Identification and localisation of SERCA 2 isoforms in mammalian sperm

Upon binding to the egg's zona pellucida, capacitated spermatozoa will undergo a calcium-dependent exocytotic event called acrosome reaction. During this process, Ca2+ depletion from internal stores is followed by an important rise in [Ca2+]i due to a massive Ca2+ influx. Previous reports have shown that the acrosome can act as a Ca2+ store and that depletion of thapsigargin-sensitive stores induces acrosome exocytosis in capacitated spermatozoa from different mammalian species. The effect of thapsigargin, a specific inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCAs), suggests the presence and implication of SERCA in the active Ca2+ uptake during mammalian sperm capacitation. Although the presence of a thapsigargin-sensitive Ca2+-ATPase has been debated, the aim of this study was to clearly determine whether SERCAs are present in mammalian spermatozoa. Using three different anti-SERCA 2 antibodies, mono- and polyclonal, which recognised the same protein, we successfully identified and localised SERCA 2 in human, mouse and bovine sperm. Western blot analysis suggests that more than one SERCA 2 splice variant are present, one detected in the fraction containing the outer acrosomal membranes and another one present in the subcellular fraction containing the sperm midpiece. These results were confirmed by indirect immunofluorescence where SERCA 2 was observed in the acrosome and midpiece regions of human sperm. SERCA 2 immunohistochemical studies on human testis and PCR-amplification of mRNA encoding for each SERCA 2 splice variant in spermatogenic cells support the presence of this Ca2+-ATPase family in mature spermatozoa. In this paper, we clearly demonstrate, for the first time, the presence of SERCA 2 in mammalian sperm.     

Influence of sperm immobilization on onset of Ca(2+) oscillations after ICSI

Sperm immobilization prior to intracytoplasmic sperm injection (ICSI) is thought to be necessary for efficient fertilization. A variety of methods of sperm immobilization (pipetting, squeezing and piezo application) are currently employed in ICSI. The effect of differences in immobilization method on the timing of initial Ca(2+) oscillations of oocytes in ICSI was investigated. Motile spermatozoa were immobilized in eosin Y solution using pipetting, squeezing and piezo application. Complete staining of the sperm head was achieved after 220.7, 42.2 and 5.0 s respectively. Oscillations after ICSI were measured fluorometrically for each method. The onset of Ca(2+) oscillations was observed at 4.8 to 80.4 min after ICSI. Ca(2+) oscillations developed earlier with the piezo method (14.4 +/- 6.4 min) than other methods (pipetting, 43.1 +/- 20.2 min, P < 0.01; squeezing, 18.4 +/- 3.8 min, P = NS). The piezo method produced the earliest staining of the sperm head and may have caused the most damage to the sperm membrane. A more rapid onset of Ca(2+) oscillations was also observed with the piezo method. The method of sperm immobilization may be important for the rapid release of sperm factors that initiate oocyte activation. This study also showed that Ca(2+) oscillations develop earlier in human oocytes treated by ICSI than indicated in previous reports.     

Effect of fibronectin on proteasome activity, acrosome reaction, tyrosine phosphorylation and intracellular calcium concentrations of human sperm

