Capacitative Ca2+ entry contributes to the Ca2+ influx induced by thyrotropin-releasing hormone (TRH) in GH3 pituitary cells

Treatment of GH₃ cells with either hypothalamic peptide thyrotropin-releasing hormone (TRH), the endomembrane Ca²⁺-ATPase inhibitor thapsigargin or the Ca²⁺ ionophore ionomycin mobilized, with different kinetics, essentially all of the Ca²⁺ pool from the intracellular Ca²⁺ stores. Any of the above-described treatments induced a sustained increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), which was dependent on extracellular Ca²⁺ and was prevented by Ni²⁺ but not by dihydropyridines (DHPs), suggesting that it was due to capacitative Ca²⁺ entry via activation of a plasma membrane pathway which opened upon the emptying of the intracellular Ca²⁺ stores. The increase of the plasma membrane permeability to Ca²⁺ correlated negatively with the filling degree of the intracellular Ca²⁺ stores and was reversed by refilling of the stores. The mechanism of capacitative Ca²⁺ entry into GH₃ cells differed from similar mechanisms described in several types of blood cells in that the pathway was poorly permeable to Mn²⁺ and not sensitive to cytochrome P₄₅₀ inhibitors. In GH₃ cells, TRH induced a transient [Ca²⁺]_i increase due to Ca²⁺ release from the stores (phase 1) followed by a sustained [Ca²⁺]_i increase due to Ca²⁺ entry (phase 2). At the single-cell level, phase 2 was composed of a DHP-insensitive sustained [Ca²⁺]_i increase, due to activation of capacitative Ca²⁺ entry, superimposed upon which DHP-sensitive [Ca²⁺]_i oscillations took place. The two components of the TRH-induced Ca²⁺ entry differed also in that [Ca²⁺]_i oscillations remained for several minutes after TRH removal, whereas the sustained [Ca²⁺]_i increase dropped quickly to prestimulatory levels, following the same time course as the refilling of the stores. The drop was prevented when the refilling was inhibited by thapsigargin. It is concluded that, even though the mechanisms of capacitative Ca²⁺ entry may show differences from cell to cell, it is also present and may contribute to the regulation of physiological functions in excitable cells such as GH₃. There, capacitative Ca²⁺ entry cooperates with voltage-gated Ca²⁺ channels to generate the [Ca²⁺]_i increase seen during phase 2 of TRH action. This contribution of capacitative Ca²⁺ entry may be relevant to the enhancement of prolactin secretion induced by TRH.     

Mechanisms of vancomycin-induced histamine release from rat peritoneal mast cells

The mechanisms of vancomycin (VCM)-induced histamine release were studied with rat peritoneal mast cells. VCM (>1×10^–3 M) released histamine from the isolated mast cells in a dose-dependent and noncytotoxic manner. In the absence of extracellular Ca²⁺, the histamine release was reduced markedly. When the intracellular Ca²⁺ was depleted, it was further decreased. The Fura-2-loaded single mast cells showed a biphasic increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) by VCM: the first transient and the second sustained components. In the absence of extracellular Ca²⁺, the transient component was unchanged, while the sustained component was eliminated completely. The IP₃ content in the mast cells increased within 10 s after the application of VCM. These results suggest that VCM releases histamine from rat peritoneal mast cells via an IP₃ production and increase in [Ca²⁺]_i.     

Effects of caffeine on intracellular calcium,calcium current and calcium-dependent potassium current in anterior pituitary GH3 cells

