Effects of levobupivacaine and bupivacaine on intracellular calcium signaling in cultured rat dorsal root ganglion neurons

Bupivacaine and levobupivacaine have been shown to be effective in the treatment of pain as local anesthetics, although the mechanisms mediating their antinociceptive actions are still not well understood. The aim of this study was to investigate the effects of bupivacaine and levobupivacaine on intracellular calcium ([Ca²⁺]_i) signaling in cultured rat dorsal root ganglion (DRG) neurons. DRG neuronal cultures loaded with 5?μM Fura-2/AM and [Ca²⁺]_i transients for stimulation with 30?mM KCl (Hi K⁺) were assessed by using fluorescent ratiometry. DRGs were excited at 340 and 380?nm, emission was recorded at 510?nm, and responses were determined from the change in the 340/380 ratio (basal-peak) for individual DRG neurons. Data were analyzed by using Student’s t-test. Levobupivacaine and bupivacaine attenuated the KCl-evoked [Ca²⁺]_i transients in a reversible manner. [Ca²⁺]_i increase evoked by Hi K⁺ was significantly reduced to 99.9?±?5.1% (n?=?18) and 62.5?±?4.2% (n?=?15, P?<?0.05) after the application of 5 and 50?µM levobupivacaine, respectively. Bupivacaine also inhibited Hi K⁺-induced [Ca²⁺]_i responses, reduced to 98.7?±?4.8% (n?=?10) and 69.5?±?4.5% (n?=?9, P?<?0.05) inhibition of fluorescence ratio values of Hi K⁺-induced responses at 5 and 50?μM, respectively. Our results indicate that bupivacaine and levobupivacaine, with no significant differences between both agents, attenuated KCl-evoked calcium transients in a reversible manner. The inhibition of calcium signals in DRG neurons by levobupivacaine and bupivacaine might contribute to the antinociceptive effects of these local anesthetics.     

Changes in Calcium/Calmodulin Level and Mitochondrial Membrane Potential in Cochlear Neurons of Newborn Mice Infected with Murine Cytomegalovirus

We sought to elucidate the pathogenesis of hearing loss in newborns due to congenital cytomegalovirus. We used the model of murine cytomegalovirus (MCMV) infection and evaluated concentrations of free calcium, calmodulin levels, and mitochondrial membrane potential in cochlear neurons of infected newborn mice. MCMV infection was established by intracranial inoculation of newborn mice with viral suspension (20 μl of MCMV TCID₅₀—10⁴ IU/0.1 ml); the mice in control group were injected 0.9 % NaCl. Concentration of intracellular free calcium concentration ([Ca²⁺] _i ), mitochondrial membrane potential, and the mRNA level of calmodulin (CaM) in the cochlear neurons were evaluated, when the mice were 1 month old. Compared with control group, intracellular [Ca²⁺] _i and CaM mRNA levels significantly (p < 0.05; both comparisons) increased, while the mitochondrial membrane potential significantly (p < 0.05) decreased in the MCMV-infected group. In conclusion, alteration of [Ca²⁺] _i and CaM levels and mitochondrial membrane potentials in cochlear neurons may be the pathological basis of sensorineural hearing loss associated with MCMV infection.     

Oxytocin induced a biphasic increase in the intracellular Ca2+ concentration of porcine myometrial cells: Participation of a pertussis toxin—insensitive G-protein,inositol 1,4,5-trisphosphate—sensitive Ca2+ pool,and Ca2+ channels