BACKGROUND: Previously we showed that the human sperm proteasome plays significant roles during mammalian fertilization. Here we studied the effect of fibronectin (Fn), an extracellular matrix protein present in the cumulus oophorus of the oocyte, on proteasome activity, acrosome reaction, intracellular calcium concentration ([Ca(2+)](i)) and protein tyrosine phosphorylation of human sperm. METHODS: Aliquots of motile sperm were incubated for 15 min (T0), 5 h (T5) and 18 h (T18), at 37 degrees C, 5% CO(2) and 95% air with Fn (0-100 microg/ml). The chymotrypsin- and trypsin-like activity of the proteasome was measured using the fluorogenic substrates, Suc-Leu-Leu-Val-Tyr-AMC and Boc-Gln-Ala-Arg-AMC, respectively. At T18, sperm aliquots were incubated for 15 min with Fn and/or progesterone in the presence or absence of epoxomicin (a proteasome inhibitor). The percentage of viable acrosome reacted sperm was evaluated using the Fluorescein isothiocyanate (FITC)-labeled Pisum sativum agglutinin. Tyrosine phosphorylation was evaluated by western blot and [Ca(2+)](i) using fura 2. RESULTS: Fn stimulated both enzymatic activities of the proteasome and the acrosome reaction of human sperm. Progesterone enhanced and epoxomicin drastically inhibited the effect of Fn. Fn treatment also increased the [Ca(2+)](i). Western blot analysis revealed that Fn increased tyrosine protein phosphorylation and that some proteasome subunits became tyrosine phosphorylated upon Fn treatment. CONCLUSIONS: These results suggest that Fn activates the proteasome and induces the acrosome reaction in human sperm. This effect may involve binding with specific receptors (integrins) on the sperm surface and the activation of tyrosine kinases.     

Initiation site of Ca(2+) entry evoked by endoplasmic reticulum Ca(2+) depletion in mouse parotid and pancreatic acinar cells

Purpose

In non-excitable cells, which include parotid and pancreatic acinar cells, Ca²⁺ entry is triggered via a mechanism known as capacitative Ca²⁺ entry, or store-operated Ca²⁺ entry. This process is initiated by the perception of the filling state of endoplasmic reticulum (ER) and the depletion of internal Ca²⁺ stores, which acts as an important factor triggering Ca²⁺ entry. However, both the mechanism of store-mediated Ca²⁺ entry and the molecular identity of store-operated Ca²⁺ channel (SOCC) remain uncertain.

Materials and Methods

In the present study we investigated the Ca²⁺ entry initiation site evoked by depletion of ER to identify the localization of SOCC in mouse parotid and pancreatic acinar cells with microfluorometeric imaging system.

Results

Treatment with thapsigargin (Tg), an inhibitor of sarco/ endoplasmic reticulum Ca²⁺-ATPase, in an extracellular Ca²⁺ free state, and subsequent exposure to a high external calcium state evoked Ca²⁺ entry, while treatment with lanthanum, a non-specific blocker of plasma Ca²⁺ channel, completely blocked Tg-induced Ca²⁺ entry. Microfluorometric imaging showed that Tg-induced Ca²⁺ entry started at a basal membrane, not a apical membrane.

Conclusion

These results suggest that Ca²⁺ entry by depletion of the ER initiates at the basal pole in polarized exocrine cells and may help to characterize the nature of SOCC.     

The leading role of membrane Ca(2+)-ATPase in recovery of Ca(2+) homeostasis after glutamate shock

Combined blockade of Na⁺/Ca²⁺ exchange, Ca²⁺ uptake by mitochondria and endoplasmic reticulum usually does not prevent recovery of the basal level of intracellular Ca²⁺ after 1-min action of glutamate (100 M) or K⁺ (50 mM). However, replacement of Ca²⁺ with Ba²⁺, which cannot be transported by Ca²⁺-ATPase, considerably delayed the decrease in intracellular Ba²⁺ after its rise caused by glutamate or potassium application in all examined cells, which attest to an important role of Ca²⁺-ATPase in Ca²⁺ extrusion after the action of glutamate or K⁺.     

Effect of Ca2+/Mg2+-free medium on the biopsy procedure for preimplantation genetic diagnosis and further development of human embryos

In this study, the use of Ca²⁺/Mg²⁺-free medium for biopsy ofhuman embryos at the 4- to 10-cell stage on the third day ofdevelopment was evaluated. When compared with control mediumcontaining normal concentrations of Ca²⁺ and Mg²⁺ ions, theuse of Ca²⁺/Mg²⁺ -free medium allows an easier removal of blastomeresas illustrated by a lower rate of cell lysis as well as by ashorter time needed to perform the procedure. Subsequent embryodevelopment to the blastocyst stage is not affected by the choiceof biopsy medium, not even when embryos are exposed to the mediumfor 45 min. The use of Ca²⁺/Mg²⁺-free medium thus allows foran easier biopsy procedure during preimplantation genetic diagnosis,while it does not result in a loss of developmental potentialof the embryo to the blastocyst stage.     