Caffeine elicits physiological responses in a variety of cell types by triggering the mobilization of Ca²⁺ from intracellular organelles. Here we investigate the effects of caffeine on intracellular Ca²⁺ concentration ([Ca²⁺]_i) and ionic currents in anterior pituitary cells (GH₃) cells. Caffeine has a biphasic effect on Ca²⁺-activated K⁺ current [I _K(Ca)]: it induces a transient increase superimposed upon a sustained inhibition. While the transient increase coincides with a rise in [Ca²⁺]_i, the sustained inhibition of I _K(Ca) is correlated with a sustained inhibition of the L-type Ca²⁺ current. The L-type Ca²⁺ current is also inhibited by other agents that mobilize intracellular Ca²⁺, including thyrotropin releasing hormone (TRH) and ryanodine, but in a matter distinct from caffeine. Unlike the caffeine effect, the TRH-induced inhibition washes-out under whole-cell patch-clamp conditions and is eliminated by intracellular Ca²⁺ chelators. Likewise, the ryanodine-induced inhibition desensitizes while the caffeine-induced inhibition does not. Simultaneous [Ca²⁺]_i and Ca²⁺ current measurements show that caffeine can inhibit Ca²⁺ current without changing [Ca²⁺]_i. Single-channel recordings show that caffeine reduces mean open time without affecting single-channel conductance of L-type channels. Hence the effects of caffeine on ion channels in GH₃ cells are attributable both to mobilization of intracellular Ca²⁺ and to a direct effect on the gating of L-type Ca²⁺ channels.     

Voltage-dependent Ca2+ influx in the epithelial cell line HT29: simultaneous use of intracellular Ca2+ measurements and nystatin perforated patch-clamp technique

Indirect evidence has accumulated indicating a voltage dependence of the agonist-stimulated Ca²⁺ influx into epithelial cells. Manoeuvres expected to depolarise the membrane voltage during agonist stimulation resulted in: (1) a decrease of the sustained phase of the adenosine triphosphate (ATP, 10^–5 mol/l)-induced intracellular Ca²⁺ transient, (2) a reduced fura-2 Mn²⁺-quenching rate, and (3) prevention of the refilling of the agonist-sensitive store. To quantify the change in intracellular Ca²⁺ as a function of membrane voltage, we measured simultaneously the intracellular Ca²⁺ activity ([Ca²⁺]_i) with fura-2 and the electrical properties using the nystatin perforated patch-clamp technique in single HT₂₉ cells. Ca²⁺ influx was either stimulated by ATP (10^–5 mol/l) or thapsigargin (TG, 10^–8 mol/l). After [Ca²⁺]_i reached the sustained plateau phase we clamped the membrane voltage in steps of 10 mV in either direction. A stepwise depolarisation resulted in a stepwise reduction of [Ca²⁺]_i. Similarly a stepwise hyperpolarisation resulted in a stepwise increase of [Ca²⁺]_i (ATP: 27.5±10 nmol/l per 10 mV, n=6; TG: 19 ±7.9 nmol/l per 10 mV, n=12). The summarised data show a linear relationship between the fluorescence ratio 340/380 nm change and the applied holding voltage. In unstimulated cells the same voltage-clamp protocol did not change [Ca²⁺]_i (n=9). Under extracellular Ca²⁺-free conditions [Ca²⁺]_i remained unaltered when changing the membrane voltage. These data provide direct evidence that the Ca²⁺ influx in epithelial cells is membrane voltage dependent. Our data indicate that small changes in membrane voltage lead to substantial changes in [Ca²⁺]_i. This may be due either to a change of driving force for Ca²⁺ into the cell, or may reflect voltage-dependent regulation of the respective Ca²⁺ entry mechanism.     

Intracellular calcium in mammalian brain cells: fluorescence measurements with quin2

Summary Dispersed brain cells from 12–14 day old mouse embryos were loaded with the Ca²⁺-sensitive fluorescent probe, quin2 and shown to have a resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) of 158 nM (SE ± 5) in the presence of 1 mM [Ca²⁺]_o. When external [Ca²⁺] was raised from 0 to 1 mM there was an increase of [Ca²⁺]_i of 70 nM; with further additions of Ca to >10 mM [Ca²⁺]_o the level of [Ca²⁺]_i increased by <25 nM. Releasable intracellular Ca²⁺ stores, estimated from the increase in [Ca²⁺] produced by 4Br A23187 in the absence of extracellular Ca²⁺, were 24 fmol/10⁶ cells. A small increase in [Ca²⁺]_i could be produced by the mitochondrial inhibitor, carbonyl cyanide m-chlorophenylhydrazone (CCCP). When extracellular K⁺ was raised by 10–20 mM, intracellular Ca²⁺ levels increased from 152 (SE ± 7) to 204 nM (SE ± 10). These K⁺-induced increases in [Ca²⁺]_i were blocked by verapamil, did not occur in the absence of extracellular Ca²⁺, and presumably reflect the activation of voltage-dependent Ca²⁺ channels. N-methyl-D-aspartic acid (NMDA) evoked an increase in [Ca²⁺]_i, while the kainate-like lathyrus sativus neurotoxin, L-3-oxalyl-amino-2aminopropionic acid (L-3,2-OAP) did not; this is consistent with previous observations of different and respectively Ca²⁺-dependent and -independent mechanisms of action of these excitatory amino acids.     

