Effects of caffeine on the influx of extracellular calcium in GH4C1 pituitary cells

Caffeine increases intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in a variety of cell types by triggering the mobilization of Ca²⁺ from intracellular Ca²⁺ stores. Caffeine also can change [Ca²⁺]_i by affecting Ca²⁺ influx through voltage-operated Ca²⁺ channels (VOCCs). In the present study, we investigated the effects of caffeine on Ca²⁺ entry in GH₄C₁ pituitary cells. Pretreatment of the cells with caffeine attenuated the high K⁺-evoked influx of ⁴⁵Ca²⁺ in a dose-dependent manner. This inhibition was not secondary to the caffeine-evoked elevation of [Ca²⁺]_i because caffeine was able to inhibit VOCCs also in the presence of the intracellular Ca²⁺ chelator BAPTA. However, the inhibitory effect of caffeine on ⁴⁵Ca²⁺ entry appeared to be dependent on the degree of depolarization of the plasma membrane. Only in cells depolarized with relatively high concentrations of K⁺ (20, 35, and 50 mM) was the caffeine-induced inhibition observed. A similar inhibitory effect of caffeine on the high K⁺-evoked calcium and barium entry was observed in experiments using Fura 2. Neither IBMX, forskolin nor dibutyryl cAMP reduced the enhanced [Ca²⁺]_i induced by 50 mM K⁺, suggesting that the effect of caffeine was not due to increased intracellular cAMP. Furthermore, high doses of caffeine inhibited the plateau level of the TRH-induced increase in [Ca²⁺]_i, which is caused partly by influx of Ca²⁺ through VOCCs. The inhibitory effect of caffeine was, in part, due to an hyperpolarization of the plasma membrane observed at high doses of caffeine. On the other hand, low doses of caffeine enhanced depolarization-evoked Ba²⁺ entry as well as the TRH-evoked plateau level of [Ca²⁺]_i. We conclude that caffeine has a dual effect on Ca²⁺ entry through activated VOCCs in GH₄C₁ cells: at low concentrations caffeine enhances Ca²⁺ entry, whereas high concentrations of caffeine block Ca²⁺ entry. J. Cell. Physiol. 171:52–60, 1997. © 1997 Wiley-Liss, Inc.     

Imaging of caffeine-inducible release of intracellular calcium in cultured embryonic mouse telencephalic neurons

To gain a better understanding of Ca²⁺-induced Ca²⁺ release in central neurons, we have studied the increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by application of caffeine to cells cultured from embryonic mouse telencephalon (hippocampus or cortex). The magnitudes and distributions of changes in [Ca²⁺]_i in neuron somata were measured by quantitative video microscopy. We observed that application of caffeine to pyramidally shaped neurons typically initiated an increase in [Ca²⁺]_i in the cytoplasmic region between the nucleus and the base of a major dendrite. [Ca²⁺] in this region increased over a period of 3 to 6 s and was followed by, with a slight delay, a surge of Ca²⁺ that moved across the soma and into or over the nucleus. Similar Ca²⁺ that moved across the soma and into or over the nucleus. Similar Ca²⁺ responses to caffeine were observed in Ca²⁺-containing and nominally Ca²⁺-free external solutions, suggesting that caffeine was inducing Ca²⁺ release from intracellular stores. Ca²⁺ responses to caffeine were potentiated by inducing a tonic Ca²⁺ influx through N-methyl-D-aspartate (NMDA)-type glutamate receptors activated by 0.3 μM glutamate and multiple responses to caffeine could be elicited by using this Ca²⁺ influx to refill the intracellular stores. Ryanodine inhibition of caffeine-induced Ca²⁺ release was use- and concentration-dependent; the median effective concentration EC₅₀ for ryanodine declined from 22 μM for the first application of caffeine to 20 nM for the fourth. We conclude, based on these responses to caffeine, that ryanodine-sensitive mechanisms of intracellular Ca²⁺ release are active in hippocampal and cortical neurons and may be involved in generation of directed Ca²⁺ waves that engulf the nucleus. © 1995 John Wiley & Sons, Inc.     