This study investigated the underlying mechanisms of oxytocin (OT)-induced increases in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in acutely dispersed myometrial cells from prepartum sows. A dosedependent increase in [Ca²⁺]_i was induced by OT (0.1 nM to 1 μM) in the presence and absence of extracellular Ca²⁺ ([Ca²⁺]_e). [Ca²⁺]_i was elevated by OT in a biphasic pattern, with a spike followed by a sustained plateau in the presence of [Ca²⁺]_e. However, in the absence of [Ca²⁺]_e, the [Ca²⁺]_i response to OT became monophasic with a lower amplitude and no plateau, and this monophasic increase was abolished by pretreatment with ionomycin, a Ca²⁺ ionophore. Administration of OT (1 μM) for 15 sec increased inositol 1,4,5-trisphosphate (IP₃) formation by 61%. Pretreatment with pertussis toxin (PTX, 1 μg/ml) for 2 hr failed to alter the OT-induced increase in [Ca²⁺]_i and IP₃ formation. U-73122 (30 nM to 3 μM), a phospholipase C (PLC) inhibitor, depressed the rise in [Ca²⁺]_i by OT dose dependently. U-73122 (3 μM) also abolished the OT-induced IP₃ formation. Thapsigargin (2 μM), an inhibitor of Ca²⁺-ATPase in the endoplasmic reticulum, did not increase [Ca²⁺]_i. However, it did time-dependently inhibit the OT-induced increase in [Ca²⁺]_i. Nimodipine (1 μM), a Voltage-dependent Ca²⁺ channel (VDCC) blocker, inhibited the OT-induced plateau by 26%. La³⁺ (1 μM), a nonspecific Ca²⁺ channel blocker, abrogated the OT-induced plateau. In whole-cell patch-clamp studies used to evaluate VDCC activities, OT (0.1 μM) increased Ca²⁺ Current (I_ca) by 40% with no apparent changes in the current-voltage relationship. The OT-induced increase in I_ca reached the maximum in 5 min, and the increase was abolished by nimodipine (1 μM). These results suggested that (1) activation of OT receptors in porcine myometrium evokes a cascade in the PTX-insensitive G-protein–PLC-IP₃ signal transduction, resulting in an increase in [Ca²⁺]_i; (2) the OT-induced increase in [Ca²⁺]_i is characterized by a biphasic pattern, in which the spike is predominately contributed by the intracellular Ca²⁺ release from the IP₃-sensitive pool, and to a lesser extent by Ca²⁺ influx, whereas the plateau is from increased Ca²⁺ influx; and (3) the influx is via VDCC and receptor-operated Ca²⁺ channels. © 1995 Wiley-Liss, Inc.     

Effects of apelin-13 in mice model of experimental pain and peripheral nociceptive signaling in rat sensory neurons

Objective: Apelin-13 is an endogenous peptide with potential analgesic action, although the sites of its analgesic effects remain uncertain and the results are even controversial with regard to its pain modulating action. This study evaluated the possible pain-modulating action of peripherally administered apelin-13 using heat-induced, withdrawal latency to the thermal stimuli, acute pain model in mice. Involvement of peripheral mechanisms was tested, by using the intracellular calcium concentrations as a key signal for nociceptive transmission, in 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 acetoxymethyl ester (1?µM). Intracellular calcium responses in individual DRG neurons were quantified by ratiometric calcium imaging technique. Results: Peripheral injection of a single dose of apelin-13 (100?mg/kg and 300?mg/kg) significantly decreases the latency to painful stimuli in a dose and time-dependent manner (p?<?0.01, p?<?0.05, respectively, n?=?8 each). Apelin-13 (0.1?µM and 1?µM) did not produce a significant effect on cytoplasmic Ca²⁺ ([Ca²⁺]_i) responses, evoked by membrane depolarization, in cultured rat DRG neurons. Conclusion: Together these results indicate that apelin-13 can cause increased pain sensitivity after peripheral administration, but this effect does not involve calcium mediated signaling in primary sensory neurons.     