The intracellular Ca2+ concentration optimal for T cell activation is quite different after ionomycin or CD3 stimulation

The relationship between the initial increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i) (measured at the single-cell level with an imaging system) and the ensuing proliferation was examined in a human T cell clone stimulated by a phorbol ester in combination with ionomycin, thapsigargin or an anti-CD3 mAb (monoclonal antibody against the CD3 molecule, UCHT1). From the responses to various ionomycin concentrations, one can define a range of [Ca²⁺]_i values (400–900 nM) which appears optimal for T cell proliferation; lower [Ca²⁺]_i values are suboptimal, higher values are cytotoxic. It was then examined if the [Ca²⁺]_i requirements were similar following anti-CD3 stimulation. [Ca²⁺]_i oscillations elicited by a concentration of UCHT1 (1/1,000) optimal for mitogenicity fall precisely within the 400–900 nM range. However, very low concentrations of UCHT1 (1/100,000) which evoke barely detectable [Ca²⁺]_i responses still cause the cells to proliferate. The possibility that the lower [Ca²⁺]_i requirements observed following anti-CD3 stimulation was due to [Ca²⁺]_i oscillations was tested under conditions which prevented the appearance of these oscillations. It turns out that an oscillatory Ca²⁺signal is not more mitogenic than a sustained augmentation of [Ca²⁺]_i. Finally, it was examined if overstimulation via CD3 could have toxic consequences similar to those elicited after ionomycin overstimulation. Large transient [Ca²⁺]_i responses can be observed following anti-CD3 stimulation in appropriate conditions, and namely in T cells pretreated with interleukin-2. These [Ca²⁺]_i augmentations are not cytotoxic. A role for the plasmalemmal Ca²⁺ pump in the prevention of cytotoxicity can be demonstrated. In conclusion, the correspondence between the [Ca²⁺]_i response and cell proliferation is entirely different following stimulation by ionomycin and by anti-CD3. In addition, cell proliferation evoked by very low UCHT1 concentration might reveal the existence of a yet unidentified activation pathway.     

Four types of Ca2+ signals in naive CD8+ cytotoxic T cells after stimulation with T cell agonists,partial agonists and antagonists

Stimulation of T cells via the T cell receptor (TCR) leads to an increase intracellular in free Ca²⁺ levels ([Ca²⁺]_i) and the activation of the MAP kinase signaling pathway. This study analyzes for the first time Ca²⁺ fluxes in naive cytotoxic T cells stimulated with full agonists, partial agonists, or antagonists. Four different types of Ca²⁺ responses could be observed. Full agonists triggered a strong and sustained increase in [Ca²⁺]_i. In contrast, partial T cell agonists induced either a strong but transient Ca²⁺ flux or very low to no increases in [Ca²⁺]_i, while T cell antagonists failed to induce any measurable Ca²⁺ flux. The ability of peptides to induce elevated [Ca²⁺]_i perfectly paralleled their ability to trigger TCR internalization and T cell activation. Thus, stimulation of naive cytotoxic T cells with a panel of defined altered peptide ligands reveals a consistent picture, where Ca²⁺ fluxes predict agonist, partial agonist and antagonist properties of peptides.     