Participation of Intracellular Ca2+ Stores in Ca2+ Signalling in Neurons of the Thalamic Laterodorsal Nucleus of Rats

Experiments were carried out on isolated neurons of the thalamic nucleus lateralis dorsalis (LD) from 12-day-old rats. According to the morphological characteristics, LD neurons were classified as relay thalamo-cortical units and interneurons. The concentration of free Ca²⁺ ions in the cytoplasm ([Ca²⁺] _i ) was measured by a fluorescent calcium indicator, fura-2AM. Application of 30 mM caffeine caused a transient change in the [Ca²⁺] _i in 8 of 15 and in 6 of 11 of the thalamo-cortical units and interneurons under study, respectively. After stimulation of a cell with application of 50 mM KCl, a caffeine-induced increase in the [Ca²⁺] _i was observed in all tested neurons. To study the contribution of Ca²⁺-induced Ca²⁺ release (CICR) to the calcium transient evoked by depolarization of the neuronal membrane, caffeine in a subthreshold concentration was pre-applied. After 50 mM KCl had been added to the medium following pre-application of 0.5 mM caffeine, the calcium transient amplitude in thalamo-cortical neurons increased by 51 ± 7% (n = 16). In interneurons this effect was not observed (n = 11). The data obtained allow us to hypothesize that CICR contributes to the depolarization-evoked calcium transient only in the relay (thalamo-cortical) neurons. Differences in the pattern of calcium signalling, which were detected in two types of neurons of the thalamic LD, can be a factor determining distinctions in the physiological characteristics of these neurons.     

Caffeine-induced Ca2+ oscillations in type I horizontal cell of carp retina: A mathematical model

Oscillations in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) have been observed in a variety of cell types. In the present study, we constructed a mathematical model to simulate the caffeine-induced [Ca²⁺]_i oscillations based on experimental data obtained from isolated type I horizontal cell of carp retina. The results of model analysis confirm the notion that the caffeine-induced [Ca²⁺]_i oscillations involve a number of cytoplasmic and endoplasmic Ca²⁺ processes that interact with each other. Using this model, we evaluated the importance of store-operated channel (SOC) in caffeine-induced [Ca²⁺]_i oscillations. The model suggests that store-operated Ca²⁺ entry (SOCE) is elicited upon depletion of the endoplasmic reticulum (ER). When the SOC conductance is set to 0, caffeine-induced [Ca²⁺]_i oscillations are abolished, which agrees with the experimental observation that [Ca²⁺]_i oscillations were abolished when SOC was blocked pharmacologically, verifying that SOC is necessary for sustained [Ca²⁺]_i oscillations.     

Caffeine-induced Ca2+ oscillations in type I horizontal cell of carp retina: A mathematical model

Oscillations in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) have been observed in a variety of cell types. In the present study, we constructed a mathematical model to simulate the caffeine-induced [Ca²⁺]_i oscillations based on experimental data obtained from isolated type I horizontal cell of carp retina. The results of model analysis confirm the notion that the caffeine-induced [Ca²⁺]_i oscillations involve a number of cytoplasmic and endoplasmic Ca²⁺ processes that interact with each other. Using this model, we evaluated the importance of store-operated channel (SOC) in caffeine-induced [Ca²⁺]_i oscillations. The model suggests that store-operated Ca²⁺ entry (SOCE) is elicited upon depletion of the endoplasmic reticulum (ER). When the SOC conductance is set to 0, caffeine-induced [Ca²⁺]_i oscillations are abolished, which agrees with the experimental observation that [Ca²⁺]_i oscillations were abolished when SOC was blocked pharmacologically, verifying that SOC is necessary for sustained [Ca²⁺]_i oscillations.     