Vanadate increases cytosolic free calcium in rat aortic smooth muscle cells

Although several studies have shown that vanadate evokes vasoconstriction whether it elevates cytosolic free calcium, [Ca²⁺]_i, in vascular smooth muscle (VSM) cells has not been investigated. The present study shows that acute additions of low concentrations of vanadate (10–200) to cultured aortic smooth muscle cells (ASMC) produced a rapid and a concentrationdependent increase in [Ca²⁺]_i with an EC₅₀ (mean ± SEM) value of 42 ± 11 μM. Inclusion of vanadate (200 μM) led to a significant increase (p < 0.05) in the peak [Ca²⁺]_i level to 190 ± 23 nM from a basal level of 102 ± 2 nM. At concentrations > 200 μM, vanadate caused quenching of fura-2 fluorescence. For example, addition of 1 mM vanadate led to an apparent decrease in fluorescence by about 50 % (due to a quenching effect), followed by a transient rise. H₂O₂, which is used in the preparation of peroxide forms of vanadate, pervanadate (PV), also produced a rise in [Ca²⁺]_i. These data suggest that vanadate promotes vascular tone by elevating [Ca²⁺]_i in ASMC. However, [Ca²⁺]_i measurements made with higher concentrations of vanadate and PV, using the fura-2 method, must be interpreted with caution.     

Arachidonic acid activates release of calcium ions from reticulum via ryanodine receptor channels in C2C12 skeletal myotubes

Arachidonic acid causes an increase in free cytoplasmic calcium concentration ([Ca²⁺]_i) in differentiated skeletal multinucleated myotubes C2C12 and does not induce calcium response in C2C12 myoblasts. The same reaction of myotubes to arachidonic acid is observed in Ca²⁺-free medium. This indicates that arachidonic acid induces release of calcium ions from intracellular stores. The blocker of ryanodine receptor channels of sarcoplasmic reticulum dantrolene (20 μM) inhibits this effect by 68.7 ± 6.3% (p < 0.001). The inhibitor of two-pore calcium channels of endolysosomal vesicles trans-NED19 (10 μM) decreases the response to arachidonic acid by 35.8 ± 5.4% (p < 0.05). The phospholipase C inhibitor U73122 (10 μM) has no effect. These data indicate the involvement of ryanodine receptor calcium channels of sarcoplasmic reticulum in [Ca²⁺]_i elevation in skeletal myotubes caused by arachidonic acid and possible participation of two-pore calcium channels from endolysosomal vesicles in this process.     

Increased intracellular calcium level and impaired nutrient absorption are important pathogenicity traits in the chicken intestinal epithelium during <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> colonization

Although a high number of chickens carry Campylobacter jejuni, the mechanistic action of colonization in the intestine is still poorly understood. The current study was therefore designed to investigate the effects of C. jejuni on glucose uptake, amino acids availability in digesta, and intracellular calcium [Ca²⁺]_i signaling in the intestines of broiler chickens. For this, we compared: control birds (n?=?60) and C. jejuni-infected birds (n?=?60; infected orally with 1?×?10⁸ CFU of C. jejuni NCTC 12744 at 14 days of age). Our results showed that glucose uptake was reduced due to C. jejuni infection in isolated jejunal, but not in cecal mucosa at 14 days postinfection (dpi). The decrease in intestinal glucose absorption coincided with a decrease in body weight gain during the 2-week post-infectious period. A reduction in the amount of the amino acids (serine, proline, valine, leucine, phenylalanine, arginine, histidine, and lysine) in ileal digesta of the infected birds at 2 and/or 7 dpi was found, indicating that Campylobacter utilizes amino acids as a carbon source for their multiplication. Applying the cell-permeable Ca²⁺ indicator Fluo-4 and two-photon microscopy, we revealed that [Ca²⁺]_i was increased in the jejunal and cecal mucosa of infected birds. The muscarinic agonist carbachol induced an increase in [Ca²⁺]_i in jejunum and cecum mucosa of control chickens, a response absent in the mucosa of infected chickens, demonstrating that the modulation of [Ca²⁺]_i by Campylobacter might be involved in facilitating the necessary cytoskeletal rearrangements that occur during the bacterial invasion of epithelial cells. In conclusion, this study demonstrates the multifaceted interactions of C. jejuni with the gastrointestinal mucosa of broiler chickens. For the first time, it could be shown that a Campylobacter infection could interfere with intracellular Ca²⁺ signaling and nutrient absorption in the small intestine with consequences on intestinal function, performance, and Campylobacter colonization. Altogether, these findings indicate that Campylobacter is not entirely a commensal and can be recognized as an important factor contributing to an impaired chicken gut health.     

