Taurine prevents intracellular calcium overload during calcium paradox of cultured cardiomyocytes

Summary The effect of taurine on the cellular distribution of [Ca²⁺]_i, during the calcium paradox was examined by digital imaging of a single fura-2-loaded cell. Cardiomyocytes superfused with control medium containing 2mM Ca²⁺ exhibited typical transients associated with spontaneous beating. When the cells were exposed to Ca²⁺-free buffer, immediate cessation of both spontaneous contractions and calcium transients was observed as [Ca²⁺]; rapidly fell to a level of 3–6 × 10^–8M. Subsequent restoration of medium calcium increased [Ca²⁺]_i to level 4–7 times normal. Large increases in [Ca²⁺]_i were observed in most cells and were associated with the development of contracture and bleb formation.Taurine pretreatment (20mM) caused no significant effect on [Ca²⁺]_i during Ca²⁺ depletion. However, it inhibited excessive accumulation of [Ca²⁺]_i during the Ca²⁺ repletion. Moreover, taurine treated cells recovered their Ca²⁺-transients and beating pattern earlier than non-treated cells. Finally morphological abnormalities commonly associated with calcium overload were attenuated by taurine treatment.     

An Interaction Between ATP and High K+: Mutual Impairment of ATP- and High K+-Evoked [Ca2+]i Increase in NG 108–15 Cells

The interaction between ATP- and high K⁺-evoked increase in intracellular free calcium concentration ([Ca²⁺]_i) was investigated to gain an insight into the mechanism of interaction of ATP with voltage-sensitive calcium channels. [Ca²⁺]_i was measured in the neuronal model, neuroblastoma × glioma hybrid cells (NG 108–15), using the fluorescence indicator fura-2. In the presence of 1.8 mM extracellular Ca²⁺, ATP induced a rapid, concentration-dependent increase in [Ca²⁺]_i. High K⁺ (50 mM) evoked a [Ca²⁺]_i rise from 109 ± 11 nM to 387 ± 81 nM (n = 16). The application of either of these two [Ca²⁺]_i-increase provoking agents in sequence with the other caused impairment of the latter effect. The mutual desensitization of the responses to ATP and high K⁺ strongly suggests that both agents rely at least in part on the same source of Ca²⁺ for elevation of [Ca²⁺]_i in NG 108–15 cells.     

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)     

Spinorphin inhibits membrane depolarization- and capsaicin-induced intracellular calcium signals in rat primary nociceptive dorsal root ganglion neurons in culture

Abstract

Objective: Spinorphin is a potential endogenous antinociceptive agent although the mechanism(s) of its analgesic effect remain unknown. We conducted this study to investigate, by considering intracellular calcium concentrations as a key signal for nociceptive transmission, the effects of spinorphin on cytoplasmic Ca²⁺ ([Ca²⁺]_i) transients, evoked by high-K⁺ (30?mM) depolariasation or capsaicin, and to determine whether there were any differences in the effects of spinorphin among subpopulation of cultured rat dorsal root ganglion (DRG) neurons. Methods: DRG neurons were cultured on glass coverslips following enzymatic digestion and mechanical agitation, and loaded with the calcium sensitive dye fura-2 AM (1?µM). Intracellular calcium responses in individual DRG neurons were quantified using standard fura-2 based ratiometric calcium imaging technique. All data were analyzed by using unpaired t test, p?<?0.05 defining statistical significance. Results: Here we found that spinorphin inhibited cytoplasmic Ca²⁺ ([Ca²⁺]_i) transients, evoked by depolarization and capsaicin selectively in medium and small cultured rat DRG neurons. Spinorphin (10–300?µM) inhibited the Ca²⁺ signals in concentration dependant manner in small- and medium diameter DRG neurons. Capsaicin produced [Ca²⁺]_i responses only in small- and medium-sized DRG neurons, and pre-treatment with spinorphin significantly attenuated these [Ca²⁺]_i responses. Conclusion: Results from this study indicates that spinorphin significantly inhibits [Ca²⁺]_i signaling, which are key for the modulation of cell membrane excitability and neurotransmitter release, preferably in nociceptive subtypes of this primary sensory neurons suggesting that peripheral site is involved in the pain modulating effect of this endogenous agent.     

