCS-1 was originally discovered in Drosophila as an overexpression mutant (Frequenin), having an increased frequency of Ca²⁺-evoked neurotransmission. NCS-1 is N-terminally myristoylated, can bind intracellular membranes, and has a Ca²⁺ affinity of 0.3 μM. Over 10 years ago it was discovered that NCS-1 overexpression enhances Ca²⁺-evoked secretion in bovine adrenal chromaffin cells. The mechanism was unclear, but there was no apparent direct effect on the exocytotic machinery. It was revealed, again in chromaffin cells, that NCS-1 regulates voltage-gated Ca²⁺ channels (Cavs) in G-Protein Coupled Receptor (GPCR) signaling pathways. This work in chromaffin cells highlighted NCS-1 as an important modulator of neurotransmission. NCS-1 has since been shown to regulate and/or directly interact with many proteins including Cavs (P/Q, N, and L), TRPC1/5 channels, GPCRs, IP3R, and PI4 kinase type IIIβ. NCS-1 also affects neuronal outgrowth having roles in learning and memory affecting both short- and long-term synaptic plasticity. It is not known if NCS-1 affects neurotransmission and synaptic plasticity via its effect on PIP2 levels, and/or via a direct interaction with Ca²⁺ channels or their signaling complexes. This review gives a historical account of NCS-1 function, examining contributions from chromaffin cells, PC12 cells and other models, to describe how NCS-1’s regulation of Ca²⁺ channels allows it to exert its physiological effects.     

Comparison of Ca<Superscript>2+</Superscript> Currents of Chromaffin Cells from Normotensive Wistar Kyoto and Spontaneously Hypertensive Rats

Spontaneously hypertensive rats (SHR) are widely used as model to investigate the pathophysiological mechanisms of essential hypertension. Catecholamine plasma levels are elevated in SHR, suggesting alterations of the sympathoadrenal axis. The residual hypertension in sympathectomized SHR is reduced after demedullation, suggesting a dysfunction of the adrenal medulla. Intact adrenal glands exposed to acetylcholine or high K⁺ release more catecholamine in SHR than in normotensive Wistar Kyoto (WKY) rats, and adrenal chromaffin cells (CCs) from SHR secrete more catecholamines than CCs from WKY rats. Since Ca²⁺ entry through voltage-gated Ca²⁺ channels (VGCC) triggers exocytosis, alterations in the functional properties of these channels might underlie the enhanced catecholamine release in SHR. This study compares the electrophysiological properties of VGCC from CCs in acute adrenal slices from WKY rats and SHR at an early stage of hypertension. No significant differences were found in the macroscopic Ca²⁺ currents (current density, I–V curve, voltage dependence of activation and inactivation, kinetics) between CCs of SHR and WKY rats, suggesting that Ca²⁺ entry through VGCC is not significantly different between these strains, at least at early stages of hypertension. Ca²⁺ buffering, sequestration and extrusion mechanisms, as well as Ca²⁺ release from intracellular stores, must now be evaluated to determine if alterations in their function can explain the enhanced catecholamine secretion reported in CCs from SHR.     

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.     

Down-Modulation of Ca<Superscript>2+</Superscript> Channels by Endogenously Released ATP and Opioids: from the Isolated Chromaffin Cell to the Slice of Adrenal Medullae

Modifications in Ca²⁺ influx may lead to profound changes in the cell activity associated with Ca²⁺-dependent processes, from muscle contraction and neurotransmitter release to calcium-mediated cell death. Therefore, calcium entry into the cell requires fine regulation. In this context, understanding of the modulation of voltage-dependent Ca²⁺ channels seems to be critical. The modulatory process results in the enhancement or decrement of calcium influx that may regulate the local and global cytosolic Ca²⁺ concentrations. Here, we summarize the well-established data on this matter described in isolated chromaffin cells by our laboratory and others, and the new results we have obtained in a more physiological preparation: freshly isolated slices of mouse adrenal medullae.     