TRPC3 participates in angiotensin II type 1 receptor-dependent stress-induced slow increase in intracellular Ca<Superscript>2+</Superscript> concentration in mouse cardiomyocytes

When a cardiac muscle is held in a stretched position, its [Ca²⁺] transient increases slowly over several minutes in a process known as stress-induced slow increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) (SSC). Transient receptor potential canonical (TRPC) 3 forms a non-selective cation channel regulated by the angiotensin II type 1 receptor (AT1R). In this study, we investigated the role of TRPC3 in the SSC. Isolated mouse ventricular myocytes were electrically stimulated and subjected to sustained stretch. An AT1R blocker, a phospholipase C inhibitor, and a TRPC3 inhibitor suppressed the SSC. These inhibitors also abolished the observed SSC-like slow increase in [Ca²⁺]_i induced by angiotensin II, instead of stretch. Furthermore, the SSC was not observed in TRPC3 knockout mice. Simulation and immunohistochemical studies suggest that sarcolemmal TRPC3 is responsible for the SSC. These results indicate that sarcolemmal TRPC3, regulated by AT1R, causes the SSC.     

Caffeine-induced oscillations of cytosolic Ca2+ in GH3 pituitary cells are not due to Ca2+ release from intracellular stores but to enhanced Ca2+ influx through voltage-gated Ca2+ channels

Caffeine, a well known facilitator of Ca²⁺-induced Ca²⁺ release, induced oscillations of cytosolic free Ca²⁺ ([Ca²⁺]_i) in GH₃ pituitary cells. These oscillations were dependent on the presence of extracellular Ca²⁺ and blocked by dihydropyridines, suggesting that they are due to Ca²⁺ entry through L-type Ca²⁺ channels, rather than to Ca²⁺ release from the intracellular Ca²⁺ stores. Emptying the stores by treatment with ionomycin or thapsigargin did not prevent the caffeine-induced [Ca²⁺]_i oscillations. Treatment with caffeine occluded phase 2 ([Ca²⁺]_i oscillations) of the action of thyrotropin-releasing hormone (TRH) without modifying phase 1 (Ca²⁺ release from the intracellular stores). Caffeine also inhibited the [Ca²⁺]_i increase induced by depolarization with high-K⁺ solutions (56% at 20 mM), suggesting direct inhibition of the Ca²⁺ entry through voltage-gated Ca²⁺ channels. We propose that the [Ca²⁺]_i increase induced by caffeine in GH₃ cells takes place by a mechanism similar to that of TRH, i.e. membrane depolarization that increases the firing frequency of action potentials. The increase of the electrical activity overcomes the direct inhibitory effect on voltage-gated Ca²⁺ channels with the result of increased Ca²⁺ entry and a rise in [Ca²⁺]_i. Consideration of this action cautions interpretation of previous experiments in which caffeine was assumed to increase [Ca²⁺]_i only by facilitating the release of Ca²⁺ from intracellular Ca²⁺ stores.     

Effects of 2,5-di(tert-butyl)-1,4-hydroquinone on intracellular free Ca2+ levels and histamine secretion in RBL-2H3 cells

The effects of 2,5-di(tert-butyl)-1,4-hydroquinone (DTBHQ) on the intracellular free Ca²⁺ level ([Ca²⁺]_i) and histamine secretion of rat basophilic leukemia (RBL-2H3) cells were examined. DTBHQ (0.1–10 µmol/l) alone induced rapid and sustained increases in [Ca²⁺]_i in a concentration-dependent manner. In cells sensitized with anti-dinitrophenyl IgE, DTBHQ (10 µmol/1) further increased the antigen (dinitrophenylated BSA)-induced Ca²⁺ response. In the absence of external Ca²⁺ with addition of 1 mmol/1 EGTA, both DTBHQ (10 µmol/l) and the antigen (10 µg/ml) induced transient increase in [Ca²⁺]_i. In sensitized cells, both DTBHQ (10 µmol/1) and antigen (10 µg/ml) elicited histamine secretion, although the response was far stronger in the latter case. The DTBHQ-induced histamine secretion was markedly enhanced by addition of the protein kinase C activator, phorbol 12-myristate 13-acetate (TPA) (10 ng/ml) whereas TPA alone did not cause any increase. Moreover, DTBHQ enhanced the antigen-induced histamine secretion. The results suggest that DTBHQ increases [Ca²⁺]_i and enhances antigeninduced histamine secretion while DTBHQ alone does not cause as much histamine secretion as antigen, which support the idea that calcium signals are necessary but are not sufficient for maximum histamine secretion in RBL-2H3 cells.     