Methylmercury-Induced Elevations in Intrasynaptosomal Zinc Concentrations: An 19F-NMR Study

Abstract: Methylmercury (MeHg) increases the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) and another endogenous polyvalent cation in both synaptosomes and NG108-15 cells. In synaptosomes, the elevation in [Ca²⁺]_i was strictly dependent on extracellular Ca²⁺ (Ca²⁺_e); similarly, in NG108-15 cells, a component of the elevations in [Ca²⁺]_i was Ca²⁺_e dependent. The MeHg-induced elevations in endogenous polyvalent cation concentration were independent of Ca²⁺_e in synaptosomes and NG108-15 cells. The pattern of alterations in fura-2 fluorescence suggested the endogenous polyvalent cation may be Zn²⁺. Using ¹⁹F-NMR spectroscopy of rat cortical synaptosomes loaded with the fluorinated chelator 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5F-BAPTA), we have determined unambiguously that MeHg increases the free intrasynaptosomal Zn²⁺ concentration ([Zn²⁺]_i). In buffer containing 200 µM EGTA to prevent the Ca²⁺_e-dependent elevations in [Ca²⁺]_i, the [Zn²⁺]_i was 1.37 ± 0.20 nM; following a 40-min exposure to MeHg-free buffer [Zn²⁺]_i was 1.88 ± 0.53 nM. Treatment of synaptosomes for 40 min with 125 µM MeHg yielded [Zn²⁺]_i of 2.69 ± 0.55 nM, whereas 250 µM MeHg significantly elevated [Zn²⁺]_i to 3.99 ± 0.68 nM. No Zn²⁺ peak was observed in synaptosomes treated with the cell-permeant heavy metal chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, 100 µM) following 250 µM MeHg exposure. [Ca²⁺]_i in buffer containing 200 µM EGTA was 338 ± 26 nM and was 370 ± 64 nM following an additional 40-min exposure to MeHg-free buffer. [Ca²⁺]_i was 498 ± 28 or 492 ± 53 nM during a 40-min exposure to 125 or 250 µM MeHg, respectively. None of the values of [Ca²⁺]_i differed significantly from either pretreatment levels or buffer-treated controls.     

Determination of cytoplasmic calcium concentration inDryopteris spores

Germination of Dryopteris spores is mediated by the physiologically active, far-red-absorbing form of phytochrome, Pfr, and external Ca²⁺ is necessary for the transduction of the light signal. Because knowledge about the cytoplasmic calcium ion concentration, [Ca²⁺]_i, is of great importance for understanding the role of calcium during signal transduction, this value was measured using fura-2 in fern spores undergoing the normal developmental progression into germination. Fura-2 was loaded into the spores by electroporation, which does not disrupt the normal process of germination. The intensity of the fluorescence emission of the loaded fura-2 was analysed by a microspectrophotometric assay of single spores, and successful loading could be obtained by the application of ten electrical pulses (field strength 7.5 kV · cm^–1, half-life (time constant) 230 s). Fura-2 was alternately excited by light of wavelengths 355 and 385 nm through an inverted fluorescence microscope, and the emitted fura-2 fluorescence was collected by a silicon-intensified video camera. The cytoplasmic calcium ion concentration was calculated from the ratio of the camera output obtained for both wavelengths and displayed by a pseudo-color technique. Spores responded to changes of the extracellular Ca²⁺ concentration, and this observation is considered as evidence that fura-2 is loaded into the cytoplasm. The substitution of a low external [Ca²⁺] (1 mM ethyleneglycol-bis(2-aminoethyl-ether) {ie166-01},N-tetraacetic acid (EGTA)) by 1 mM CaCl₂ caused a fast increase of [Ca²⁺]_i from approx. 50 nM to above 500 nM. In contrast, the subsequent substitution of CaCl₂ by EGTA decreased [Ca²⁺]_i again below 100 nM within 0.5 h. Furthermore, the application of ionomycin could initiate a change in [Ca2+]_i according to the Ca²⁺ gradient established between the extracellular medium and cytoplasm. In spores sown on a Ca²⁺-free medium, [Ca²⁺]_i, analysed in a buffer containing EGTA, was found to be around 50 nM during the first days of cultivation, independent of the irradiation protocol. However, if spores were grown in darkness on a Ca²⁺-containing medium and analysed in EGTA, [Ca²⁺]_i was significantly higher ( 500 nM). In red-light-irradiated spores, [Ca²⁺]_i was found to decrease with increasing time after irradiation, and was determined to be less than 100 nM when analysis was done 44 h after germination was initiated by the light treatment.Dedicated to Professor H. Mohr on the occasion of his 60th birthday     

