Increase in gap junction resistance with acidification in crayfish septate axons is closely related to changes in intracellular calcium but not hydrogen ion concentration

Summary Neutral-carrier pH-and Ca-sensitive microelectrodes were used to investigate the relationship between junctional electrical resistance and either pH_i or [Ca²⁺]_i in crayfish septate axons uncoupled by acidification. For measuring [Ca²⁺]_i a new neutral carrier sensor sensitive to picomolar [Ca²⁺] and virtually insensitive to other ions was used. Uncoupling was induced by superfusing the axons with Na-acetate solutions (pH 6.3). With acetate, the time course of changes in junctional resistance differed markedly from that of pH_i or [H⁺]_i peaked 40–90 sec before junctional resistance. The difference in shape and peak time between pH_i and junctional resistance curves caused significant hysteresis in the pH_i versus junctional resistance relationship. In addition, junctional resistance maxima reached with slow acidification rates were 3–4 times greater than those with fast acidifications of similar magnitude. With acetate, [Ca²⁺]_i, increased by approximately one order of magnitude from basal values of 0.1–0.3 m. The curves describing the time course of changes in [Ca²⁺]_i and junctional resistance matched well with each other in shape, peak time and magnitude. Both junctional resistance and [Ca²⁺]_i recovered following a single exponential decay with a time constant of 2 min. Different rates of acidification caused increases in [Ca²⁺]_i and junctional resistance comparable in magnitude. The data indicate that the increase in junctional resistance induced by acidification is more closely related to [Ca²⁺]_i than to [H⁺]_i.     

Effects of the anesthetics heptanol,halothane and isoflurane on gap junction conductance in crayfish septate axons: A calcium- and hydrogen-independent phenomenon potentiated by caffeine and theophylline,and inhibited by 4-aminopyridine

Summary This study has monitored junctional and nonjunctional resistance. [Ca²⁺] _i and [H^–] _i , and the effects of various drugs in crayfish septate axons exposed to neutral anesthetics. The uncoupling efficiency of heptanol and halothane is significantly potentiated by caffeine and theophylline. The modest uncoupling effects of isoflurane, described here for the first time, are also enhanced by caffeine. Heptanol causes a decrease in [Ca²⁺] _i and [H⁺] _i both in the presence and absence of either caffeine or theophylline. A similar but transient effect on [Ca²⁺] _i is observed with halothane. 4-Aminopyridine strongly inhibits the uncoupling effects of heptanol. The observed decrease in [Ca^2–] _i with heptanol and halothane and negative results obtained with different [Ca²⁺] _o , Ca²⁺-channel blockers (nisoldipine and Cd²⁺) and ryanodine speak against a Ca²⁺ participation. Negative results obtained with 3-isobutyl-l-methylxanthine, forskolin, CPT-cAMP, 8Br-cGMP, adenosine, phorbol ester and H7, superfused in the presence and absence of caffeine and/or heptanol. indicate that neither the heptanol effects nor their potentiation by caffeine are mediated by cyclic nucleotides, adenosine receptors and kinase C. The data suggest a direct effect of anesthetics. possibly involving both polar and hydrophobic interactions with channel proteins. Xanthines and 4-aminopyridine may participate by influencing polar interactions. The potentiating effect of xanthines on cell-to-cell uncoupling by anesthetics may provide some clues on the nature of cardiac arrhythmias in patients treated with theophylline during halothane anesthesia.     