G protein inhibition of CaV2 calcium channels

Voltage-gated Ca²⁺ channels translate the electrical inputs of excitable cells into biochemical outputs by controlling influx of the ubiquitous second messenger Ca²⁺. As such the channels play pivotal roles in many cellular functions including the triggering of neurotransmitter and hormone release by Ca_V2.1 (P/Q-type) and Ca_V2.2 (N-type) channels. It is well established that G protein coupled receptors (GPCRs) orchestrate precise regulation neurotransmitter and hormone release through inhibition of Ca_V2 channels. Although the GPCRs recruit a number of different pathways, perhaps the most prominent, and certainly most studied among these is the so-called voltage-dependent inhibition mediated by direct binding of Gβγ to the α₁ subunit of Ca_V2 channels. This article will review the basics of Ca²⁺-channels and G protein signaling, and the functional impact of this now classical inhibitory mechanism on channel function. It will also provide an update on more recent developments in the field, both related to functional effects and crosstalk with other signaling pathways, and advances made toward understanding the molecular interactions that underlie binding of Gβγ to the channel and the voltage-dependence that is a signature characteristic of this mechanism.     

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.     

Synaptically Activated Ca<Superscript>2+</Superscript> Release From Internal Stores in CNS Neurons

Synaptically activated postsynaptic [Ca²⁺]_i increases occur through three main pathways: Ca²⁺ entry through voltage-gated Ca²⁺ channels, Ca²⁺ entry through ligand-gated channels, and Ca²⁺ release from internal stores. The first two pathways have been studied intensively; release from stores has been the subject of more recent investigations.Ca²⁺ release from stores in CNS neurons primarily occurs as a result of IP₃ mobilized by activation of metabotropic glutamatergic and/or cholingergic receptors coupled to PLC. Ca²⁺ release is localized near spines in Purkinje cells and occurs as a wave in the primary apical dendrites of pyramidal cells in the hippocampus and cortex. The amplitude of the [Ca²⁺]_i increase can reach several micromolar, significantly larger than the increase due to backpropagating spikes.The large amplitude, long duration, and unique location of the [Ca²⁺]_i increases due to Ca²⁺ release from stores suggests that these increases can affect specific downstream signaling mechanisms in neurons.     

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.     

Ca<Superscript>2+</Superscript> Dynamics in the Secretory Vesicles of Neurosecretory PC12 and INS1 Cells

We have investigated the dynamics of the free [Ca²⁺] inside the secretory granules of neurosecretory PC12 and INS1 cells using a low-Ca²⁺-affinity aequorin chimera fused to synaptobrevin-2. The steady-state secretory granule [Ca²⁺] ([Ca²⁺]_SG] was around 20–40 μM in both cell types, about half the values previously found in chromaffin cells. Inhibition of SERCA-type Ca²⁺ pumps with thapsigargin largely blocked Ca²⁺ uptake by the granules in Ca²⁺-depleted permeabilized cells, and the same effect was obtained when the perfusion medium lacked ATP. Consistently, the SERCA-type Ca²⁺ pump inhibitor benzohydroquinone induced a rapid release of Ca²⁺ from the granules both in intact and permeabilized cells, suggesting that the continuous activity of SERCA-type Ca²⁺ pumps is essential to maintain the steady-state [Ca²⁺]_SG. Both inositol 1,4,5-trisphosphate (InsP₃) and caffeine produced a rapid Ca²⁺ release from the granules, suggesting the presence of InsP₃ and ryanodine receptors in the granules. The response to high-K⁺ depolarization was different in both cell types, a decrease in [Ca²⁺]_SG in PC12 cells and an increase in [Ca²⁺]_SG in INS1 cells. The difference may rely on the heterogeneous response of different vesicle populations in each cell type. Finally, increasing the glucose concentration triggered a decrease in [Ca²⁺]_SG in INS1 cells. In conclusion, our data show that the secretory granules of PC12 and INS1 cells take up Ca²⁺ through SERCA-type Ca²⁺ pumps and can release it through InsP₃ and ryanodine receptors, supporting the hypothesis that secretory granule Ca²⁺ may be released during cell stimulation and contribute to secretion.     

γ<Subscript>1</Subscript>-Dependent Down-regulation of Recombinant Voltage-gated Ca<Superscript>2+</Superscript> Channels