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.     

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)     

Thrombin and histamine activate phospholipase C in human endothelial cells via a phorbol ester-sensitive pathway

The effects of phorbol esters and synthetic diglycerides on thrombin- and histamine-stimulated increases in inositol trisphosphate (IP₃) and cytosolic free calcium ([Ca²⁺]_i) were studied in cultured human umbilical vein endothelial cells (HEC). Thrombin (0.003–3.0 U/ml) and histamine (10^?7–10^?4 M) induced rapid increases in [Ca²⁺]_i in suspended cells as monitored with the fluorescent calcium indicator fura-2. In [³H]myoinositol-labeled cells, both thrombin (3 U/ml)- and histamine (10^?4 M)-induced IP₃ increases (195% ± 6% and 98% ± 4%, respectively) occurred in less than 15 sec and were temporally correlated with [Ca²⁺]_i increases. Brief incubations (5–60 min) with different protein kinase C activators [4-β-phorbol 12-myristate 13-acetate (1–100 nM), mezerein (100 nM), and sn-1,2 dioctanoylglycerol (0.1–10 μM)] attenuated agonist-induced increases in [Ca²⁺]_i. These compounds also inhibited thrombin- and histaminestimulated IP₃ formation, thus suggesting a tight coupling between phospholipase C activation and calcium flux in cultured HEC. Overall, these observations suggest that the pathway linking receptors to phospholipase C stimulation in human endothelial cells is sensitive to protein kinase C activation.     

Bauhinia bauhinioides cruzipain inhibitor reduces endothelial proliferation and induces an increase of the intracellular Ca2+ concentration

Proteinase inhibitors, isolated from different types of Bauhinia, have an effect on apoptosis, angiogenesis and inflammation. The Bauhinia bauhinioides cruzipain inhibitor (BbCI) is a Kunitz-type inhibitor and inactivates the cysteine proteinases cruzipain and cruzain from Trypanosoma cruzi. Cruzipain and tissue kallikrein have similar biochemical properties, e.g. the proteolytic cleavage of the kininogen precursor of lys-bradykinin. Tissue kallikrein stimulation in endothelial cells causes migration and capillary tube formation. The aim of this study was to examine whether the antiproliferative effect of BbCI is dependent on changes of the intracellular calcium concentration and membrane hyperpolarization. Endothelial cells were isolated from human umbilical cord veins (HUVEC). For proliferation experiments, HUVEC were incubated with BbCI (10–100 μmol/L) for 48 h. The proliferation was detected by cell counting with a Neubauer chamber. The effect of BbCI (10–100 μM) on the membrane potential was measured with the fluorescence dye DiBAC₄(3) and the effect on [Ca⁺²] _i with the fluorescence probe Fluo-3 AM. The change of the fluorescence intensity was determined with a GENios plate reader (Tecan). The experiments showed that BbCI (10–100 μmol/L) reduces the endothelial cell proliferation significantly in a concentration-dependent manner with a maximum effect at 100 μmol/L (35.1?±?1.8% as compared to control (p?≤?0.05; n?=?45)). As compared to the control, the addition of BbCI (100 μmol/L) caused a significant increase of systolic Ca²⁺ of 28.4?±?5.0% after 30 min incubation. HUVEC treatment with BbCI (100 μmol/L) showed a weak but significant decrease of the membrane potential of 9.5?±?0.9% as compared to control (p?≤?0.05; n?=?80). BbCI influenced significantly the endothelial proliferation, the intracellular Ca²⁺ concentration and the membrane potential.     