Effect of caffeine on mucus secretion and agonist-dependent Ca2+ mobilization in human gastric mucus secreting cells

Caffeine is known to stimulate gastric acid secretion, but, the effects of caffeine on gastric mucus secretion have not been clarified. To elucidate the action of caffeine on gastric mucin-producing cells and its underlying mechanism, the effects of caffeine on mucus glycoprotein secretion and agonist-induced [Ca²⁺]_i mobilization were examined in human gastric mucin secreting cells (JR-I cells). The measurement of [Ca²⁺]_i using Indo-1 and the whole cell voltage clamp technique were applied. Mucus glycoprotein secretion was assessed by release of [³H]glucosamine. Caffeine by itself failed to increase [Ca²⁺]_i and affect membrane currents, while it dose-dependently inhibited agonist (acetylcholine (ACh) or histamine)-induced [Ca²+]_i rise, resulting in inhibiting activation of Ca²⁺-dependent K⁺ current (I_K.Ca) evoked by agonists. The effect of caffeine was reversible, and the half maximal inhibitory concentration was about 0.5 mM. But, caffeine did not suppress [Ca²⁺]_i rise and activation of I_K.Ca induced by A23187 or inositol trisphosphate (IP₃). Theophylline or 3-isobutyl-1-methyl-xanthine (IBMX) did not mimic the effect of caffeine. Caffeine failed to stimulate mucus secretion, while it significantly decreased ACh-induced mucus secretion. These results indicate that caffeine selectively inhibits agonist-mediated [Ca²⁺]_i rise in human gastric epithelial cells, probably through the blockade of receptor-IP₃ signaling pathway, which may affect the mucin secretion. © 1997 Elsevier Science B.V. All rights reserved.     

Excitation-contraction coupling in heart muscle

We have investigated the links between electrical excitation and contraction in mammalian heart muscle. Using isolated single cells from adult rat ventricle, a whole-cell voltage-clamp technique and quantitative fluorescence microscopy, we have measured simultaneously calcium current (I_ca) and [Ca²⁺]_i (with fura-2). We find that the voltage-dependence of Ica and the [Ca ²⁺]_i-transient and the dependence of [Ca²⁺]_i-transient on depolarization-duration cannot both be readily explained by a simple calcium-induced Ca-release (CICR) mechanism. Additionally, we find that when [Ca²⁺]_i and [Na⁺]_i are at their diastolic levels, activation of the Na-Ca exchange mechanism by depolarization does not measurably trigger the release of Ca²⁺i. Finally, measuring I_ca in adult and neonatal rat heart cells and using the alkaloid ryanodine, we have carried out complementary experiments. These experiments show that there may be an action of ryanodine on I_ca that is independent of [Ca²⁺]_i and independent of a direct action of the alkaloid on the calcium channel itself. Along with experiments of others showing that ryanodine binds to the sarcoplasmic reticulum calcium-release channel/spanning protein complex, our data suggests a model to explain our findings. The model links the calcium channels responsible for Ica to the sarcoplasmic reticulum by means of one or more of the spanning protein(s). Information from the calcium channel can be communitated to the sarcoplasmic reticulum by this route and, presumably, information can move in the opposite direction from the sarcoplasmic reticulum to the calcium channel.     

Nitric oxide induces transient Ca2+ changes in endothelial cells independent of cGMP