Spatial and temporal patterns of intracellular calcium in colonic smooth muscle

Summary Intracellular calcium [Ca²⁺] _i measurements in cell suspension of gastrointestinal myocytes have suggested a single [Ca²⁺] _i transient followed by a steady-state increase as the characteristic [Ca²⁺] _i response of these cells. In the present study, we used digital video imaging techniques in freshly dispersed myocytes from the rabbit colon, to characterize the spatiotemporal pattern of the [Ca²⁺] _i signal in single cells. The distribution of [Ca²⁺] _i in resting and stimulated cells was nonhomogeneous, with gradients of high [Ca²⁺] _i present in the subplasmalemmal space and in one cell pole. [Ca²⁺] _i gradients within these regions were not constant but showed temporal changes in the form of [Ca²⁺] _i oscillations and spatial changes in the form of [Ca²⁺] _i waves. [Ca²⁺] _i oscillations in unstimulated cells (n = 60) were independent of extracellular [Ca²⁺] and had a mean frequency of 12.6 +1.1 oscillations per min. The baseline [Ca²⁺], was 171 ± 13 nm and the mean oscillation amplitude was 194 ± 12 nm. Generation of [Ca²⁺] _i waves was also independent of influx of extracellular Ca²⁺. [Ca²⁺] _i waves originated in one cell pole and were visualized as propagation mostly along the subplasmalemmal space or occasionally throughout the cytoplasm. The mean velocity was 23 +3 m per sec (n = 6). Increases of [Ca²⁺] _i induced by different agonists were encoded into changes of baseline [Ca²⁺] _i and the amplitude of oscillations, but not into their frequency. The observed spatiotemporal pattern of [Ca²⁺] _i regulation may be the underlying mechanism for slow wave generation and propagation in this tissue. These findings are consistent with a [Ca²⁺] _i regulation whereby cell regulators modulate the spatiotemporal pattern of intracellularly generated [Ca²⁺] _i oscillations.The authors thank Debbie Anderson for excellent technical assistance with the electron microscopy and Dr. M. Regoli for providing the NK-1 agonist [Sar⁹,Met(O₂)¹¹]-SP. This work was supported by National Institutes of Health Grants DK 40919 and DK 40675 and Veterans Administration Grant SMI.     

Regulatory Volume Decrease by SV40-transformed Rabbit Corneal Epithelial Cells Requires Ryanodine-sensitive Ca2+-induced Ca2+ Release

The relationship between relative cell volume and time-dependent changes in intracellular Ca²⁺ concentration ([Ca²⁺] _i ) during exposure to hypotonicity was characterized in SV-40 transformed rabbit corneal epithelial cells (tRCE) (i). Light scattering measurements revealed rapid initial swelling with subsequent 97% recovery of relative cell volume (characteristic time (τ _vr ) was 5.9 min); (ii). Fura2-fluorescence single-cell imaging showed that [Ca²⁺] _i initially rose by 216% in 30 sec with subsequent return to near baseline level after another 100 sec. Both relative cell volume recovery and [Ca²⁺] _i transients were inhibited by either: (a) Ca²⁺-free medium; (b) 5 mm Ni²⁺ (inhibitor of plasmalemma Ca²⁺ influx); (c) 10 μm cyclopiazonic acid, CPA (which causes depletion of intracellular Ca²⁺ content); or (d) 100 μm ryanodine (inhibitor of Ca²⁺ release from intracellular stores). To determine the temporal relationship between an increased plasmalemma Ca²⁺ influx and the emptying of intracellular Ca²⁺ stores during the [Ca²⁺] _i transients, Mn²⁺ quenching of fura2-fluorescence was quantified. In the presence of CPA, hypotonic challenge increased plasmalemma Mn²⁺ permeability 6-fold. However, Mn²⁺ permeability remained unchanged during exposure to either: 1.100 μm ryanodine; 2.10 μm CPA and 100 μm ryanodine. This report for the first time documents the time dependence of the components of the [Ca²⁺] _i transient required for a regulatory volume decrease (RVD). The results show that ryanodine sensitive Ca²⁺ release from an intracellular store leads to a subsequent increase in plasmalemma Ca²⁺ influx, and that both are required for cells to undergo RVD. Received: 7 November 1996/Revised: 6 January 1997     