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.     

Physiology of spontaneous [Ca2+]i oscillations in the isolated vasopressin and oxytocin neurones of the rat supraoptic nucleus

The magnocellular vasopressin (AVP) and oxytocin (OT) neurones exhibit specific electrophysiological behaviour, synthesise AVP and OT peptides and secrete them into the neurohypophysial system in response to various physiological stimulations. The activity of these neurones is regulated by the very same peptides released either somato-dendritically or when applied to supraoptic nucleus (SON) preparations in vitro. The AVP and OT, secreted somato-dendritically (i.e. in the SON proper) act through specific autoreceptors, induce distinct Ca²⁺ signals and regulate cellular events. Here, we demonstrate that about 70% of freshly isolated individual SON neurones from the adult non-transgenic or transgenic rats bearing AVP (AVP-eGFP) or OT (OT-mRFP1) markers, produce distinct spontaneous [Ca²⁺]_i oscillations. In the neurones identified (through specific fluorescence), about 80% of AVP neurones and about 60% of OT neurones exhibited these oscillations. Exposure to AVP triggered [Ca²⁺]_i oscillations in silent AVP neurones, or modified the oscillatory pattern in spontaneously active cells. Hyper- and hypo-osmotic stimuli (325 or 275 mOsmol/l) respectively intensified or inhibited spontaneous [Ca²⁺]_i dynamics. In rats dehydrated for 3 or 5 days almost 90% of neurones displayed spontaneous [Ca²⁺]_i oscillations. More than 80% of OT-mRFP1 neurones from 3 to 6-day-lactating rats were oscillatory vs. about 44% (OT-mRFP1 neurones) in virgins. Together, these results unveil for the first time that both AVP and OT neurones maintain, via Ca²⁺ signals, their remarkable intrinsic in vivo physiological properties in an isolated condition.     

Modulation of the epithelial calcium channel, ECaC, by intracellular Ca

We have studied the modulation by intracellular Ca²⁺of the epithelial Ca²⁺channel, ECaC, heterologously expressed in HEK 293 cells. Whole-cell and inside-out patch clamp current recordings were combined with FuraII-Ca²⁺measurements:1. Currents through ECaC were dramatically inhibited if Ca²⁺was the charge carrier. This inhibition was dependent on the extracellular Ca²⁺concentration and occurred also in cells buffered intracellularly with 10 mM BAPTA.2. Application of 30 mM [Ca²]_einduced in non-Ca²] buffered HEK 293 cells at −80 m V an increase in intracellular Ca²⁺([Ca²]_i) with a maximum rate of rise of 241 ±15nM/s (n= 18 cells) and a peak value of 891 ± 106 nM. The peak of the concomitant current with a density of 12.3 ± 2.6 pA/pF was closely correlated with the peak of the first-time derivative of the Ca²⁺transient, as expected if the Ca²⁺transient is due to influx of Ca²⁺. Consequently, no Ca²⁺] signal was observed in cells transfected with the Ca²⁺impermeable ECaC mutant, D542A, in which an aspartate in the pore region was neutralized.3. Increasing [Ca²⁺]_iby dialyzing the cell with pipette solutions containing various Ca²⁺] concentrations, all buffered with 10 mM BAPTA, inhibited currents through ECaC carried by either Na⁺or Ca²⁺] ions. Half maximal inhibition of Ca²⁺currents in the absence of monovalent cations occurred at 67 nM (n between 6 and 8), whereas Na⁺currents in the absence of Ca²⁺] and Mg²⁺were inhibited with an IC₅₀of 89 nM (n between 6 and 10). Currents through ECaC in the presence of 1 mM Ca²⁺and Na⁺, which are mainly carried by Ca²⁺, are inhibited by [Ca²]_iwith an IC₅₀of 82 nM (n between 6 and 8). Monovalent cation currents through the Ca²⁺impermeable D542A ECaC mutant were also inhibited by an elevation of [Ca²]_i(IC₅₀= 123 nM, n between 7 and 18).4. The sensitivity of ECaC currents in inside-out patches for [Ca²]_iwas slightly shifted to higher concentrations as compared with whole cell measurements. Half-maximal inhibition occurred at 169 nM if Na⁺was the charge carrier (n between 4 and 11) and 228 nM at 1 mM [Ca²]_e(n between 4 and 8).5. Recovery from inhibition upon washout of extracellular Ca²⁺(whole-cell configuration) or removal of Ca²⁺from the inner side of the channel (inside-out patches) was slow in both conditions. Half-maximal recovery was reached after 96 ± 34 s (n= 15) in whole-cell mode and after 135 ± 23 s (n= 17) in inside-out patches.6. We conclude that influx of Ca²⁺through ECaC and [Ca²]_iinduce feedback inhibition of ECaC currents, which is controlled by the concentration of Ca²⁺in a micro domain near the inner mouth of the channel. Slow recovery seems to depend on dissociation of Ca²⁺from an internal Ca²⁺binding site at ECaC.     