Carboxyeosin decreases the rate of decay of the [Ca2+]i transient in uterine smooth muscle cells isolated from pregnant rats

 In myometrial smooth muscle cells the rate of decline of intracellular calcium ([Ca²⁺]_i) is determined by Ca²⁺ extrusion from the cell and uptake into intracellular stores. The relative quantitative contribution of these processes however, has not been established. We therefore examined the effect of the sarcolemmal Ca²⁺ pump inhibitor, carboxyeosin, on the rate of the [Ca²⁺]_i transient decline in myocytes isolated from pregnant rat uterus. Indo-1 was used in conjunction with the whole-cell patch-clamp technique to measure [Ca²⁺]_i simultaneously with transmembrane calcium current (I _Ca). [Ca²⁺]_i transients were elicited by repetitive membrane depolarization to simulate the natural pattern of uterine electrical activity. The rate of [Ca²⁺]_i removal was calculated from the falling phase of the [Ca²⁺]_i transient. Pre-treatment of the cells with 2 μM carboxyeosin led to a marked decrease in the rate of [Ca²⁺]_i transient decay, suggesting that the sarcolemmal Ca²⁺ pump is involved in the calcium extrusion process. Removal of the extracellular Na also decreased the rate of [Ca²⁺]_i decay, indicating an important role for the Na⁺/Ca²⁺ exchange. When both the sarcolemmal Ca²⁺ pump and Na⁺/Ca²⁺ exchange were inhibited the cell failed to restore [Ca²⁺]_i after the stimulation. Comparison of the rate constants of [Ca²⁺]_i decay in control conditions and after carboxyeosin treatment shows that approximately 30% of [Ca²⁺]_i decay is due to the sarcolemmal calcium pump activity. The remaining 70% can be attributed to the activity of Na⁺/Ca²⁺ exchanger and the intracellular calcium stores. Received: 17 July 1998 / Received after revision: 23 September 1998 / Accepted: 25 September 1998     

Endothelin-1 inhibits voltage-sensitive Ca2+ channels in cultured rat cerebellar granule neurones via the ET-A receptor

 In this study, we have investigated the effect of the vasoconstrictor peptide endothelin-1 (ET-1) on voltage-sensitive Ca²⁺ channels in rat cerebellar granule neurones using the patch-clamp technique. Using amphotericin B perforated-patch recording of whole-cell currents, the Ca²⁺ channel current was inhibited by 28.4±6.4% by 400 nM ET-1, but was unaffected when experiments were repeated using the whole-cell, ruptured-patch configuration. In cell-attached patches, 400 nM ET-1 inhibited unitary L-type Ca²⁺ channel currents (I _Ba) by 85±5%. ET-1 decreased the open probability (NP _o) and the frequency of channel opening and increased the mean closed time of channels. No effects on the mean open time or the time constants for channel opening or closure were observed. L-type Ca²⁺ channel inhibition was dose dependent with an IC₅₀ of 19 nM. The effect of ET-1 was prevented by the combined endothelin-A and -B receptor antagonist PD145065 (10 μM), indicating a receptor-mediated effect. The ET-A receptor antagonist BQ-123 (10 μM) prevented Ca²⁺ channel inhibition by ET-1, while the ET-B receptor agonist sarafotoxin 6c (500 nM) had no effect. The inhibition by ET-1 was not due to a change in the voltage of channel activation. Fura-2 Ca²⁺ imaging showed that no substantial rise in intracellular Ca²⁺ levels occurred during ET-1 application excluding a Ca²⁺-dependent inhibition of the channels. Thus in cultured rat cerebellar granule neurones, ET-1 inhibits L-type Ca²⁺ channels via activation of the ET-A receptor. Inhibition may be mediated by an as yet unidentified cytoplasmic second messenger. Received: 13 March 1998 / Received after revision and accepted: 14 May 1998     

Extracellular Ca2+-dependent and independent calcium transient in fetal myotubes

Spatio-temporal changes in the intracellular calcium concentration [Ca²⁺]_i of dissociated mice myotubes from 14-day and 18-day-old fetuses were studied using digital imaging analysis of the Ca²⁺ indicator fura-2. Myotubes from 18-day-old fetuses displayed a transient [Ca²⁺]_i increase upon electrical stimulation either in nominally calcium-free external solution or in Krebs solution containing 100 M lanthanum. Thus, at this developmental stage, membrane depolarization appears to increase [Ca²⁺]_i by stimulating Ca²⁺ release from the sarcoplasmic reticulum independently of extracellular Ca²⁺ influx. Similarly, myotubes from 14-day-old fetuses also showed a calcium transient upon electrical stimulation in Krebs solution. However, in 46% of these myotubes the calcium transient was abolished when Ca²⁺ entry through calcium channels was suppressed.     