Oscillations of cytosolic free calcium in bombesin-stimulated HIT-T15 cells

The mechanism underlying the generation of cytosolic free Ca²⁺ ([Ca²⁺_i) oscillations by bombesin, a receptor agonist activating phospholipase C, in insulin secreting HIT-T15 cells was investigated. At 25 μM, 61% of cells displayed [Ca²⁺]_i oscillations with variable patterns. The bombesin-induced [Ca²⁺]_i oscillations could last more than 1 h and glucose was required for maintaining these [Ca²⁺ fluctuations. Bombesin-evoked [Ca²⁺]_i oscillations were dependent on extracellular Ca²⁺ entry and were attenuated by membrane hype rpolarization or by L-type Ca²⁺ channel blockers. These [Ca²⁺]_i oscillations were apparently not associated with fluctuations in plasma membrane Ca²⁺ permeability as monitored by the Mn²⁺ quenching technique. 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) and 4-chloro-m-cresol, which interfere with intracellular Ca²⁺ stores, respectively, by inhibiting Ca²⁺-ATPase of endoplasmic reticulum and by affecting Ca²⁺-induced Ca²⁺ release, disrupted bombesin-induced [Ca²⁺]_i oscillations. 4-chloro-m-resol raised [Ca²⁺]_i by mobilizing an intracellular Ca²⁺ pool, an effect not altered by ryanodine. Caffeine exerted complex actions on [Ca²⁺]_i It raised [Ca²⁺]_i by promoting Ca²⁺ entry while inhibiting bombesin-elicited [Ca²⁺]_i oscillations. Our results suggest that in bombesin-elicited [Ca²⁺]_i oscillations in HIT-T15 cells: (i) the oscillations originate primarily from intracellular Ca²⁺ stores; and (ii) the Ca²⁺ influx required for maintaining the oscillations is in part membrane potential-sensitive and not coordinated with [Ca²⁺]_i oscillations. The interplay between intracellular Ca²⁺ stores and voltage-sensitive and voltage-insensitive extracellular Ca²⁺ entry determines the [Ca²⁺]_i oscillations evoked by bombesin.     

Stimulant-evoked depolarization and increase in [Ca2+] i in insulin-secreting cells is dependent on external Na+

Summary The patch-clamp technique and measurements of single cell [Ca²⁺] _i have been used to investigate the importance of extracellular Na⁺ for carbohydrate-induced stimulation of RINm5F insulin-secreting cells. Using patch-clamp whole-cell (current-clamp) recordings the average cellular transmembrane potential was estimated to be –60±1 mV (n=83) and the average basal [Ca²⁺] _i 102±6nm (n=37). When challenged with either glucose (2.5–10mm) ord-glyceraldehyde (10mm) the cells depolarized, which led to the initiation of Ca²⁺ spike potentials and a sharp rise in [Ca²⁺] _i . Similar effects were also observed with the sulphonylurea compound tolbutamide (0.01–0.1mm). Both the generation of the spike potentials and the increase in [Ca²⁺] _i were abolished when Ca²⁺ was removed from the bathing media. When all external Na⁺ was replaced with N-methyl-d-glucamine, in the continued presence of either glucose,d-glyceraldehyde or tolbutamide, a membrane repolarization resulted, which terminated Ca²⁺ spike potentials and attenuated the rise in [Ca²⁺] _i . Tetrodotoxin (TTX) (1–2 m) was also found to both repolarize the membrane and abolish secretagogue-induced rises in [Ca²⁺] _i .     

Ca as second messenger in PTTH-stimulated prothoracic glands of the silkworm, Bombyx mori

Measurements of Ca²⁺ influx and [Ca²⁺]_i changes in Fura-2/AM-loaded prothoracic glands (PGs) of the silkworm, Bombyx mori, were used to identify Ca²⁺ as the actual second messenger of the prothoracicotropic hormone (PTTH) of this insect. Dose-dependent increases of [Ca²⁺]_i in PG cells were recorded in the presence of recombinant PTTH (rPTTH) within 5 minutes. The rPTTH-mediated increases of [Ca²⁺]_i levels were dependent on extracellular Ca²⁺. They were not blocked by the dihydropyridine derivative, nitrendipine, an antagonist of high-voltage-activated (HVA) Ca²⁺ channels, and by bepridil, an antagonist of low-voltage-activated (LVA) Ca²⁺ channels. The trivalent cation La³⁺, a non-specific blocker of plasma membrane Ca²⁺ channels, eliminated the rPTTH-stimulated increase of [Ca²⁺]_i levels in PG cells and so did amiloride, an inhibitor of T-type Ca²⁺ channels. Incubation of PG cells with thapsigargin resulted in an increase of [Ca²⁺]_i levels, which was also dependent on extracellular Ca²⁺ and was quenched by amiloride, suggesting the existence of store-operated plasma membrane Ca²⁺ channels, which can also be inhibited by amiloride. Thapsigargin and rPTTH did not operate independently in stimulating increases of [Ca²⁺]_i levels and one agent’s mediated increase of [Ca²⁺]_i was eliminated in the presence of the other. TMB-8, an inhibitor of intracellular Ca²⁺ release from inositol 1,4,5 trisphosphate (IP₃)-sensitive Ca²⁺ stores, blocked the rPTTH-stimulated increases of [Ca²⁺]_i levels, suggesting an involvement of IP₃ in the initiation of the rPTTH signaling cascade, whereas ryanodine did not influence the rPTTH-stimulated increases of [Ca²⁺]_i levels. The combined results indicate the presence of a cross-talk mechanism between the [Ca²⁺]_i levels, filling state of IP₃-sensitive intracellular Ca²⁺ stores and the PTTH-receptor’s-mediated Ca²⁺ influx.     