Involvement of Intracellular or Extracellular Calcium in Activation of Tyrosine Hydroxylase Gene Expression in PC12 Cells

Abstract: The relationship between elevations in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) by different mechanisms and tyrosine hydroxylase (TH) gene expression was examined. Depolarization by an elevated K⁺ concentration triggered rapid and sustained increases in [Ca²⁺]_i from a basal level of ～50 to 110–150 nM and three- to fourfold elevations in TH mRNA levels, requiring extracellular calcium but not inositol 1,4,5-trisphosphate (IP₃). On the other hand, bradykinin or thapsigargin, both of which induce release of intracellular calcium stores via IP₃ or inhibition of Ca²⁺-ATPase, rapidly elevated [Ca²⁺]_i to >200 nM and increased TH gene expression (three-to fivefold). Confocal imaging showed that the elevations in [Ca²⁺]_i in each case occurred throughout the cyto- and nucleoplasm. The initial rise in [Ca²⁺]_i due to either bradykinin or thapsigargin, which did not require extracellular calcium, was sufficient to initiate the events leading to increased TH expression. Consistent with this, the effects of bradykinin on TH expression were inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid or 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester which chelates or inhibits the release of intracellular calcium, respectively. Bradykinin required a rise in [Ca²⁺]_i for <10 min, as opposed to 10–30 min for depolarization to increase TH mRNA levels. These results demonstrate that although each of these treatments increased TH gene expression by raising [Ca²⁺]_i, there are important differences among them in terms of the magnitude of elevated [Ca²⁺]_i, requirements for extracellular calcium or release of intracellular calcium stores, and duration of elevated [Ca²⁺]_i, indicating the involvement of different calcium signaling pathways leading to regulation of TH gene expression.     

Oxidative Stress and Ca2+ Signals Involved on Cadmium-Induced Apoptosis in Rat Hepatocyte

Cadmium (Cd) is an important industrial and environmental pollutant. In animals, the liver is the major target organ of Cd toxicity. In this study, rat hepatocytes were treated with 2.5～10 μM Cd for various durations. Studies on nuclear morphology, chromatin condensation, and apoptotic cells demonstrate that Cd concentrations ranging within 2.5～10 μM induced apoptosis. The early-stage marker of apoptosis, i.e., decreased mitochondrial membrane potential, was observed as early as 1.5 h at 5 μM Cd. Significant (P?P?2+ concentration ([Ca²⁺] _i ) of Cd-exposed cells significantly increased (P?2+] _i may play an important role in apoptosis. Overall, these results showed that oxidative stress and Ca²⁺ signaling were critical mediators of the Cd-induced apoptosis of rat hepatocytes.     