Phosphatidylserine externalization in human sperm induced by calcium ionophore A23187: relationship with apoptosis, membrane scrambling and the acrosome reaction

BACKGROUND: Translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane is a modification of the lipid architecture occurring in sperm. This is one of the earliest signs of apoptosis that can be monitored by the calcium-dependent binding of annexin V. METHODS AND RESULTS: Flow cytometric analysis of annexin V binding was performed. Calcium ionophore A23187 led to a significant increase in the proportion of living sperm with PS exposure: 7.3 3.2% of cells in the untreated ejaculate versus 47.5 5.6% of cells after 1 h of incubation with A23187. Conversely, diminution of mitochondrial membrane potential [DiOC6(3)/propidium iodide (PI) assay], caspase activation [fluorescein isothiocyanate (FITC)-Val-Ala-Asp-fluoromethylketone (VAD-FMK)/PI assay], increased plasma membrane permeability (Yo-Pro-1/PI assay) and increased DNA fragmentation [TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay], which are among the main signs of apoptosis, were not observed in sperm, even after 4 h of incubation with A23187. However, A23187 significantly increased the proportion of sperm with plasma membrane scrambling and with a reacted acrosome, as detected with the merocyanine 540 probe (M540) and the monoclonal anti-human CD46-PE antibody respectively. CONCLUSIONS: Our results suggest that PS exposure in human sperm, as induced by A23187, is mainly related to the acrosome reaction rather than to apoptosis.     

Fertilization and early embryology: Intracytoplasmic sperm injection of mouse oocytes with 5 mM Ca2+ at different intervals

The objective of this investigation was to determine whetherintracytoplasmic sperm injection (ICSI) can be performed inthe mouse. Metaphase II oocytes were obtained from F1 hybridmice (C57BLx CBA) by i.p. injections of 10 IU pregnant mare'sserum gonadotrophin (PMSG) and human chorionic gonadotrophin(HCG) administered 48 h apart. Oocytes with cumulus oophoruswere retrieved 13–14 h post HCG. Cumulus was dispersedwith 0.1% hyaluronidase. Mouse spermatozoa were obtained fromthe cauda epididymides of males of the same strain. The spermatozoawere processed by the standard swim-up procedure. The harvestedspermatozoa were then incubated for 1.5 h to allow capacitation.Healthy oocytes were injected with 3–4 pi 5 mM Ca²⁺, followedby one live morphologically normal spermatozoon into the cytoplasmat intervals of 0, 0.5, 1, 2 and 3 h. The proportion of 2-cellembryos that developed from oocytes injected with Ca²⁺ and spermatozoaranged between 29.5 and 36.5% in all groups, with no statisticaldifference between treatments. Chromosomal analysis showed thattwo-thirds of the ICSI-derived 2-cell embryos were diploid.The proportion of parthenogenetically activated embryos in theICSI groups was similar to that in the control group (8–10%)which was injected with Ca²⁺ and polyvinyl pyrrolidone only.The proportion of blastocysts that developed in culture fromthe ICSI-derived 2-cell embryos was of the order of 36–42%.Some blastocysts were used for cell number counts. There wasa significant increase in total and inner cell mass counts ofblastocysts in which the spermatozoon was injected at 2 and3 h following Ca²⁺. The remaining blastocysts were transferredto day 3 pseudopregnant mice, of which 33% subsequently becamepregnant. Of the blastocysts transferred, 16–25% developedto term in vivo. No deformities were observed in the pups. Webelieve this is the first report of live-birth following mouseICSI.     

Does Ca/Mg-free medium have an effect on the survival of the preimplantation mouse embryo after biopsy?