(1) Voltage-gated Ca²⁺ (Ca_V) channels are multi-subunit membrane complexes that allow depolarization-induced Ca²⁺ influx into cells. The skeletal muscle L-type Ca_V channels consist of an ion-conducting Ca_V1.1 subunit and auxiliary α₂δ−1, β₁ and γ₁ subunits. This complex serves both as a Ca_V channel and as a voltage sensor for excitation–contraction coupling. (2) Though much is known about the mechanisms by which the α₂δ−1 and β₁ subunits regulate Ca_V channel function, there is far less information on the γ₁ subunit. Previously, we characterized the interaction of γ₁ with the other components of the skeletal Ca_V channel complex, and showed that heterologous expression of this auxiliary subunit decreases Ca²⁺ current density in myotubes from γ₁ null mice. (3) In the current report, using Western blotting we show that the expression of the Ca_V1.1 protein is significantly lower when it is heterologously co-expressed with γ₁. Consistent with this, patch-clamp recordings showed that transient transfection of γ₁ drastically inhibited macroscopic currents through recombinant N-type (Ca_V2.2/α₂δ−1/β₃) channels expressed in HEK-293 cells. (4) These findings provide evidence that co-expression of the auxiliary γ₁ subunit results in a decreased expression of the ion-conducting subunit, which may help to explain the reduction in Ca²⁺ current density following γ₁ transfection.     

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.     

A two-step model for acetylcholine control of exocytosis via nicotinic receptors

The view that Ca²⁺ entry through voltage-dependent Ca²⁺ channels (VDCC) and through nicotinic receptors for acetylcholine (nAChRs) causes equal catecholamine release responses in chromaffin cells, was reinvestigated here using new protocols. We have made two-step experiments consisting in an ACh prepulse followed by a depolarizing pulse (DP). In voltage-clamped bovine chromaffin cells an ACh prepulse caused a slow-rate release but augmented 4.5-fold the much faster exocytotic response triggered by a subsequent depolarizing pulse (measured with capacitance and amperometry). If the ACh prepulse was given with mecamylamine or in low external Ca²⁺, the secretion increase disappeared. This suggests a two-step model for the effects of ACh: (1) meager Ca²⁺ entry through nAChRs mostly serves to keep loaded with vesicles the secretory machine; and (2) in this manner, the cell is prepared to respond with an explosive secretion of catecholamine upon depolarization and fast high Ca²⁺ entry through VDCC.     

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.     

Ca2+ influx and clearance at hyperpolarized membrane potentials modulate spontaneous and stimulated exocytosis in neuroendocrine cells

Neuroendocrine adrenal chromaffin cells release neurohormones catecholamines in response to Ca²⁺ entry via voltage-gated Ca²⁺ channels (VGCCs). Adrenal chromaffin cells also express non-voltage-gated channels, which may conduct Ca²⁺ at negative membrane potentials, whose role in regulation of exocytosis is poorly understood. We explored how modulation of Ca²⁺ influx at negative membrane potentials affects basal cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and exocytosis in metabolically intact voltage-clamped bovine adrenal chromaffin cells. We found that in these cells, Ca²⁺ entry at negative membrane potentials is balanced by Ca²⁺ extrusion by the Na⁺/Ca²⁺ exchanger and that this balance can be altered by membrane hyperpolarization or stimulation with an inflammatory hormone bradykinin. Membrane hyperpolarization or application of bradykinin augmented Ca²⁺-carrying current at negative membrane potentials, elevated basal [Ca²⁺]_i, and facilitated synchronous exocytosis evoked by the small amounts of Ca²⁺ injected into the cell via VGCCs (up to 20 pC). Exocytotic responses evoked by the injections of the larger amounts of Ca²⁺ via VGCCs (> 20 pC) were suppressed by preceding hyperpolarization. In the absence of Ca²⁺ entry via VGCCs and Ca²⁺ extrusion via the Na⁺/Ca²⁺ exchanger, membrane hyperpolarization induced a significant elevation in [Ca²⁺]_i and asynchronous exocytosis. Our results indicate that physiological interferences, such as membrane hyperpolarization and/or activation of non-voltage-gated Ca²⁺ channels, modulate basal [Ca²⁺]_i and, consequently, segregation of exocytotic vesicles and their readiness to be released spontaneously and in response to Ca²⁺ entry via VGCCs. These mechanisms may play role in homeostatic plasticity of neuronal and endocrine cells.     