The protective effect of class III antiarrhythmic agents against calcium overload in cultured myocytes

Calcium ions have been implicated in the mechanisms of ventricular arrhythmias. Impairment of intercellular coupling by calcium overload is considered to facilitate ventricular fibrillation (VF) and to sup-press its self termination. According to our hypothesis, any compound that decreases intracellular calcium concentration [Ca²⁺]_i during VF can serve as defibrillating drug. In this study, we examined the effect of d-sotalol and tedisamil on calcium overload in cultured, spontaneously beating rat cardiomyocytes. The changes of [Ca²⁺]_i were measured by indo-1 method and the intercellular synchronization by image analysis. The results showed that increase in [Ca²⁺]_o from 1.9 mM to 3.9 mM increased [Ca²⁺]_i from 100 nM to 320 nM and transformed the synchronized cell movement to an asynchronous one. Administration of 5 × 10⁻⁶ M d-sotalol or 10⁻⁶ M tedisamil, decreased the [Ca²⁺]_i to its basic level and restored the synchronized activity. In summary: Our results showed that increase in [Ca²⁺]_i known to caused inhibition of intercellular coupling, that could lead to arrhythmia and fibrillation while d-sotalol or tedisamil prevented this effect. These results support our hypothesis, that class III antiarrhythmic compounds with positive inotropic effect, increase intercellular synchronization, by decreasing free [Ca²⁺]_i, most probably by increasing the Ca²⁺ uptake by the sarcoplasmic reticulum, and therefore act as a defibrillating compound.     

Oxidative stress disruption of receptor-mediated calcium signaling mechanisms

Background

Oxidative stress increases the cytosolic content of calcium in the cytoplasm through a combination of effects on calcium pumps, exchangers, channels and binding proteins. In this study, oxidative stress was produced by exposure to tert-butyl hydroperoxide (tBHP); cell viability was assessed using a dye reduction assay; receptor binding was characterized using [³H]N-methylscopolamine ([³H]MS); and cytosolic and luminal endoplasmic reticulum (ER) calcium concentrations ([Ca²⁺]_i and [Ca²⁺]_L, respectively) were measured by fluorescent imaging.

Results

Activation of M3 muscarinic receptors induced a biphasic increase in [Ca²⁺]_i: an initial, inositol trisphosphate (IP3)-mediated release of Ca²⁺ from endoplasmic reticulum (ER) stores followed by a sustained phase of Ca²⁺ entry (i.e., store-operated calcium entry; SOCE). Under non-cytotoxic conditions, tBHP increased resting [Ca²⁺]_i; a 90 minute exposure to tBHP (0.5-10 mM ) increased [Ca²⁺]_i from 26 to up to 127 nM and decreased [Ca²⁺]_L by 55%. The initial response to 10 μM carbamylcholine was depressed by tBHP in the absence, but not the presence, of extracellular calcium. SOCE, however, was depressed in both the presence and absence of extracellular calcium. Acute exposure to tBHP did not block calcium influx through open SOCE channels. Activation of SOCE following thapsigargin-induced depletion of ER calcium was depressed by tBHP exposure. In calcium-free media, tBHP depressed both SOCE and the extent of thapsigargin-induced release of Ca²⁺ from the ER. M3 receptor binding parameters (ligand affinity, guanine nucleotide sensitivity, allosteric modulation) were not affected by exposure to tBHP.

Conclusions

Oxidative stress induced by tBHP affected several aspects of M3 receptor signaling pathway in CHO cells, including resting [Ca²⁺]_i, [Ca²⁺]_L, IP3 receptor mediated release of calcium from the ER, and calcium entry through the SOCE. tBHP had little effect on M3 receptor binding or G protein coupling. Thus, oxidative stress affects multiple aspects of calcium homeostasis and calcium dependent signaling.     