The way in which the different technical aspects of clinicalpreimplantation genetic diagnosis affect the survival of thebiopsied embryos is not well understood. One of these aspectsis the influence of Ca²⁺/Mg²⁺-free medium on the developmentalcapacity of the biopsied embryo and the biopsied cell itself.Therefore, we used an experimental design involving 4-cell mouseembryos to evaluate the effect of performing the biopsy procedureunder such conditions. Our results indicate that the use ofCa²⁺/Mg²⁺-free medium has no detrimental effect (at least inmouse embryos) on the viability of the biopsied embryos andtheir biopsied cells when used during relatively short timesof exposure.     

Andrology: Modulation of prostaglandin synthesis in mammalian sperm acrosome reaction

Exogenous arachidonic acid induces the acrosome reaction andthe production of the prostaglandins PGE₂ and PGF₂ in bovinespermatozoa. Exogenous PGE₂ also induces the acrosome reactionand PGF₂ synthesis. To understand better the role of PGE₂ inthe induction of PGF₂ synthesis through modulation of phospholipaseA₂, inhibitors of this enzyme were used. The effects of PGE₂were blocked by phospholipase A₂ inhibitors and this inhibitionwas reversed by addition of arachidonic acid. These data indicatethat PGE₂ activates phospholipase A₂ to produce arachidonicacid. To determine whether protein kinase C modulates phospholipaseA₂ activity in this process, staurosporin, an inhibitor of proteinkinase C, was used. The effect of PGE₂ on PGF₂ production isinhibited by staurosporin and this inhibition was reversed byaddition of arachidonic acid indicating that protein kinaseC is involved in phospholipase A₂ activation. The effect ofexogenous arachidonic acid or PGE₂ on the acrosome reactionis blocked by lipoxygenase inhibitors but not by inhibitorsof cyclo-oxygenase, indicating that lipoxygenase products areinvolved in the mechanism of the acrosome reaction. The presenteddata shed light on the cross-talk between cyclo-oxygenase andlipoxygenase and their involvement in the sperm acrosome reaction.It is suggested that cyclo-oxygenase products modulate the activityof lipoxygenase which is a key enzyme in the mechanism leadingto the acrosome reaction. Stimulation of cyclo-oxygenase tosynthesize PGE₂ activates phospholipase A₂ to release arachidonicacid which is the substrate for lipoxygenase activity. Thus,the role of cyclo-oxygenase products is probably to reactivatethe phospholipase A₂ which was initially activated via the signaltransduction cascade.     

Ghrelin increases intracellular Ca(2+) concentration in the various hormone-producing cell types of the rat pituitary gland

Ghrelin, isolated from the stomach as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), has potent growth hormone release ability in vivo and in vitro. Although GHS-R is abundantly expressed in the pituitary gland, there is no direct evidence of a relationship between hormone-producing cells and functional GHS-R in the pituitary gland. The aim of this study was to determine which anterior pituitary cells respond to ghrelin stimulation in male rats. We performed Fura-2 Ca(2+) imaging analysis using isolated pituitary cells, and performed immunocytochemistry to identify the type of pituitary hormone-producing cells. In Fura-2 Ca(2+) imaging analysis, ghrelin administration increased the intracellular Ca(2+) concentration in approximately 50% of total isolated anterior pituitary cells, and 20% of these cells strongly responded to ghrelin. Immunocytochemical analysis revealed that 82.9±1.3% of cells that responded to ghrelin stimulation were GH-immunopositive. On the other hand, PRL-, LH-, and ACTH-immunopositive cells constituted 2.0±0.3%, 12.6±0.3%, and 2.5±0.8% of ghrelin-responding pituitary cells, respectively. TSH-immunopositive cells did not respond to ghrelin treatment. These results suggest that ghrelin directly acts not only on somatotrophs, but also on mammotrophs, gonadotrophs, and corticotrophs in the rat pituitary gland.     