Characteristics of cytosolic Ca2+ elevation induced by muscarinic receptor activation in single adrenal chromaffin cells of the guinea pig

In Fura-2 loaded-single guinea pig adrenal chromaffin cells, muscarine, nicotine and KCl all caused an early peak rise in intracellular Ca concentration ([Ca²⁺]_i) followed by a sustained rise. In Ca²⁺-free solution, muscarine, but neither nicotine nor KCl, caused a transient increase in [Ca²⁺]_i, which was partially reduced by preceding application of caffeine or by treatment with ryanodine plus caffeine. In voltage-clamped cells at a holding potential of −60 mV, the muscarine-induced [Ca²⁺]_i, rise, especially its sustained phase, decreased in magnitude. intracellular application of inositol 1,4,5-trisphosphate caused a transient increase in [Ca²⁺]_i and inhibited the following [Ca²⁺]_i response to muscarine without affecting responses to nicotine and a depolarizing pulse. Muscarine evoked membrane depolarization following brief hyperpolarization in most cells tested. There was a significant positive correlation between the amplitude of the depolarization and the magnitude of the sustained rise in [Ca²⁺]_i. Muscarine-induced sustained [Ca²⁺]_i rise was much greater in the current-clamp mode than that in the voltage-clamp mode. The sustained phase of [Ca²⁺]_i rise and Mn²⁺ influx in response to muscarine were suppressed by a voltage-dependent Ca²⁺ channel blocker, methoxyverapamil. These results suggest that stimulation of muscarinic receptors causes not only extracellular Ca²⁺ entry, but also Ca²⁺ mobilization from inositol 1,4,5-trisphosphate-sensitive intracellular stores. Voltage-dependent Ca²⁺ channels may function as one of the Ca²⁺ entry pathways activated by muscarinic receptor in guinea pig adrenal chromaffin cells.     

Caffeine-Induced Ca2+ Transients and Exocytosis in Paramecium Cells. A Correlated Ca2+ Imaging and Quenched-Flow/Freeze-Fracture Analysis

Caffeine causes a [Ca²⁺] _i increase in the cortex of Paramecium cells, followed by spillover with considerable attenuation, into central cell regions. From [Ca²⁺]^rest _i ∼50 to 80 nm, [Ca²⁺]^act _i rises within ≤3 sec to 500 (trichocyst-free strain tl) or 220 nm (nondischarge strain nd9–28°C) in the cortex. Rapid confocal analysis of wildtype cells (7S) showed only a 2-fold cortical increase within 2 sec, accompanied by trichocyst exocytosis and a central Ca²⁺ spread during the subsequent ≥2 sec. Chelation of Ca²⁺ _o considerably attenuated [Ca²⁺] _i increase. Therefore, caffeine may primarily mobilize cortical Ca²⁺ pools, superimposed by Ca²⁺ influx and spillover (particularly in tl cells with empty trichocyst docking sites). In nd cells, caffeine caused trichocyst contents to decondense internally (Ca²⁺-dependent stretching, normally occurring only after membrane fusion). With 7S cells this usually occurred only to a small extent, but with increasing frequency as [Ca²⁺] _i signals were reduced by [Ca²⁺] _o chelation. In this case, quenched-flow and ultrathin section or freeze-fracture analysis revealed dispersal of membrane components (without fusion) subsequent to internal contents decondensation, opposite to normal membrane fusion when a full [Ca²⁺] _i signal was generated by caffeine stimulation (with Ca²⁺ _i and Ca²⁺ _o available). We conclude the following. (i) Caffeine can mobilize Ca²⁺ from cortical stores independent of the presence of Ca²⁺ _o . (ii) To yield adequate signals for normal exocytosis, Ca²⁺ release and Ca²⁺ influx both have to occur during caffeine stimulation. (iii) Insufficient [Ca²⁺] _i increase entails caffeine-mediated access of Ca²⁺ to the secretory contents, thus causing their decondensation before membrane fusion can occur. (iv) Trichocyst decondensation in turn gives a signal for an unusual dissociation of docking/fusion components at the cell membrane. These observations imply different threshold [Ca²⁺] _i -values for membrane fusion and contents discharge. Received: 23 May 1997/Revised: 18 August 1997     