Mechanism of exocrine pancreatic insufficiency in streptozotocin-induced diabetes mellitus in rat: Effect of cholecystokinin-octapeptide

This study investigated the effects of cholecystokinin-octapeptide (CCK-8) on pancreatic juice flow and its contents, and on cytosolic calcium (Ca²⁺) and magnesium (Mg²⁺) levels in streptozotocin (STZ)-induced diabetic rats compared to healthy age-matched controls. Animals were rendered diabetic by a single injection of STZ (60 mg kg^–1, I.P.). Age-matched control rats obtained an equivalent volume of citrate buffer. Seven weeks later, animals were either anaesthetised (1 g kg^–1 urethane; IP) for the measurement of pancreatic juice flow or humanely killed and the pancreas isolated for the measurements of cytosolic Ca²⁺ and Mg²⁺ levels. Non-fasting blood glucose levels in control and diabetic rats were 92.40 ± 2.42 mg dl^–1 (n= 44) and >500 mg dl^–1 (n= 27), respectively. Resting (basal) pancreatic juice flow in control and diabetic anaesthetised rats was 0.56 ± 0.05 ul min^–1 (n= 10) and 1.28 ± 0.16 ul min^–1 (n= 8). CCK-8 infusion resulted in a significant (p < 0.05) increase in pancreatic juice flow in control animals compared to a much larger increase in diabetic rats. In contrast, CCK-8 evoked significant (p < 0.05) increases in protein output and amylase secretion in control rats compared to much reduced responses in diabetic animals. Basal [Ca²⁺]_i in control and diabetic fura-2-loaded acinar cells was 109.40 ± 15.41 nM (n= 15) and 130.62 ± 17.66 nM (n= 8), respectively. CCK-8 (10^–8M) induced a peak response of 436.55 ± 36.54 nM (n= 15) and 409.31 ± 34.64 nM (n= 8) in control and diabetic cells, respectively. Basal [Mg²⁺]_i in control and diabetic magfura-2-loaded acinar cells was 0.96 ± 0.06 nM (n= 18) and 0.86 ± 0.04 nM (n= 10). In the presence of CCK-8 (10^–8) [Mg²⁺]_i in control and diabetic cells was 0.80 ± 0.05 nM (n= 18) and 0.60 ± 0.02 nM (n= 10), respectively. The results indicate that diabetes-induced pancreatic insufficiency may be associated with derangements in cellular Ca²⁺ and Mg²⁺ homeostasis. (Mol Cell Biochem 261: 83–89, 2004)     

Effects of cryopreservation on the intracellular calcium concentration of human spermatozoa and its response to progesterone

The intracellular free calcium concentration [Ca²⁺]_i of sperm from 23 ejaculates was measured before and after cryopreservation using the fluorescent probe Fura-2. Spermatozoa were treated with 3.18 μM progesterone so that the regulation of [Ca²⁺]_i in a dynamic situation could be studied. [Ca²⁺]_i (nM) was 290 ± 13 in fresh spermatozoa vs. 550 ± 26 in cryopreserved samples (mean ± S.E.M. P < 0.0001 paired t-test). Progesterone at a dose of 3.18 μM stimulated a large and rapid increase in [Ca²⁺]_i to a peak value > 1 μM after 10–20 seconds. [Ca²⁺]_i then declined to a slightly raised basal level over the next 30–40 seconds. This phenomenon occurred in all the fresh samples, but about half the frozen thawed samples failed to respond. The peak [Ca²⁺] attained by frozen samples which did respond after the addition of progesterone was similar to that observed with fresh sperm. The calcium channel blocker verapamil (200 μM) completely inhibited the transient rise in [Ca²⁺]_i produced by progesterone, but 100 μM verapamil had only a partial effect. We conclude that (1) cryopreservation causes a substantial elevation of the [Ca²⁺]_i in human spermatozoa and (2) damage to the plasma membrane during cryopreservation may result in the loss of the progesterone receptor. Both factors may contribute to the loss of fertility after cryopreservation. © 1994 Wiley-Liss, Inc.