The pathway for refilling intracellular Ca2+ stores passes through the cytosol in human leukaemia cells

The pathway for refilling the intracellular Ca²⁺ stores of HL60 and U937 human leukaemia cells loaded with fura-2 has been investigated. On addition of external Ca²⁺ to cells with empty stores there was an increase in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) which preceded the refilling of the stores. The increase in [Ca²⁺]_i was faster than the refilling, by 3-to 15-fold, depending on the cell type. In measurements in single HL60 cells we found that the refilling of the stores correlated with the extent of the [Ca²⁺]_i increase on addition of external Ca²⁺. The cells showing no [Ca²⁺]_i increase were unable to refill their stores. The addition of Ni²⁺ to the extracellular medium prevented both the [Ca²⁺]_i increase and the refilling of the stores. These results indicate that the limiting step for store refilling is the entry of Ca²⁺ from the extracellular medium to the cytosol. Hence, we conclude that extracellular Ca²⁺ cannot gain access directly to the intracellular Ca²⁺ stores in these cells, but must first enter the cytosol and be taken up from there into the stores.     

alpha-SNAP and NSF are required in a priming step during the human sperm acrosome reaction

The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacological stimuli it undergoes a special type of Ca2+-dependent exocytosis termed the acrosome reaction (AR), which is an absolute prerequisite for fertilization. Aided by a streptolysin-O permeabilization protocol developed in our laboratory, we have previously demonstrated requirements for Rab3A, N-ethylmaleimide-sensitive factor (NSF), several soluble NSF-attachment protein receptor (SNARE) proteins, and synaptotagmin VI in the human sperm AR. Here, we show that alpha-soluble NSF-attachment protein (alpha-SNAP), a protein essential for most fusion events through its interaction with NSF and the SNARE complex, exhibits a direct role in the AR. First, the presence of alpha-SNAP is demonstrated by the Western blot of human sperm protein extracts. Immunostaining experiments reveal an acrosomal localization for this protein. Second, the Ca2+ and Rab3A-triggered ARs are inhibited by anti-alpha-SNAP antibodies. Third, bacterially expressed alpha-SNAP abolishes exocytosis in a fashion that depends on its interaction with NSF. Fourth, we show a requirement for alpha-SNAP/NSF in a prefusion step early in the exocytotic pathway, after the tethering of the acrosome to the plasma membrane and before the efflux of intra-acrosomal Ca2+. These results suggest a key role for alpha-SNAP/NSF in the AR, and strengthen our understanding of the molecular players involved in the vesicle-to-plasma membrane fusion taking place during exocytosis.     

Glutamate transporter blockade affects Ca(2+) responses in astrocytes

Brief pretreatment of astrocytes in culture with glutamate (500 microM for 20 min), was earlier shown to significantly enhance the Ca(2+) responses to a depolarizing pulse. It is known that malfunction of glutamate transporters increases extracellular glutamate concentration. We hypothesized that pretreatment of astrocytes with glutamate in conditions where the glutamate transporter activity is blocked should cause further elevation of the Ca(2+) responses to a depolarizing pulse. To test the hypothesis we pretreated astrocytes in culture (primary rat astrocyte cultures) with glutamate (500 microM) and glutamate transport inhibitor, threo-beta-hydroxy-aspartate (200 microM, TBHA) or glutamate (500 microM) in Na(+) free extracellular solution for 20 min. The Ca(2+) responses were elicited by depolarization of the astrocyte to evoke voltage-gated Ca(2+) currents. Paradoxical attenuation of the Ca(2+) transients was observed when the glutamate pretreatment was done in conditions that blocked glutamate transport, accompanied by faster rise and decay times. When the experiments were done on astrocyte pairs that were pretreated with glutamate and TBHA, we observed attenuated Ca(2+) responses in the adjoining cell when compared with the depolarized cell. The results were contrary to our earlier observation of heightened responses in the adjoining cell of the astrocyte pair, in cells pretreated with glutamate alone. The attenuated Ca(2+) responses in astrocytes would imply decrease in the vesicular release of glutamate and ATP. Extracellular glutamate concentration dependent regulation of the Ca(2+) signaling mechanism thus seems to operate in astrocytes, which may be important in regulating the neurotoxic accumulation of glutamate in the extracellular space and the synapse.     