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.     

Calcium-dependent control of volume regulation in renal proximal tubule cells: II. Roles of dihydropyridine-sensitive and-insensitive Ca2+ entry pathways

Summary The Ca^2– entry pathways in the basolateral plasma membrane of the isolated, nonperfused proximal straight tubule (PST) of rabbit kidney were investigated using fura-2 fluorescence microscopy. Under isotonic conditions, reduction of bath [Ca^2–] from 1 mM to 1 M caused intracellular free calcium concentration ([Ca²⁺]_i) to fall close to zero. Treatment with 10 M verapamil, a calcium channel blocker, had a similar effect. Treatment with verapamil or low Ca²⁺ also induced fluctuations in cell volume. However, isotonic treatment with 10 M nifedipine, a dihydropyridine (DHP)-type calcium channel blocker, did not affect [Ca²⁺]_i or cell volume, indicating that the endogenous Ca²⁺ entry pathway is verapamil-sensitive but DHP-insensitive. When cells were exposed to hypotonic solutions in the presence of 1 mM Ca²⁺, they swelled and underwent normal RVD while [Ca²⁺]_i increased transiently to a peak before decreasing to a late phase plateau level above the baseline level (see McCarty, N.A., O'Neil, R.G. 1991.J. Membrane Biol. 123:149–160). When cells were swollen in the presence of verapamil or low bath [Ca²⁺], RVD was abolished and [Ca²⁺]_i fell well below the baseline during the late phase response. In contrast, when cells were swollen in the presence of nifedipine, RVD and the late phase rise in [Ca²⁺]_i were abolished, but [Ca²⁺]_i did not fall below the baseline level in the late phase, indicating that nifedipine inhibited the swelling-induced Ca²⁺ entry but that Ca²⁺ entry by another pathway was undisturbed. It was concluded that PST cells are characterized by two Ca²⁺ permeability pathways in the basolateral membrane. Under both isotonic and hypotonic conditions, Ca²⁺ entry occurs at a slow rate via a verapamil-sensitive, DHP-insensitive baseline Ca²⁺ entry pathway. Cell swelling activates a separate DHP-sensitive, verapamil-sensitive Ca²⁺ entry pathway, which is responsible for the supply of Ca ions to the Ca²⁺-dependent mechanism by which cell volume regulation is achieved.     