Ca²⁺ changes induced by nitric oxide (NO^·) were investigated in cultured human endothelial cells. Sodium nitroprusside (SNP) (1–100 μmol/L) and S-Nitroso-N-acetylpenicillamine (SNAP) (100 μmol/L) were used as NO^· donors. The cytoplasmatic Ca²⁺ concentration was calculated using ratiometric FURA2 fluorescence measurements. Both NO^· donors caused transient oscillatory Ca²⁺ changes, which were not detectable in the presence of oxyhemoglobin (50 μmol/L). Digital ratio imaging revealed initiation sites within cells where Ca²⁺ increases started spreading, which indicates that nonuniformly distributed targets might be involved in these reactions. Calcium was released from intracellular stores as indicated by experiments performed in Ca²⁺-free buffer. L-type Ca²⁺-channel blocker diltiazem (100 μmol/L) was not able to block these responses. NO^·-induced Ca²⁺ release from intracellular stores caused capacitative Ca²⁺ entry. Both thapsigargin (1 μmol/L) and cyclopiazonic acid (10 μmol/L) inhibited the SNP response completely, whereas neither ryanodine (up to 100 μmol/L) nor dantrolene (100 μmol/L) was able to inhibit Ca²⁺ changes induced by SNP, indicating that primarily inositol 1,4,5-triphosphate (IP₃)-dependent stores are released upon stimulation with NO^·. A small inhibitory effect of ATP- and SNP-induced peak [Ca²⁺]_i increase was measured in the presence of both caffeine (20 mmol/L) and procaine (1 mmol/L). Evidence is presented that cGMP is not involved in NO^·-induced Ca²⁺ signals, as neither inhibitors of guanylate cyclase (methylene blue and LY (83583) nor cell permeant analogues of cGMP altered or simulated [Ca²⁺]_i changes. An inhibitor of cGMP-dependent protein kinase was also ineffective. We therefore propose that endothelial cells have specific targets proximal or at IP₃ receptors to induce Ca²⁺ changes in endothelial cells stimulated with NO^·. J. Cell. Physiol. 172:296–305, 1997. © 1997 Wiley-Liss, Inc.     

Agonist-Evoked Ca2+ Mobilization from Stores Expressing Inositol 1,4,5-Trisphosphate Receptors and Ryanodine Receptors in Cerebellar Granule Neurones

Abstract: The mechanisms involved in Ca²⁺ mobilization evoked by the muscarinic cholinoceptor (mAChR) agonist carbachol (CCh) and N-methyl-d -aspartate (NMDA) in cerebellar granule cells have been investigated. An initial challenge with caffeine greatly reduced the subsequent intracellular Ca²⁺ concentration ([Ca²⁺]_i) response to CCh (to 45 ± 19% of the control), and, similarly, a much reduced caffeine response was detectable after prior stimulation with CCh (to 27 ± 6% of the control). CCh-evoked [Ca²⁺]_i responses were inhibited by preincubation with thapsigargin (10 µM), 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ; 25 µM), ryanodine (10 µM), or dantrolene (25 µM). BHQ pretreatment was found to have no effect on the sustained phase of the NMDA-evoked [Ca²⁺]_i response. Both CCh (1 mM) and 1-aminocyclopentane-1S,3R-dicarboxylic acid (ACPD; 200 µM) evoked a much diminished increase in [Ca²⁺]_i in granule cells pretreated with CCh for 24 h compared with vehicle-treated control cells (CCh, 23 ± 14%; ACPD, 27 ± 1% of respective control values). In contrast, a 24-h CCh pretreatment decreased the subsequent inositol 1,4,5-trisphosphate (InsP₃) response to CCh to a much greater extent compared with responses evoked by metabotropic glutamate receptor (mGluR) agonists; this suggests that the former effect on Ca²⁺ mobilization represents a heterologous desensitization of the mGluR-mediated response distal to the pathway second messenger. Furthermore, [Ca²⁺]_i responses to caffeine and NMDA were unaffected by a 24-h pretreatment with CCh. This study indicates that ryanodine receptors, as well as InsP₃ receptors, appear to be crucial to the mAChR-mediated [Ca²⁺]_i response in granule cells. As BHQ apparently differentiates between the CCh- and NMDA-evoked responses, it is possible that the directly InsP₃-sensitive pool is physically different from the ryanodine receptor pool. Also, activation of InsP₃ receptors may not contribute significantly to NMDA-evoked elevation of [Ca²⁺]_i in cerebellar granule cells. A model for the topographic organization of cerebellar granule cell Ca²⁺ stores is proposed.     