重组人内抑素对佐剂性关节炎大鼠成纤维样滑膜细胞内钙离子稳态的影响

目的 观察重组人内抑素(rhEndestatin)对佐剂性关节炎大鼠成纤维样滑膜细胞(AA FLSs)内钙离子(Ca2+)稳态的影响,探讨rhEndomafin促进AA FLSs凋亡的离子机制,为RA药物治疗及寻找其治疗新靶点提供实验依据.方法雄性SD大鼠12只,体质量140～160 g,分为正常组(n=3)和从模型组(n=9).模型组大鼠制备从模型,体外培养AA FLSs,应用Ca2+荧光指示剂Fluo-3/AM孵育培养的细胞,激光扫描共焦显微镜检测有、无细胞外Ca2+,而rhEndestatin作用所致AA FLSs胞内Ca2+荧光强度发生动态变化,可以判断rhEndostatin对AA FLSs胞内Ca2+浓度([Ca2+]I)的影响.结果在胞外有Ca2+的情况下,rhEndostatin可引起静态AA FLS[Ca2+];快速增加,rhEndostatin作用10s后,[Ca2+];急剧增加达峰值,继之随时间缓慢下降,停止加药50 s后,[Ca2+]I尚未回复到加药前基础水平;而在无细胞外钙环境中,rhEndostatin未引起AA FLSs[Ca2+]I变化.结论 rhEndestafin可促进AA FLSs胞外Ca2+内流,引起胞内Ca2+超载,从而促进从FLSs凋亡.     

Spatial and temporal control of intracellular free Ca2+ in chick sensory neurons

Digital imaging of fura-2 fluorescence and the voltage-clamp technique were combined to study cytoplasmic free Ca²⁺ concentration, [Ca]_i, in neurons cultured from chick dorsal root ganglia. Depolarizing pulses raised [Ca]_i to a new steady-state level which was achieved earlier in neurites than in the soma. The rise in [Ca]_i during stimulated bursting or rhythmic activity was also faster in neurites. After stimulation [Ca]_i recovered monoexponentially in the soma and biexponentially in neurites. Application of 50 mM KCl produced membrane depolarization and a concomitant increase of [Ca]_i. During wash-out [Ca]_i often declined to an intermediate steady-state level at which it stayed for several minutes. Thereafter the resting level of [Ca]_i was quickly restored. [Ca]_i recovery was delayed after treating the cell with 2 M thapsigargin, an inhibitor of the Ca²⁺ pump of internal Ca²⁺ stores. Caffeine (10 mM) transiently increased [Ca]_i. A second caffeine application produced smaller [Ca]_i changes due to the prior depletion of Ca²⁺ stores, which could be replenished by brief exposure to KCl. Thapsigargin (2 M) transiently increased [Ca]_i both in the standard and Ca²⁺-free solution. [Ca]_i transients due to caffeine and thapsigargin started in the cell interior, in contrast to [Ca]_i changes evoked by membrane depolarization, which were noticed first at the cell edge. Caffeine and thapsigargin induced a transient inward current which persisted in the presence of 1 mM La³⁺ and in Ca²⁺-free solutions, but which was greatly diminished in Na⁺-free solutions. The effects of caffeine and thapsigargin were mutually exclusive both in the generation of [Ca]_i transients and in the inward current induction.     

Effects of intracellular Ca2+ chelating compounds on inward currents caused by Ca2+ release from sarcoplasmic reticulum in guinea-pig atrial myocytes