Calcium and the regulation of mammalian ciliary beating

Summary This report summarizes our recent work on the role of intracellular Ca²⁺ ([Ca²⁺]_i) in regulating mammalian ciliary beat frequency (CBF). CBF from a single ovine cilium and [Ca²⁺]_i from the same cell were measured by digital video phase contrast microscopy and fura-2 ratiometric imaging video microscopy, respectively. Cells were stimulated with two exposures to 10 M acetylcholine (ACh). CBF was recorded during the first and [Ca²⁺]_i during the second stimulation. ACh increased [Ca²⁺]_i and CBF transiently with indistinguishable kinetics and, early in culture, even induced [Ca²⁺]_i oscillations and ciliary frequency modulations with the same peak-to-peak time interval. Cells treated with 1 M thapsigargin, an inhibitor of the endoplasmic-reticulum Ca²⁺-ATPase, showed transient [Ca²⁺]_i and CBF increases, again with similar kinetics, which often remained at an elevated plateau. Application of ACh to cells pretreated with thapsigargin produced decreases in both [Ca²⁺]_i and CBF. Finally, changing extracellular Ca²⁺-concentrations induced corresponding changes in [Ca²⁺]_i that were associated with kinetically similar CBF changes. These data strongly suggested that [Ca²⁺]_i is a critical signal to regulate CBF in mammalian tracheal epithelial cells. In an initial effort to provide constraints on the number and type of reactions that link changes in [Ca²⁺]_i to changes in CBF, simultaneous recordings of both signals from a single cell were analyzed. Such recordings provided higher resolution of the kinetic responses of CBF and [Ca²⁺]_i to ACh as well as they allowed direct assessment of the coupling between [Ca²⁺]_i and CBF. Simultaneous measurements revealed that [Ca²⁺]_i and CBF were perfectly correlated within the CBF measurement time resolution, except for the period of the fastest changes in both signals during the initial ACh exposure. There, changes in CBF lagged the changes in [Ca²⁺]_i by 1–3 ciliary beat cycles (ca. 150–450 ms).     

Sodium-calcium exchange in mammalian heart: current-voltage relation and intracellular calcium concentration

Membrane currents and changes in intracellular calcium ion concentration ([Ca²⁺]_i) have been recorded that can be attributed to the operation of an electrogenic, voltage-dependent sodium-calcium (Na-Ca) exchanger in mammalian heart cells. Single guinea-pig ventricular myocytes under voltage clamp were perfused internally with the fluorescent Ca²⁺-indicator, fura-2, and changes in [Ca²⁺]_i and membrane current that resulted from Na-Ca exchange were isolated through the use of various organic channel blockers (verapamil, TTX), impermeant ions (Cs⁺, Ni²⁺), and inhibitors of sarcoplasmic reticulum (ryanodine). The I-V relation of Na-Ca exchange was obtained from the Ni²⁺-sensitive current elicited by ramp repolarization from +90 mV to –80 mV. Ramps were sufficiently rapid that little change in [Ca²⁺]_i occured during the ramp. The (constant) [Ca²⁺]_i during the ramp was varied over the range 100 nM to 1000 nM by varying the amplitude and duration of a pre-pulse to the ramp. The reversal potential of the Ni²⁺-sensitive ramp current varied linearly with 1n([Ca²⁺])_i. The I-V relations at different [Ca²⁺]_i over the range –60 mV to +140 mV were in reasonable accord with the predictions of a simple, simultaneous scheme of Na-Ca exchange, on the basis that only [Ca²⁺]_i had changed. The relationship between [Ca²⁺]_i and current at a constant membrane voltage was also in accord with this scheme. We suggest that Ca²⁺-fluxes through the exchanger during the cardiac action potential can be understood quantitatively by considering the binding of Ca²⁺ to the exchanger during the [Ca²⁺]_i-transient and the effects of membrane voltage on the exchanger.     