Calcium mobilization and muscle contraction induced by acetylcholine in swine trachealis

The roles of Ca²⁺ mobilization in development of tension induced by acetylcholine (ACh, 0.1–100 µM) in swine tracheal smooth muscle strips were studied. Under control conditions, ACh induced a transient increase in free cytosolic calcium concentration ([Ca²⁺]_i) that declined to a steady-state level. The peak increase in [Ca²⁺]_i correlated with the magnitude of tension at each [ACh] after a single exposure to ACh, while the steady-state [Ca²⁺]_i did not. Removal of extracellular Ca²⁺ had little effect on peak [Ca²⁺]_i but greatly reduced steady-state increases in [Ca²⁺]_i and tension. Verapamil inhibited steady-state [Ca²⁺]_i only at [ACh]<1 µM. After depletion of internal Ca²⁺ stores by 10 min exposure to ACh in Ca²⁺-free solution and then washout of ACh for 5 min in Ca²⁺-free solution, simultaneous re-exposure to ACh in the presence of 2.5 mM Ca²⁺ increased [Ca²⁺]_i to the control steady-state level without overshoot. The tension attained was the same as control for each [ACh] used. Continuous exposure to successively increasing [ACh] (0.1–100 µM) also reduced the overshoot of [Ca²⁺]_i at 10 and 100 µM ACh, yet tension reached control levels at each [ACh] used. We conclude that the steady-state increase in [Ca²⁺]_i is necessary for tension maintenance and is dependent on Ca²⁺ influx through voltage-gated calcium channels at 0.1 µM ACh and through a verapamil-insensitive pathway at 10 and 100 µM. The initial transient increase in calcium arises from intracellular stores and is correlated with the magnitude of tension only in muscles that have completely recovered from previous exposure to agonists.     

Characterization of the 1,25-dihydroxycholecalciferol-stimulated calcium influx pathway in CaCo-2 cells

The present studies were conducted to investigate the mechanisms underlying the 1,25-dihydroxycholecalciferol (1,25(OH)₂D₃)-induced increase in intracellular Ca²⁺ ([Ca²⁺] _i ) in individual CaCo-2 cells. In the presence of 2mm Ca²⁺, 1,25(OH)₂D₃-induced a rapid transient rise in [Ca²⁺] _i in Fura-2-loaded cells in a concentration-dependent manner, which decreased, but did not return to baseline levels. In Ca²⁺-free buffer, this hormone still induced a transient rise in [Ca²⁺] _i , although of lower magnitude, but [Ca²⁺] _i then subsequently fell to baseline. In addition, 1,25(OH)₂D₃ also rapidly induced⁴⁵Ca uptake by these cells, indicating that the sustained rise in [Ca²⁺] _i was due to Ca²⁺ entry. In Mn²⁺-containing solutions, 1,25(OH)₂D₃ increased the rate of Mn²⁺ influx which was temporally preceded by an increase in [Ca²⁺] _i . The sustained rise in [Ca²⁺] _i was inhibited in the presence of external La³⁺ (0.5mm). 1,25(OH)₂D₃ did not increase Ba²⁺ entry into the cells. Moreover, neither high external K⁺ (75mm), nor the addition of Bay K 8644 (1 μm), an L-type, voltage-dependent Ca²⁺ channel agonist, alone or in combination, were found to increase [Ca²⁺] _i , 1,25(OH)₂D₃ did, however, increase intracellular Na⁺ in the absence, but not in the presence of 2mm Ca²⁺, as assessed by the sodium-sensitive dye, sodium-binding benzofuran isophthalate. These data, therefore, indicate that CaCo-2 cells do not express L-type, voltage-dependent Ca²⁺ channels. 1,25(OH)₂D₃ does appear to activate a La³⁺-inhibitable, cation influx pathway in CaCo-2 cells.     