A patch-clamp study of the Ca2+ mobilization from internal stores in bovine aortic endothelial cells. I. Effects of caffeine on intracellular Ca2+ stores

Summary The effects of agents known to interfere with Ca²⁺ release processes of endoplasmic reticulum were investigated in bradykinin (BK)-stimulated bovine aortic endothelial cells (BAE cells), via the activation of Ca²⁺-activated potassium channels [K(Ca²⁺) channels]. In cell-attached patch experiments, the external application of caffeine (1 mm) caused a brief activation of K(Ca²⁺) channels in Ca²⁺-free and Ca²⁺-containing external solutions. The application of BK (10 nm) during cell stimulation by caffeine (1–20 mm) invariably led to a drastic channel activation which was maintained during a recording period longer than that observed in caffeine-free conditions. In addition, the cell exposure to caffeine (20 mm) during the BK stimulation enhanced systematically the channel activation process. Since a rapid inhibition of BK-evoked channel activity was also produced by removing caffeine from the bath medium, it is proposed that the sustained single-channel response recorded in the concomittant presence of both agents was due to their synergic action on internal stores and/or the external Ca²⁺ entry pathway resulting in an increased [Ca²⁺]_i. In addition, the local anesthetic, procaine, depressed the initial BK-induced K(Ca²⁺) channel activity and completely blocked the secondary phase of the channel activation process related to the external Ca²⁺ influx into stimulated cells. In contrast, this blocking effect of procaine was not observed on the initial caffeine-elicited channel activity and could not suppress the external Ca²⁺-dependent phase of this channel activation process. Our results confirm the existence of at least two pharmacologically distinct types of Ca²⁺-release from internal stores in BAE cells: an inositol 1,4,5-triphosphate (InsP₃)-dependent and a caffeine-induced Ca²⁺-release process.The authors would like to thank Dr. A. Diarra for his contribution to the fluorescence measurements and Diane Vallerand for preparing cell cultures. These data were presented in part at the 14th Scientific Meeting of the International Society of Hypertension (Madrid, Spain, June 14–18, 1992), and have been published in abstract form in the Journal of Hypertension (1992). Dominique Thuringer is a fellow of the Heart and Stroke Foundation of Canada. Rémy Sauvé is a senior fellow from the Fonds de la Recherche en Santé du Québec. This work was supported by a grant from the Medical Research Council of Canada.     

Role of LTD4 in the Regulatory Volume Decrease Response in Ehrlich Ascites Tumor Cells

Stimulation with leukotriene D₄ (LTD₄) (3–100 nm) induces a transient increase in the free intracellular Ca²⁺ concentration ([Ca²⁺] _i ) in Ehrlich ascites tumor cells. The LTD₄-induced increase in [Ca²⁺] _i is, however, significantly reduced in Ca²⁺-free medium (2 mm EGTA), and under these conditions stimulation with a low LTD₄ concentration (3 nm) does not result in any detectable increase in [Ca²⁺] _i . Addition of LTD₄ (3–100 nm) moreover accelerates the KCl loss seen during Regulatory Volume Decrease (RVD) in cells suspended in a hypotonic medium. The LTD₄-induced (100 nm) acceleration of the RVD response is also seen in Ca²⁺-free medium and also at 3 nm LTD₄, indicating that LTD₄ can open K⁺- and Cl⁻-channels without any detectable increase in [Ca²⁺] _i . Buffering cellular Ca²⁺ with BAPTA almost completely blocks the LTD₄-induced (100 nm) acceleration of the RVD response. Thus, the reduced [Ca²⁺] _i level after BAPTA-loading or buffering of [Ca²⁺] _i seems to inhibit the LTD₄-induced stimulation of the RVD response even though the LTD₄-induced cell shrinkage is not necessarily preceded by any detectable increase in [Ca²⁺] _i . The LTD₄ receptor antagonist L649,923 (1 μm) completely blocks the LTD₄-induced increase in [Ca²⁺] _i and inhibits the RVD response as well as the LTD₄-induced acceleration of the RVD response. When the LTD₄ receptor is desensitized by preincubation with 100 nm LTD₄, a subsequent RVD response is strongly inhibited. In conclusion, the present study supports the notion that LTD₄ plays a role in the activation of the RVD response. LTD₄ seems to activate K⁺ and Cl⁻ channels via stimulation of a LTD₄ receptor with no need for a detectable increase in [Ca²⁺] _i . Received: 25 September 1995/Revised: 25 January 1996     