The role of cytosolic free calcium in the regulation of pyruvate dehydrogenase in synaptosomes

Calcium homeostasis and mitochondrial oxidative metabolism interact closely in brain and both processes are impaired during hypoxia. Since the regulation of the pyruvate dehydrogenase complex (PDHC) may link these two processes, the relation of cytosolic free calcium ([Ca²⁺]_i) to the activation state of PDHC (PDH_a) was assessed in isolated nerve terminals (i.e. synaptosomes) under conditions that alter [Ca²⁺]_i. K⁺ depolarization elevated [Ca²⁺]_i and PDH_a and both responses required external calcium. Treatment with KCN, an in vitro model of hypoxia decreased ATP and elevated [Ca²⁺]_i and PDH_a. Furthermore, in the presence of KCN, PDH_a became more sensitive to K⁺ depolarization as indicated by larger changes in PDH_a than in [Ca²⁺]_i. The calcium ionophore Br-A23187 elevated [Ca²⁺]_i, but did not affect PDH_a. K⁺ depolarization elevated [Ca²⁺]_i and PDH_a even if [Ca²⁺]_i was elevated by prior addition of ionophore or KCN. Previous in vivo studies by others show that PDH_a is altered during and after ischemia. The current in vitro results suggest that hypoxia, only one component of ischemia, is sufficient to increase PDH_a. These data also further support the notion that PDH_a is regulated by [Ca²⁺]_i as well as by other factors such as ATP. Our results are consistent with the concept that PDH_a in nerve endings may be affected by [Ca²⁺]_i and that these two processes are clearly linked.Abbreviations (PDH_a) Activation state of the pyruvate dehydrogenase complex - ([Ca²⁺]_i) cytosolic free calcium concentrations - (MOPS) 3(N-morpholino)propanesulfonic acid - (fura-2AM) fura-2 acetoxymethyl ester - (AABS) p-(p-aminophenylazo)benzene sulfonic acid - (PDHC) pyruvate dehydrogenase complex - (TES) N-tris{[hydroxymethyl]methyl}-2-amino-ethanesulfonic acid - (SNK-test) Student-Newman-Keuls test     

Effect of Celecoxib on Ca2+ Fluxes and Proliferation in MDCK Renal Tubular Cells

The effect of celecoxib on renal tubular cells is largely unexplored. In Madin Darby canine kidney (MDCK) cells, the effect of celecoxib on intracellular Ca^{2 +} concentration ([Ca^{2 +}]_i) and proliferation was examined by using the Ca^{2 +}-sensitive fluorescent dye fura-2 and the viability detecting fluorescent dye tetrazolium, respectively. Celecoxib (≥1 μ M) caused an increase of [Ca^{2 +}]_i in a concentration-dependent manner. Celecoxib-induced [Ca^{2 +}]_i increase was partly reduced by removal of extracellular Ca^{2 +}. Celecoxib-induced Ca^{2 +} influx was independently suggested by Mn^{2 +} influx-induced fura-2 fluorescence quench. In Ca^{2 +}-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca^{2 +}-ATPase, caused a monophasic [Ca^{2 +}]_i increase, after which celecoxib only induced a tiny [Ca^{2 +}]_iincrease; conversely, pretreatment with celecoxib completely inhibited thapsigargin-induced [Ca^{2 +}]_i increases. U73122, an inhibitor of phospholipase C, abolished ATP (but not celecoxib)-induced [Ca^{2 +}]_i increases. Overnight incubation with 1 or 10 μ M celecoxib decreased cell viability by 80% and 100%, respectively. These data indicate that celecoxib evokes a [Ca^{2 +}]_i increase in renal tubular cells by stimulating both extracellular Ca^{2 +} influx and intracellular Ca^{2 +} release and is highly toxic to renal tubular cells in vitro.     

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 .     