The δ-Opioid Receptor Regulates Activity of Ryanodine Receptors in the Human Neuroblastoma Cell Line SK-N-BE

Abstract: Recent studies have demonstrated that opioid agonists affect the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) either by regulating plasma membrane Ca²⁺-channel activity or by mobilizing intracellular Ca²⁺ stores. The present report documents the [Ca²⁺]_i increase induced by opioid agonists in a human neuroblastoma cell line, SK-N-BE, expressing δ-opioid receptors. In the presence, as well as in the absence, of extracellular Ca²⁺, opioid agonists enhanced significantly [Ca²⁺]_i, whereas carbachol, known to mobilize specifically inositol 1,4,5-trisphosphate-sensitive intracellular Ca²⁺ stores, acted only in the presence of extracellular Ca²⁺. The opioid-induced increase in [Ca²⁺]_i was not affected by treatments modifying the trimeric G_i, G_o, and G_s protein transduction mechanisms or the activity of adenylyl cyclase. The Ca²⁺-ATPase pump-inhibiting sesquiterpene lactone, thapsigargin, did not modify the opioid-induced [Ca²⁺]_i response, whereas it abolished the effects of carbachol. The Ryana speciosa alkaloid, ryanodine, at concentrations known to block endoplasmic reticulum ryanodine receptors, decreased significantly the response to opioids without affecting the effects of carbachol. Thus, our results suggest that, in SK-N-BE cells, δ-opioid receptors mobilize Ca²⁺ from intracellular ryanodine-sensitive stores and the mechanism involved is independent of G_i/G_o and G_s proteins and protein kinase A activation.     

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.     

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.     

Role of protein kinase C in the regulation of cytosolic Ca2+ in A431 cells: Separation of growth factor and bradykinin pathways

Summary Calcium signaling systems in nonexcitable cells involve activation of Ca²⁺ entry across the plasma membrane and release from intracellular stores as well as activation of Ca²⁺ pumps and inhibition of passive Ca²⁺ pathways to ensure exact regulation of free cytosolic Ca²⁺ concentration ([Ca²⁺] _i ). A431 cells loaded with fura-2 cells were used as a model system to examine regulation of Ca²⁺ entry and intracellular release. Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-) both stimulated Ca²⁺ entry and release while bradykinin appeared only to release Ca²⁺ from intracellular stores. The possible role of protein kinase C (PKC) in modulating the [Ca²⁺] _i response to these agonists was examined by four methods. Low concentrations of TPA (2×10^–10 m) had no effect on Ca²⁺ release due to EGF, TGR- or bradykinin but resulted in a rapid return of [Ca²⁺] _i to baseline levels for EGF or TGF-. Addition of the PKC inhibitor staurosporine (1 and 10nm)_completely inhibited the action of TPA on EGF-induced [Ca²⁺] _i changes. An inhibitor of diglyceride kinase (R59022) mimicked the action of TPA. Down-regulation of PKC by overnight incubation with 0.1 or 1 m TPA produced the converse effect, namely prolonged Ca²⁺ entry following stimulation with EGF or TGF-. To show that one effect of TPA was on Ca²⁺ entry, fura-2 loaded cells were suspended in Mn²⁺ rather than Ca²⁺ buffers. Addition of EGF or TGF- resulted in Ca²⁺ release and Mn²⁺ entry. TPA but not the inactive phorbol ester, 4--phorbol-12,13-didecanoate, inhibited the Mn²⁺ influx. Thus, PKC is able to regulate Ca²⁺ entry due to EGF or TGF- in this cell type. A431 cells treated with higher concentrations of TPA (5×10^–8 m) inhibited not only Ca²⁺ entry but also Ca²⁺ release due to EGF/TGF- but had no effect on bradykinin-mediated Ca²⁺ release, suggesting differences in the regulation of the intracellular stores responsive to these two classes of agonists. Furthermore, sequential addition of EGF or TGF- gave a single transient of [Ca²⁺] _i , showing a common pool of Ca²⁺ for these agonists. In contrast, sequential addition of EGF (or TGF-) and bradykinin resulted in two [Ca²⁺] _i transients equal in size to those obtained with a single agonist. Ionomycin alone was able to fully deplete intracellular Ca²⁺ stores, whereas ionomycin following either EGF (or TGF-) or bradykinin gave an elevation of the [Ca²⁺] _i signal equal to that of the second agonist. These data indicate that there are separate pools of intracellular Ca²⁺ for EGF-mediated Ca²⁺ release which also respond differently to TPA.     