Effects of DK-002, a synthesized (6aS,cis)-9,10-Dimethoxy-7,11b-dihydro-indeno[2,1-c]chromene-3,6a-diol, on platelet activity

In the present study, the mechanism of antiplatelet activity of DK-002, a synthesized (6aS,cis)-9,10-Dimethoxy-7,11b-dihydro-indeno[2,1-c]chromene-3,6a-diol, was investigated. DK-002 inhibited the thrombin, collagen, and ADP-induced rat platelet aggregation in a concentration-dependent manner, with IC₅₀ values of 120, 27, and 47 μM, respectively. DK-002 also inhibited thrombin-induced dense granule secretion, thromboxane A₂ synthesis, and [Ca²⁺]_i elevation in platelets. DK-002 did not show any significant effect on ADP-induced inhibition of cyclic AMP elevation by prostaglandin E₁, but DK-002 was confirmed to inhibit ADP-induced [Ca²⁺]_i elevation and shape change. DK-002 inhibited 4-bromo-A23187-induced [Ca²⁺]_i elevation in the presence of creatine phosphate/creatine phosphokinase (CP/CPK, a ADP scavenging system) and indomethacin (a specific inhibitor of cyclooxygenase). DK-002 also inhibited Ca²⁺ mobilization in thrombin- or 4-bromo-A23187-stimulated platelets through its inhibitory effects on both Ca²⁺ release from intracellular stores and Ca²⁺ influx, in the presence of CP/CPK and indomethacin. Taken together, the present study shows that DK-002 has inhibitory effects on stimulation of platelets, and suggests that its antiplatelet activity might be related to the inhibitory mechanism on Ca²⁺ mobilization in stimulated platelets.     

Characterization of Ryanodine-sensitive Ca2+ Release from Microsomal Vesicles of Rat Parotid Acinar Cells: Regulation by Cyclic ADP-ribose

We have measured ryanodine (caffeine)-sensitive ⁴⁵Ca²⁺ release from isolated microsomal vesicles of endoplasmic reticulum prepared from rat parotid acinar cells. After a steady state of ATP-dependent ⁴⁵Ca²⁺ uptake, the addition of caffeine (40 mm), ryanodine (10∼500 μm) or an NAD⁺ metabolite, cyclic ADP-ribose (cADPR, 4 μm) released about 10% of the ⁴⁵Ca²⁺ that had been taken up. The ⁴⁵Ca²⁺ release was not inhibited by heparin, an antagonist of IP₃ receptor. The effects of caffeine, ryanodine and cADPR on ⁴⁵Ca²⁺ release were also tested in the presence of thapsigargin (TG), an inhibitor of microsomal Ca²⁺-ATPase. When caffeine (10∼40 mm), ryanodine (10 μm) or cADPR (1∼10 μm) was added in the medium with 100 nm TG, a significant ⁴⁵Ca²⁺ release was seen, while higher concentrations of ryanodine (>100 μm) did not cause any ⁴⁵Ca²⁺ release in the presence of TG. The initial rate of caffeine (40 mm)-induced ⁴⁵Ca²⁺ release was increased by a pretreatment with 10 μm ryanodine, whereas the caffeine-induced ⁴⁵Ca²⁺ release was strongly inhibited by the presence of a higher concentration (500 μm) of ryanodine. cADPR-induced ⁴⁵Ca²⁺ release was also inhibited by 500 μm ryanodine. Caffeine (40 mm)- or cADPR (4 μm)-induced ⁴⁵Ca²⁺ release was abolished by a presence of ruthenium red (50∼100 μm). The presence of a low concentration (0.5 μm) of cADPR shifted the dose-response curve of caffeine-induced ⁴⁵Ca²⁺ release to the left. These results indicate the presence of a ryanodine sensitive Ca²⁺ release mechanism in the endoplasmic reticulum of rat parotid acinar cells that is distinct from the IP₃-sensitive Ca²⁺ channel and is activated by caffeine, cADPR and a low concentration (10 μm) of ryanodine, but is inhibited by higher concentrations (>100 μm) of ryanodine and ruthenium red. The properties of the ryanodine-sensitive mechanism are similar to that of the ryanodine receptor as described in muscle cells. Received: 11 June 1996/Revised: 14 November 1996     