Acute effects of taurine on intracellular calcium in normal and diabetic cardiac myocytes

The present study investigated the acute effects of taurine on intracellular Ca²⁺ ([Ca²⁺]_i) in normal and diabetic cardiac myocytes. [Ca²⁺]_i was monitored using fura-2 in single myocytes isolated from control or streptozotocin-treated rats and paced at frequencies between 0.33 Hz and 2.0 Hz in the absence or presence of 20 mM taurine. Increasing stimulus frequency resulted in significant increases in resting and peak [Ca²⁺]_i, and amplitude of the Ca²⁺ transient in both control and diabetic myocytes. The amplitude of the Ca²⁺ transient and the extent of its increase with increasing frequency was, however, significantly lower in the diabetic myocytes. Taurine significantly increased resting [Ca²⁺]_i, peak [Ca²⁺]_i, and the amplitude of the Ca²⁺ transient in both control and diabetic myocytes at all stimulus frequencies examined. The degree of potentiation of the Ca²⁺ transient decreased with increasing stimulus frequency in control cells but not in diabetic cells. In the absence of taurine the decay of the Ca²⁺ transient was significantly slower in diabetic than control myocytes. Taurine was without significant effect on the time course of the Ca²⁺ transient decay in control cells, however, in diabetic cells it significantly accelerated the rate of decay. The data demonstrate directly that taurine is able to increase [Ca²⁺]_i and the amplitude of the Ca²⁺ transient in both normal and diabetic cardiac myocytes. In addition several of the effects of taurine appeared to be more pronounced in diabetic than control cells. Received: 13 January 1999 / Received after revision: 12 March 1999 / Accepted: 14 April 1999     

Oxytocin mobilizes calcium from a unique heparin-sensitive and thapsigargin-sensitive store in single myometrial cells from pregnant rats

Intracellular free Ca²⁺ concentration ([Ca²⁺]_i) was monitored using the fluorescence from the dye Fura-2-AM in single myometrial cells from pregnant rats. Oxytocin and acetylcholine applied to the cell evoked an initial peak in [Ca²⁺]_i followed by a smaller sustained rise which was rapidly terminated upon removal of acetylcholine or persisted after oxytocin removal. A Ca²⁺ channel blocker (oxodipine) and external Ca²⁺ removal decreased both the transient and sustained rises in [Ca²⁺]_i suggesting that Ca²⁺ influx through L-type Ca²⁺ channels participated in the global Ca²⁺ response induced by oxytocin. However, the initial peak in [Ca²⁺]_i produced by oxytocin was mainly due to Ca²⁺ store release: it was abolished by inclusion of heparin [which blocks inositol 1,4,5-trisphosphate (InsP ₃) receptors] in the pipette (whole-cell recording mode of patch-clamp) and external application of thapsigargin (which blocks sarcoplasmic reticulum Ca²⁺-ATPases). In contrast, the transient Ca²⁺ response induced by oxytocin was unaffected by ryanodine. Moreover, caffeine failed to induce a rise in [Ca²⁺]_i but reduced the oxytocin-induced transient Ca²⁺ response. The later sustained rise in [Ca²⁺]_i produced by oxytocin was due to the entry of Ca²⁺ into the cell as it was suppressed in external Ca²⁺-free solution. The Ca²⁺ entry pathway is permeable to Mn²⁺ ions, in contrast to that described in various vascular and visceral smooth muscle cells. Oxytocin-induced Ca²⁺ release is blocked by the oxytocin antagonist d(CH₂)₅[Tyr(Me)²,Thr⁴,Tyr-NH ₂ ⁹ ]OVT. The prolonged increase in [Ca²⁺]_i after oxytocin removal is rapidly terminated by addition of the oxytocin antagonist suggesting that oxytocin dissociation from its receptor is very slow. The oxytocin stimulation of [Ca²⁺]_i was insensitive to incubation with pertussis toxin, and blocked by a pipette solution containing anti-q/₁₁ antibody. These data show that myometrial cells possess an unique heparin-sensitive and thapsigargin-sensitive store that can be mobilized by activation of oxytocin receptors which couples with a Gq/G₁₁-protein to activate phospholipase C.     