Influence of endogenous and exogenous RNases on the variation of pollen cytosolic-free Ca2+ in Pyrus serotina Rehd

The objective of this study was to examine whether S-RNase plays a specific role in the pre-germinated Pyrus pollen. Effects of exogenous RNase and endogenous S-RNase on concentration of cytosolic-free calcium ([Ca²⁺]_i) variation of pre-germinated Pyrus pollen were studied. [Ca²⁺]_i variation caused by different RNases were complex. In 1 h after being cultured, exogenous RNase, RNase T₁ and RNase A, and endogenous incompatible ‘Hohsui’ RNase promoted the [Ca²⁺]_i of ‘Hohsui’ pollen. Acid proteins of ‘Hohsui’ had no remarkable influence on the [Ca²⁺]_i of self-pollen. Endogenous compatible ‘Kohsui’ RNase reduced the [Ca²⁺]_i of ‘Hohsui’ pollen, but compatible ‘Hohsui’ RNase can stimulate the [Ca²⁺]_i of ‘Kohsui’ pollen. RNase T₁, RNase A and incompatible ‘Kohsui’ S-RNase can also make ‘Kohsui’ pollen [Ca²⁺]_i increase. Different from ‘Hohsui’ pollen, acid proteins of ‘Hohsui’ pull down the ‘Kohsui’ pollen [Ca²⁺]_i remarkably. Conclusion can be made that during the prophase of pollen germination, endogenous S-RNase has no specific effect on pollen [Ca²⁺]_i changes.     

The effect of hyposmotic and isosmotic cell swelling on the intracellular [Ca2+] in lactating rat mammary acinar cells

The effect of hyposmotic and isosmotic cell swelling on the free intracellular calcium concentration ([Ca²⁺]_i) in rat mammary acinar cells has been examined using the fura-2 dye technique. A hyposmotic shock (40% reduction) increased the [Ca²⁺]_i in rat mammary acinar cells in a fashion which was transient; the [Ca²⁺]_i returned to a value similar to that found under isomotic conditions within 180 sec. The increase in the [Ca²⁺]_i was dependent upon the extent of the osmotic shock. The hyposmotically-activated increase in the [Ca²⁺]_i could not be attributed to a reduction in extracellular Na⁺ or a change in the ionic strength of the incubation medium. Thapsigargin (1 M) enhanced the hyposmotically-activated increase in the [Ca²⁺]_i. Isosmotic swelling of rat mammary acinar cells, using urea, had no significant effect on the [Ca²⁺]_i. Similarly, a hyperosmotic shock did not affect the [Ca²⁺]_i in rat mammary acinar cells. It appears that the effect of cell swelling on the [Ca²⁺]_i in rat mammary acinar cells depends on how the cells are swollen (hyposmotic vs. isosmotic). This finding may have important physiological implications given that it is predicted that mammary cell volume will change in vivo under isomotic conditions.     

Effects of elevated cytosolic calcium on ACh-induced swine tracheal smooth muscle contraction

Increased intracellular calcium concentration ([Ca²⁺]_i) is required for smooth muscle contraction. In tracheal and other tonic smooth muscles, contraction and elevated [Ca²⁺]_i are maintained as long as an agonist is present. To evaluate the physiological role of steady-state increases in Ca²⁺ on tension maintenance, [Ca²⁺]_i was elevated using ionomycin, a Ca²⁺ ionophore or charybdotoxin, a large-conductance calcium-activated potassium channel (K_Ca) blocker prior to or during exposure of tracheal smooth muscle strips to Ach (10^–9 to 10^–4 M). Ionomycin (5 µM) in resting muscles induced increases in [Ca²⁺]_i to 500±230 nM and small increases in force of 2.6±2.3 N/cm². This tension is only 10% of the maximal tension induced by ACh. Charybdotoxin had no effect on [Ca²⁺]_i or tension in resting muscle. After pretreatment of muscle with ionomycin, the concentration-response relationship for ACh-induced changes in tension shifted to the left (EC₅₀=0.07±0.05 µM ionomycin; 0.17±0.07 µM, control, p<0.05). When applied to the muscles during steady-state responses to submaximal concentrations of ACh, both ionomycin and charybdotoxin induced further increases in tension. The same magnitude increase in tension occurs after ionomycin and charybdotoxin treatment, even though the increase in [Ca²⁺]_i induced by charybdotoxin is much smaller than that induced by ionomycin. We conclude that the resting muscle is much less sensitive to elevation of [Ca²⁺]_i when compared to muscles stimulated with ACh. Steady-state [Ca²⁺]_i limits tension development induced by submaximal concentrations of ACh. The activity of K_Ca moderates the response of the muscle to ACh at concentrations less than 1 µM.     