Effects of Small Molecule Modulators on ATP Binding to Skeletal Ryanodine Receptor

Calcium release for muscle contraction in skeletal muscle is mediated in part by the ryanodine receptor 1, RyR1, Ca²⁺-channel and is strongly affected by intrinsic modulators like Ca²⁺, Mg²⁺ and ATP. We showed differential effects on ATP binding in the presence of Ca²⁺ or Mg²⁺ ions using ESR spectroscopy and a spin-labeled ATP analog, SL-ATP (Dias et al. Biochemistry 45: 9408–9415, 2006). We here report the effects of RyR1 modulators like ryanodine, caffeine and dantrolene on the ATP binding of RyR1 using the same technique. We present evidence that the exogenous effectors induce changes within RyR1 that lead to different ATP binding characteristics: In the presence of the activating modulator, caffeine, or in the presence of ryanodine, which causes a half-open state of the channel, binding of eight ATP per RyR1 was observed, even in the presence of inhibitory Ca²⁺, suggestive of a stable “open” channel conformation. In the presence of the inhibitory modulator dantrolene, ATP binding affinity decreased in the presence of activating Ca²⁺, while in the presence of inhibitory Ca²⁺, ATP binding affinity increased, but at the same time the number of accessible sites decreased to four, suggestive of a closed conformation of the channel. The results imply that modulation of ATP binding to RyR1 as well as the overall number of accessible ATP binding sites on the channel are crucial for regulation and are in direct correlation with the modified activity of the channel induced by pharmacological agents.     

Platelet-Activating Factor Evokes Ca2+ Transients After the Blockade of Ryanodine Receptor by Dantrolene in RAW 264.7 Macrophages

In the present study we studied platelet-activating factor (PAF)-, and ATP-induced increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i) using RAW 264.7 macrophages filled with fura-2/AM and imaged with fluorescence video microscopy. We found that the prevalence of detectable [Ca²⁺]_i responses to PAF application was significantly higher in the presence of dantrolene. Dantrolene itself significantly decreased basal [Ca²⁺]_i of macrophages compared to control cases after a 20-min incubation period. In the dantrolene-treated cells even the peak [Ca²⁺]_i in response to PAF (as an average of all cells) was below the baseline of control suggesting that decreased [Ca²⁺]_i plays a permissive role in the Ca²⁺ rise induced by PAF in macrophages. In contrast to the effect of PAF, neither the amplitude of response to ATP nor the frequency of responding cells changed significantly during dantrolene treatment in our experiments. These cells were able to respond to a standard immune stimulus as well: lipopolysaccharide (LPS) was able to increase [Ca²⁺]_i. Our data indicate that the effectiveness of PAF to increase [Ca²⁺]_i in RAW 264.7 macrophages depends on the resting [Ca²⁺]_i. It has also been shown in this study that PAF and ATP differently regulate Ca²⁺ homeostasis in macrophages during inflammatory response and therefore they possibly differently modulate cytokine production by macrophages.     

Homeostatic and stimulus-induced coupling of the L-type Ca2+ channel to the ryanodine receptor in the hippocampal neuron in slices