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.     

External bioenergy-induced increases in intracellular free calcium concentrations are mediated by Na+/Ca2+ exchanger and L-type calcium channel

External bioenergy (EBE, energy emitted from a human body) has been shown to increase intracellular calcium concentration ([Ca²⁺]_i, an important factor in signal transduction) and regulate the cellular response to heat stress in cultured human lymphoid Jurkat T cells. In this study, we wanted to elucidate the underlying mechanisms. A bioenergy specialist emitted bioenergy sequentially toward tubes of cultured Jurkat T cells for one 15-minute period in buffers containing different ion compositions or different concentrations of inhibitors. [Ca²⁺]_i was measured spectrofluorometrically using the fluorescent probe fura-2. The resting [Ca²⁺]_i in Jurkat T cells was 70 ± 3 nM (n = 130) in the normal buffer. Removal of external calcium decreased the resting [Ca²⁺]_i to 52 ± 2 nM (n = 23), indicating that [Ca²⁺] entry from the external source is important for maintaining the basal level of [Ca²⁺]_i. Treatment of Jurkat T cells with EBE for 15 min increased [Ca²⁺]_i by 30 ± 5% (P 0.05, Student t-test). The distance between the bioenergy specialist and Jurkat T cells and repetitive treatments of EBE did not attenuate [Ca²⁺]_i responsiveness to EBE. Removal of external Ca²⁺ or Na⁺, but not Mg²⁺, inhibited the EBE-induced increase in [Ca²⁺]_i. Dichlorobenzamil, an inhibitor of Na⁺/Ca²⁺ exchangers, also inhibited the EBE-induced increase in [Ca²⁺]_i in a concentration-dependent manner with an IC50 of 0.11 ± 0.02 nM. When external [K⁺] was increased from 4.5 mM to 25 mM, EBE decreased [Ca²⁺]_i. The EBE-induced increase was also blocked by verapamil, an L-type voltage-gated Ca²⁺ channel blocker. These results suggest that the EBE-induced [Ca²⁺]_i increase may serve as an objective means for assessing and validating bioenergy effects and those specialists claiming bioenergy capability. The increase in [Ca²⁺]_i is mediated by activation of Na⁺/Ca²⁺ exchangers and opening of L-type voltage-gated Ca²⁺ channels. (Mol Cell Biochem 271: 51–59, 2005)     

Exocytosis Coupled to Mobilization of Intracellular Calcium by Muscarine and Caffeine in Rat Chromaffin Cells

Abstract: We used cultured rat chromaffin cells to test the hypothesis that Ca²⁺ entry but not release from internal stores is utilized for exocytosis. Two protocols were used to identify internal versus external Ca²⁺ sources: (a) Ca²⁺ surrounding single cells was transiently displaced by applying agonist with or without Ca²⁺ from an ejection pipette. (b) Intracellular stores of Ca²⁺ were depleted by soaking cells in Ca²⁺-free plus 1 mM EGTA solution before transient exposure to agonist plus Ca²⁺. Exocytosis from individual cells was measured by microelectrochemical detection, and the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured by indo-1 fluorescence. KCl (35 mM) and nicotine (10 µM) caused an immediate increase in [Ca²⁺]_i and secretion in cells with or without internal Ca²⁺ stores, but only when applied with Ca²⁺ in the ejection pipette. Caffeine (10 mM) and muscarine (30 µM) evoked exocytosis whether or not Ca²⁺ was included in the pipette, but neither produced responses in cells depleted of internal Ca²⁺ stores. Pretreatment with ryanodine (0.1 µM) inhibited caffeine- but not muscarine-stimulated responses. Elevated [Ca²⁺]_i and exocytosis exhibited long latency to onset after stimulation by caffeine (2.9 ± 0.38 s) or muscarine (2.2 ± 0.25 s). However, the duration of caffeine-evoked exocytosis (7.1 ± 0.8 s) was significantly shorter than that evoked by muscarine (33.1 ± 3.5 s). The duration of caffeine-evoked exocytosis was not affected by changing the application period between 0.5 and 30 s. An ～20-s refractory period was found between repeated caffeine-evoked exocytotic bursts even though [Ca²⁺]_i continued to be elevated. However, muscarine or nicotine could evoke exocytosis during the caffeine refractory period. We conclude that muscarine and caffeine mobilize different internal Ca²⁺ stores and that both are coupled to exocytosis in rat chromaffin cells. The nicotinic component of acetylcholine action depends primarily on influx of external Ca²⁺. These results and conclusions are consistent with our original observations in the perfused adrenal gland.     