Uncoupling secretion and tip growth in lily pollen tubes: evidence for the role of calcium in exocytosis

Cytoplasmic calcium concentration ([Ca²⁺]_i) and extracellular calcium (Ca²⁺_o) influx has been studied in pollen tubes of Lilium longliflorum in which the processes of cell elongation and exocytosis have been uncoupled by use of Yariv phenylglycoside ((β-D-Glc)₃). Growing pollen tubes were pressure injected with the ratio dye fura-2 dextran and imaged after application of (β-D-Glc)₃, which binds arabinogalactan proteins (AGPs). Application of (β-D-Glc)₃ inhibited growth but not secretion. Ratiometric imaging of [Ca²⁺]_i revealed an initial spread in the locus of the apical [Ca²⁺]_i gradient and substantial elevations in basal [Ca²⁺]_i followed by the establishment of new regions of elevated [Ca²⁺]_i on the flanks of the tip region. Areas of elevated [Ca²⁺]_i corresponded to sites of pronounced exocytosis, as evidenced by the formation of wall ingrowths adjacent to the plasma membrane. Ca²⁺_o influx at the tip of (β-D-Glc)₃-treated pollen tubes was not significantly different to that of control tubes. Taken together these data indicate that regions of elevated [Ca²⁺]_i, probably resulting from Ca²⁺_o influx across the plasma membrane, stimulate exocytosis in pollen tubes independent of cell elongation.     

Outgrowth morphology and intracellular calcium of crustacean neurons displaying distinct morphologies in primary culture

Peptidesecreting neurons from crustacean X-organ regenerating in defined culture possess different ionic current profiles correlated with two distinct morphological types, veiling and branching; voltage-dependent Ca²⁺ current is prominent in neurons consistently extending large veils, but is small in neurons that repetitively branch. Intracellular free calcium ([Ca²⁺]_i) have been implicated in regulation of neurite outgrowth underlying the establishment of distinct morphologies. Here, basal [Ca²⁺]_i was measured by fura-2 fluorescence ratio imaging from these morphologically distinct neurons and compared. Both morphological tapes can extend out processes over a [Ca²⁺]_i range (approximately 50 to 300 nM) that is much greater than that reported for neurons of other phyla. Application of high k⁺ saline led to increases in [Ca²⁺]_i in soma, neurite, and lamellipodium of veiling neurons. Increase were great for veiling than branching neurons. These observations were consistent with the previous voltage clamp data for calcium currents. Media altered to perturb [Ca²⁺]_i were used to assess the role of [Ca²⁺]_i in veiling or branching outgrowth programs. Outgrowth of veiling cells was arrested addition of 100 μMCD²⁺, a calcium channel blocker. Outgrowth resumed following brief exposures to Cd²⁺. Branching neurons were unaffected by Cd²⁺. Cd²⁺ at lower levels (10 μM) had no effect on outgrowth of either neuronal type, whereas at higher levels (1 mM), outgrowth of both types was arrested. Reduction of extracellular sodium to 0.001 of normal concentration stopped veiling outgrowth, but branching outgrowth continued, although it was less robust. Addition of tetrodotoxin (1 μM) did not alter outgrowth of either neuronal type relative to controls. Thus, peptidergic neurons of differing intrinsic morphologies maintain similar basal [Ca²⁺]_i levels under identical culture conditions, yet show differing sensitivities to manipulations influencing [Ca²⁺]_i with respect to regenerative outgrowth, but not its form. 1994 John Wiley & Sons, Inc.     