Mechanisms of Extracellular NO and Ca<Superscript>2+</Superscript> Regulating the Growth of Wheat Seedling Roots

Our previous studies suggested the cross talk of nitric oxide (NO) with Ca²⁺ in regulating stomatal movement. However, its mechanism of action is not well defined in plant roots. In this study, sodium nitroprusside (SNP, a NO donor) showed an inhibitory effect on the growth of wheat seedling roots in a dose-dependent manner, which was alleviated through reducing extracellular Ca²⁺ concentration. Analyzing the content of Ca²⁺ and K⁺ in wheat seedling roots showed that SNP significantly promoted Ca²⁺ accumulation and inhibited K⁺ accumulation at a higher concentration of extracellular Ca²⁺, but SNP promoted K⁺ accumulation in the absence of extracellular Ca²⁺. To gain further insights into Ca²⁺ function in the NO-regulated growth of wheat seedling roots, we conducted the patch-clamped protoplasts of wheat seedling roots in a whole cell configuration. In the absence of extracellular Ca²⁺, NO activated inward-rectifying K⁺ channels, but had little effects on outward-rectifying K⁺ channels. In the presence of 2 mmol L⁻¹ CaCl₂ in the bath solution, NO significantly activated outward-rectifying K⁺ channels, which was partially alleviated by LaCl₃ (a Ca²⁺ channel inhibitor). In contrast, 2 mmol L⁻¹ CaCl₂ alone had little effect on inward or outward-rectifying K⁺ channels. Thus, NO inhibits the growth of wheat seedling roots likely by promoting extracellular Ca²⁺ influx excessively. The increase in cytosolic Ca²⁺ appears to inhibit K⁺ influx, promotes K⁺ outflux across the plasma membrane, and finally reduces the content of K⁺ in root cells.     

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.     

Ca<Superscript>2+</Superscript>-mediated Potentiation of the Swelling-induced Taurine Efflux from HeLa Cells: On the Role of Calmodulin and Novel Protein Kinase C Isoforms

The present work sets out to investigate how Ca²⁺ regulates the volume-sensitive taurine-release pathway in HeLa cells. Addition of Ca²⁺-mobilizing agonists at the time of exposure to hypotonic NaCl medium augments the swelling-induced taurine release and subsequently accelerates the inactivation of the release pathway. The accelerated inactivation is not observed in hypotonic Ca²⁺-free or high-K⁺ media. Addition of Ca²⁺-mobilizing agonists also accelerates the regulatory volume decrease, which probably reflects activation of Ca²⁺-activated K⁺ channels. The taurine release from control cells and cells exposed to Ca²⁺ agonists is equally affected by changes in cell volume, application of DIDS and arachidonic acid, indicating that the volume-sensitive taurine leak pathway mediates the Ca²⁺-augmented taurine release. Exposure to Ca²⁺-mobilizing agonists prior to a hypotonic challenge also augments a subsequent swelling-induced taurine release even though the intracellular Ca²⁺-concentration has returned to the unstimulated level. The Ca²⁺-induced augmentation of the swelling-induced taurine release is abolished by inhibition of calmodulin, but unaffected by inhibition of calmodulin-dependent kinase II, myosin light chain kinase and calcineurin. The effect of Ca²⁺-mobilizing agonists is mimicked by protein kinase C (PKC) activation and abolished in the presence of the PKC inhibitor Gö6850 and following downregulation of phorbol ester-sensitive PKC isoforms. It is suggested that Ca²⁺ regulates the volume-sensitive taurine-release pathway through activation of calmodulin and PKC isoforms belonging to the novel subclass (nPKC).This revised version was published online in June 2005 with a corrected cover date.     

Block of a Ca<Superscript>2+</Superscript>-activated Potassium Channel by Cocaine

The primary target for cocaine is believed to be monoamine transporters because of cocaine’s high-affinity binding that prevents re-uptake of released neurotransmitter. However, direct interaction with ion channels has been shown to be important for certain pharmacological/toxicological effects of cocaine. Here I show that cocaine selectively blocks a calcium-dependent K⁺ channel in hippocampal neurons grown in culture (IC₅₀ = ∼30 μM). Single-channel recordings show that in the presence of cocaine, the channel openings are interrupted with brief closures (flicker block). As the concentration of cocaine is increased the open-time is reduced, whereas the duration of brief closures is independent of concentration. The association and dissociation rate constants of cocaine for the neuronal Ca²⁺-activated K⁺ channels are 261 ± 37 μM⁻¹s⁻¹ and 11451 ± 1467 s⁻¹. The equilibrium dissociation constant (K_B) for cocaine, determined from single-channel parameters, is 43 μM. The lack of voltage dependence of block suggests that cocaine probably binds to a site at the mouth of the pore. Block of Ca²⁺-dependent K⁺ channels by cocaine may be involved in functions that include broadening of the action potential, which would facilitate transmitter release, enhancement of smooth muscle contraction particularly in blood vessels, and modulation of repetitive neuronal firing by altering the repolarization and afterhyperpolarization phases of the action potential.