Endothelin-1 induces intracellular [Ca2+] increase via Ca2+ influx through the L-type Ca2+ channel, Ca2+-induced Ca2+ release and a pathway involving ETA receptors, PKC, PKA and AT1 receptors in cardiomyocytes

Using fura-2-acetoxymethyl ester (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly elevated the intracellular calcium level ([Ca²⁺]_i) in a dose-dependent manner and activated the L-type Ca²⁺ channel in cardiomyocytes isolated from rats. The effect of ET-1 on [Ca²⁺]_i elevation was abolished in the presence of the ET_A receptor blocker BQ123, but was not affected by the ET_B receptor blocker BQ788. ET-1-induced an increase in [Ca²⁺]_i, which was inhibited 46.7% by pretreatment with a high concentration of ryanodine (10 μmol/L), a blocker of the ryanodine receptor. The ET-1-induced [Ca²⁺]_i increase was also inhibited by the inhibitors of protein kinase A (PKA), protein kinase C (PKC) and angiotensin type 1 receptor (AT1 receptor). We found that ET-1 induced an enhancement of the amplitude of the whole cell L-type Ca²⁺ channel current and an increase of open-state probability (NPo) of an L-type single Ca²⁺ channel. BQ123 completely blocked the ET-1-induced increase in calcium channel open-state probability. In this study we demonstrated that ET-1 regulates calcium overload through a series of mechanisms that include L-type Ca²⁺ channel activation and Ca²⁺-induced Ca²⁺ release (CICR). ETA receptors, PKC, PKA and AT1 receptors may also contribute to this pathway. Supported by the National Natural Science Foundation of China (Grant No. 200830870910).     

The mechanism of protriptyline-induced Ca2+ movement and non-Ca2+-triggered cell death in PC3 human prostate cancer cells

Abstract

Protriptyline, a tricyclic anti-depressant, is used primarily to treat the combination of symptoms of anxiety and depression. However, the effect of protriptyline on prostate caner is unknown. This study examined whether the anti-depressant protriptyline altered Ca²⁺ movement and cell viability in PC3 human prostate cancer cells. The Ca²⁺-sensitive fluorescent dye fura-2 was used to measure [Ca²⁺]_i. Protriptyline evoked [Ca²⁺]_i rises concentration-dependently. The response was reduced by removing extracellular Ca²⁺. Protriptyline-evoked Ca²⁺ entry was inhibited by store-operated channel inhibitors (nifedipine, econazole and SKF96365), protein kinase C activator (phorbol 12-myristate 13 acetate, PMA) and protein kinase C inhibitor (GF109203X). Treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-tert-butylhydr-oquinone (BHQ) in Ca²⁺-free medium inhibited 60% of protriptyline-evoked [Ca²⁺]_i rises. Conversely, treatment with protriptyline abolished BHQ-evoked [Ca²⁺]_i rises. Inhibition of phospholipase C with U73122 suppressed 50% of protriptyline-evoked [Ca²⁺]_i rises. At concentrations of 50–70?µM, protriptyline decreased cell viability in a concentration-dependent manner; which were not reversed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Collectively, in PC3 cells, protriptyline evoked [Ca²⁺]_i rises by inducing phospholipase C-associated Ca²⁺ release from the endoplasmic reticulum and other stores, and Ca²⁺ influx via protein kinase C-sensitive store-operated Ca²⁺ channels. Protriptyline caused cell death that was independent of [Ca²⁺]_i rises.