The steady-state force–Ca<Superscript>2+</Superscript> relationship in intact lobster (<Emphasis Type="Italic">Homarus americanus</Emphasis>) cardiac muscle

The heart of the decapod crustacean is activated by regular impulse bursts from the cardiac ganglion. The cardiac pump function depends on ganglionic burst frequency, burst duration, and burst impulse frequency. Here, we activated isolated lobster cardiac ostial muscle (Orbicularis ostii muscle, OOM) by stimulus trains in vitro in order to characterize the response of the contractile apparatus to [Ca²⁺]_i . We employed stimulus trains that generate a steady state between the [Ca²⁺]_i and force in order to estimate the Ca²⁺ sensitivity of myofilaments. Force and [Ca²⁺]_i transients were simultaneously recorded using a silicon strain gauge and the fluorescence of iontophoretically microinjected fura-2 salt. We examined the effects of tetanus duration (TD), the interval between trains, and 6 M cyclopiazonic acid, an inhibitor of the SR Ca²⁺ pump, on the steady-state force–[Ca²⁺]_i relationship. The instantaneous force–[Ca²⁺]_i relationships appeared sigmoidal (EC₅₀ and Hill coefficient, 98.8±32.7 nM and 2.47±0.20, mean ± SD, respectively), as did the curves superimposed after 500 ms following the start of stimulation, indicating that the force–[Ca²⁺]_i relationship had reached a steady state at that time. Also, the maximum activated force (F_max) was estimated using the steady-state force–[Ca²⁺]_i relationship. Prolonged stimulus trains, decreasing the interval between recurrent trains from 5 to 2.5 s, and cyclopiazonic acid each increased the measured EC₅₀ without changing F_max. The EC₅₀ correlated strongly with averaged [Ca²⁺]_i over time. We conclude that the steady-state force–[Ca²⁺]_i relationships in the OOM indicate cooperation between force generation and Ca²⁺ binding by the myofilaments. Our data also suggest the existence of a novel Ca²⁺-dependent mechanism which reduces Ca²⁺ sensitivity and accelerates relaxation of lobster cardiac muscle myofilaments.Communicated by L.C.-H. Wang     

Ratio-imaging of Ca2+i in the self-incompatibility response in pollen tubes of Papaver rhoeas

The data presented here describe ratio-imaging of in intracellular free calcium (Ca²⁺_i) during the self-incompatibility (SI) response in pollen. Use of the ratiometric indicator, fura-2 dextran, in pollen tubes of Papaver rhoeas has provided new, detailed information about the spatial-temporal alterations in Ca²⁺_i, and has permitted calibration of alterations in the concentration of intracellular free calcium ([Ca²⁺]_i) in the SI response. Ratio images demonstrate that, like other pollen tubes, normally growing P. rhoeas pollen tubes exhibit a tip-focused gradient of Ca^2+bf_i, with levels reaching 1–2 μM at the extreme apex of the pollen tube. Non-growing pollen tubes did not exhibit this tip-focused gradient. Basal levels of Ca²⁺_i in the shank of the pollen tube were fairly consistent and had a mean value of 210 nM, with low-level fluctuations +/? 50 nM observed. Challenge with incompatible S proteins resulted in S-specific, rapid and dramatic alterations in [Ca²⁺]_i within a few seconds of challenge. Increases in [Ca²⁺]_i were visualized in the subapical/shank regions of the pollen tube and alterations in [Ca²⁺]_i in this region subsequently increased for several minutes, reaching> 1.5 μM. At the pollen tube tip, a diminution of the tip-focused gradient was observed, which following some fluctuation, was reduced to basal levels within ～1 min. Our data suggest that some of these alterations in [Ca²⁺]_i might be interpreted as a calcium wave, as the changes are not global. Although the increases in [Ca²⁺]_i in the subapical/shank region are very rapid, because tip [Ca²⁺]_i oscillates during normal growth, it is difficult to ascertain whether the increases in the shank of the pollen tube precede the decreases in [Ca²⁺]_i at the pollen tube tip.