Activation of different pathways for calcium elevation by bradykinin and ATP in rat pheochromocytoma (PC 12) cells

We have studied the pathways by which extra-cellular bradykinin and adenosine 5-triphosphate (ATP) elicit changes in intracellular free calcium ([Ca²⁺]_i) in nerve-growth-factor(NGF)- treated rat pheochromocytoma (PC 12) cells. Both substances caused a significant rise in [Ca²⁺]_i as assessed by fura-2 based micro-fluorimetry. The bradykinin-induced response consisted of an initial Ca²⁺ mobilization from an internal pool followed by a sustained increase in [Ca²⁺]_i, which was due to activation of a small inward current. The initial response always started at a localized site opposite to the cell nucleus. The inward current was partially carried by Ca²⁺ and began with a time lag of about 4 s after the start of the initial transient signal. Stepwise hyperpolarization of the plasma membrane, after activation of the inward current by bradykinin, caused a simultaneous increase in current amplitude and in [Ca²⁺]_i, due to an increase in the driving force for Ca²⁺ influx. With ATP as an agonist the onset of inward current coincided with an increase in [Ca²⁺]_i. Inward current and [Ca²⁺]_i were enhanced during hyperpolarizing steps indicating a substantial Ca²⁺ influx through ATP-activated channels. No release of Ca²⁺ from internal stores, but a large Na⁺ inward current, was observed in Ca²⁺-free external solution after addition of ATP. While the bradykinin-induced responses were much more pronounced in cell bodies than in growth cones, the ATP effects were somewhat variable in cell bodies and more homogeneous in growth cones.     

Intracellular Ca2+ signals in human-derived pancreatic somatostatin-secreting cells (QGP-1N)

Single-cell microfluorimetry techniques have been used to examine the effects of acetylcholine (0.1–100 M) on the intracellular free calcium ion concentration ([Ca²⁺]_i) in a human-derived pancreatic somatostatin-secreting cell line, QGP-1N. When applied to the bath solution, acetylcholine was found to evoke a marked and rapid increase in [Ca²⁺]_i at all concentrations tested. These responses were either sustained, or associated with the generation of complex patterns of [Ca²⁺]_i transients. Overall, the pattern of response was concentration related. In general, 0.1–10 M acetylcholine initiated a series of repetitive oscillations in cytoplasmic Ca²⁺, whilst at higher concentrations the responses consisted of a rapid rise in [Ca²⁺]_i followed by a smaller more sustained increase. Without external Ca²⁺, 100 M acetylcholine caused only a transient rise in [Ca²⁺]_i, whereas lower concentrations of the agonist were able to initiate, but not maintain, [Ca²⁺]_i oscillations. Acetylcholine-evoked Ca²⁺ signals were abolished by atropine (1–10 M), verapamil (100 M) and caffeine (20 mM). Nifedipine failed to have any significant effect upon agonist-evoked increases in [Ca²⁺]_i, whilst 50 mM KCl, used to depolarise the cell membrane, only elicited a transient increase in [Ca²⁺]_i. Ryanodine (50–500 nM) and caffeine (1–20 mM) did not increase basal Ca²⁺ levels, but the Ca²⁺-ATPase inhibitors 2,5-di(tert-butyl)-hydroquinone (TBQ) and thapsigargin both elevated [Ca²⁺]_i levels. These data demonstrate for the first time cytosolic Ca²⁺ signals in single isolated somatostatin-secreting cells of the pancreas. We have demonstrated that acetylcholine will evoke both Ca²⁺ influx and Ca²⁺ mobilisation, and we have partially addressed the subcellular mechanism responsible for these events.     