Activity-dependent increase in cytosolic calcium ([Ca²⁺]_i) is a prerequisite for many neuronal functions. We previously reported a strong direct depolarization, independent of glutamate receptors, effectively caused a release of Ca²⁺ from ryanodine-sensitive stores and induced the synthesis of endogenous cannabinoids (eCBs) and eCB-mediated responses. However, the cellular mechanism that initiated the depolarization-induced Ca²⁺-release is not completely understood. In the present study, we optically recorded [Ca²⁺]_i from CA1 pyramidal neurons in the hippocampal slice and directly monitored miniature Ca²⁺ activities and depolarization-induced Ca²⁺ signals in order to determine the source(s) and properties of [Ca²⁺]_i-dynamics that could lead to a release of Ca²⁺ from the ryanodine receptor. In the absence of depolarizing stimuli, spontaneously occurring miniature Ca²⁺ events were detected from a group of hippocampal neurons. This miniature Ca²⁺ event persisted in the nominal Ca²⁺-containing artificial cerebrospinal fluid (ACSF), and increased in frequency in response to the bath-application of caffeine and KCl. In contrast, nimodipine, the antagonist of the L-type Ca²⁺ channel (LTCC), a high concentration of ryanodine, the antagonist of the ryanodine receptor (RyR), and thapsigargin (TG) reduced the occurrence of the miniature Ca²⁺ events. When a brief puff-application of KCl was given locally to the soma of individual neurons in the presence of glutamate receptor antagonists, these neurons generated a transient increase in the [Ca²⁺]_i in the dendrosomal region. This [Ca²⁺]_i-transient was sensitive to nimodipine, TG, and ryanodine suggesting that the [Ca²⁺]_i-transient was caused primarily by the LTCC-mediated Ca²⁺-influx and a release of Ca²⁺ from RyR. We observed little contribution from N- or P/Q-type Ca²⁺ channels. The coupling between LTCC and RyR was direct and independent of synaptic activities. Immunohistochemical study revealed a cellular localization of LTCC and RyR in a juxtaposed configuration in the proximal dendrites and soma. We conclude in the hippocampal CA1 neuron that: (1) homeostatic fluctuation of the resting membrane potential may be sufficient to initiate functional coupling between LTCC and RyR; (2) the juxtaposed localization of LTCC and RyR has anatomical advantage of synchronizing a Ca²⁺-release from RyR upon the opening of LTCC; and (3) the synchronized Ca²⁺-release from RyR occurs immediately after the activation of LTCC and determines the peak amplitude of depolarization-induced global increase in dendrosomal [Ca²⁺]_i.     

The role of cytoplasmic calcium in the regulation of the resting potential in the pulmonary veins myocardium in rats and mice

We have demonstrated the phenomenon of Са²⁺-induced hyperpolarization in the myocardium of pulmonary veins (PVs) in rats. An increase in cytoplasmic calcium [Са²⁺]_i was shown to shift the resting potential (RP) in the PVs towards more negative values. The compounds inducing an increase in [Са²⁺]_i, such as isoproterenol (10 μM), caffeine (5 mM), and ryanodine (0.01 μM), caused hyperpolarization of 10 ± 2, 9 ± 1.3, and 4.1 ± 2 mV, respectively. The inhibition of calcium-dependent potassium currents (IK_Ca) did not change RP of PVs under the control conditions and did not affect the Са²⁺-induced hyperpolarization.     

DHEA attenuates catecholamine secretion from bovine adrenal chromaffin cells

Dehydroepiandrosterone (DHEA) is a putative anti-stress agent and stress is associated with the secretion of catecholamine from the adrenal gland, but the effects of DHEA on catecholamine secretion are not fully understood. Using bovine chromaffin cells, we found that DHEA inhibited catecholamine secretion and cytosolic Ca²⁺ ([Ca²⁺]_i) rise coupled with nicotinic acetylcholine receptor (nAChR) without exerting an effect on³H-nicotine binding. In the case of high K⁺ stimulation, DHEA effectively suppressed secretion without affecting [Ca²⁺]₁ rise. Trifluoperazine (TFP), a calmodulin inhibitor, was capable of counteracting the inhibition of DHEA on high K⁺-induced secretions. In permeabilized cells, DHEA suppressed the Ca²⁺-induced secretion. These results suggest that DHEA (a) acts as a channel blocker that suppresses Ca²⁺ influx and subsequent secretions associated with nAChR, or (b) affects the intracellular secretion machinery to suppress high K⁺-induced secretions without affecting the high K⁺-induced [Ca²⁺]_i rise.