白细胞介素-2对缺氧/复氧心肌细胞[Ca2+]i的作用

目的:观察白细胞介素-2(IL-2)对心肌细胞在缺氧/复氧过程中电刺激诱导的[Ca²⁺]i的作用。方法:采用酶解分离成年大鼠心室肌细胞化学缺氧模型, 以Fura-2/AM为钙探针, 用细胞内双波长钙荧光系统检测心肌[Ca²⁺]i的变化。结果:①缺氧/复氧过程中, 缺氧5min时, 心肌[Ca²⁺]i幅度降低、舒张末期[Ca²⁺]i升高, [Ca²⁺]i达峰时间(TTP)延长, 恢复时间(RT)延长。复氧10min后, 心肌[Ca²⁺]i幅度、舒张末期[Ca²⁺]i、TTP及RT逐渐回复, 但不能完全恢复到对照水平;②在缺氧期间加入IL-2(2×10⁵U/L), 复氧期间[Ca²⁺]i各参数回复减慢;③用κ-阿片受体拮抗剂nor-BNI(10^-8mol/L)预处理后, 缺氧+IL-2对复氧时[Ca²⁺]i作用的影响被减弱, 而δ-阿片受体拮抗剂纳曲吲哚(10^-6mol/L)预处理则无此作用。结论:缺氧时同时存在IL-2, 可加剧复氧时心肌[Ca²⁺]i的变化, 其机制可能是IL-2通过心肌κ-阿片受体而发挥作用。     

The effects of thapsigargin on [Ca2+]i in isolated rat mesenteric artery vascular smooth muscle cells

Summary The effects of thapsigargin were studied on single cells isolated from side branches of the rat mesenteric artery. Thapsigargin (150 nM) produced a transient increase of [Ca²⁺]_i. This transient rise of [Ca²⁺]_i was unaffected by removing external Ca²⁺ ions. This suggests that thapsigargin is releasing Ca²⁺ ions from an intracellular store. In the absence of thapsigargin both noradrenaline and caffeine also produced a transient increase of [Ca²⁺]_i. These increases were abolished by prior exposure to thapsigargin. Correspondingly, the effects of thapsigargin were abolished by prior exposure to caffeine. These results show that thapsigargin releases Ca²⁺ from the noradrenaline and caffeine-sensitive stores.     

Effects of motilin on intracellular free calcium in cultured smooth muscle cells from the antrum of neonatal rats

Aim: The aim of this study was to determine the effects of motilin on [Ca²⁺]_i regulation and its underlying molecular mechanism in cultured antral smooth muscle cells (ASMCs). Methods: Antral cells were isolated and cultured from neonatal rats, and then the [Ca²⁺]_i in these cells was evaluated by calcium fluorescent probe Fluo-3/AM on a laser scanning confocal microscope. Results: We show that motilin dose-dependently increased [Ca²⁺]_i concentration in cultured ASMCs. Pre-incubation of cells with either the calcium antagonist verapamil (10⁻⁵ mol L⁻¹) or the calcium chelator Egtazic (EGTA, 0.1 mmol L⁻¹) significantly suppressed motilin (10⁻⁶ mol L⁻¹) induced [Ca²⁺]_i increase as indicated by fluorescent intensity. Interestingly, after mixing with the non-selective intracellular calcium release blocker TMB-8 (10⁻⁵ mol L⁻¹), guanosine triphosphate regulatory protein antagonist NEM (10⁻⁵ mol L⁻¹), phospholipase C (PLC) inhibitor compound 48/80 (1.2 μg mL⁻¹) and ryanodine at high concentration (10⁻⁵ mol L⁻¹), the motilin-induced [Ca²⁺]_i increase was only partially blocked. The protein kinase C inhibitor d -sphingosine (10⁻⁶ mol L⁻¹), however, did not show any inhibitory effect on motilin-induced [Ca²⁺]_i elevation. Conclusions: Our study suggests that motilin-stimulated [Ca²⁺]_i elevation in ASMCs is probably due to sustained extracellular Ca²⁺ influx and Ca²⁺ release from Ca²⁺ stores via inositol tris-phosphate receptors and ryanodine receptors. Specifically, motilin-induced [Ca²⁺]_i release is accompanied with guanosine triphosphate-binding protein-coupled receptor–PLC–inositol tris-phosphate signalling cascades.