Ca²⁺ release from the sarcoplasmic reticulum (SR) of mammalian cardiac myocytes occuring either due to activation by a depolarization or the resulting transmembrane Ca²⁺ current (I _Ca), or spontaneously due to Ca²⁺ overload has been shown to cause inward current(s) at negative membrane potentials. In this study, the effects of different intracellular Ca²⁺ chelating compounds on I _Ca-evoked or spontaneous Ca²⁺-release-dependent inward currents were examined in dialysed atrial myocytes from hearts of adult guinea-pigs by means of whole-cell voltage-clamp. As compared to dialysis with solutions containing only a low concentration of a high affinity ethylene glycol-bis(-aminoethylether) N,N,N,N-tetraacetic acid (EGTA) like chelator (50–200 M), inward membrane currents (at –50 mV) due to evoked Ca²⁺ release, spontaneous Ca²⁺ release or Ca²⁺ overload following long-lasting depolarizations to very positive membrane potentials are prolonged if the dialysing fluid contains a high concentration of a low affinity Ca²⁺ chelating compound such as citrate or free adenosine 5-triphosphate (ATP). Without such a non-saturable Ca²⁺ chelator in the dialysing fluid, Ca²⁺-release-dependent inward currents are often oscillatory and show an irregular amplitude. With a low affinity chelator in a non-saturable concentration, discrete inward currents with constant properties can be recorded. We conclude that the variability in Ca²⁺-release-dependent inward current seen in single cells arises from spatial inhomogeneities of intracellular Ca²⁺ concentration ([Ca²⁺]_i) due to localized saturation of endogenous and exogenous high affinity Ca²⁺ buffers (e.g. [2]). This can be avoided experimentally by addition of a non-saturable buffer to the intracellular solution. This condition might be useful, if properties of Ca²⁺ release from the SR and/ or the resulting membrane current, like for example arrhythmogenic transient inward current, are to be investigated on the single cell level.     

Alteration of expression of Ca2+ signaling proteins and adaptation of Ca2+ signaling in SERCA2+/- mouse parotid acini

PURPOSE: The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), encoded by ATP2A2, is an essential component for G-protein coupled receptor (GPCR)-dependent Ca2+ signaling. However, whether the changes in Ca2+ signaling and Ca2+ signaling proteins in parotid acinar cells are affected by a partial loss of SERCA2 are not known. MATERIALS AND METHODS: In SERCA2+/- mouse parotid gland acinar cells, Ca2+ signaling, expression levels of Ca2+ signaling proteins, and amylase secretion were investigated. RESULTS: SERCA2+/- mice showed decreased SERCA2 expression and an upregulation of the plasma membrane Ca2+ ATPase. A partial loss of SERCA2 changed the expression level of 1, 4, 5-tris-inositolphosphate receptors (IP3Rs), but the localization and activities of IP3Rs were not altered. In SERCA2+/- mice, muscarinic stimulation resulted in greater amylase release, and the expression of synaptotagmin was increased compared to wild type mice. CONCLUSION: These results suggest that a partial loss of SERCA2 affects the expression and activity of Ca2+ signaling proteins in the parotid gland acini, however, overall Ca2+ signaling is unchanged.     

Agonist-induced intracellular Ca2+ transients in HT29 cells

In the present study we have investigated the mechanism of intracellular Ca²⁺ activity ([Ca²⁺]_i) changes in HT₂₉ cells induced by adenosine triphosphate (ATP), carbachol (CCH), and neurotensin (NT). [Ca²⁺]_i was measured with the fluorescent Ca²⁺ indicator fura-2 at the single-cell level or in small cell plaques with high time resolution (1–40Hz). ATP and CCH induced not only a dose-dependent [Ca²⁺]_i peak response, but also changes of the plateau phase. The [Ca²⁺]_i plateau was inversely dependent on the ATP concentration, whereas the CCH-induced [Ca²⁺]_i plateau increased at higher CCH concentrations. NT showed (from 10^–10 to 10^–7 mol/l) in most cases only a [Ca²⁺]_i spike lasting 2–3 min. The [Ca²⁺]_i plateau induced by ATP (10^–6 mol/l) and CCH (10^–5 mol/l) was abolished by reducing the Ca²⁺ activity in the bath from 10^–3 to 10^–4 mol/l (n=7). In Ca²⁺-free bathing solution the [Ca²⁺]_i peak value for all three agonists was not altered. Using fura-2 quenching by Mn²⁺ as an indicator of Ca²⁺ influx the [Ca²⁺]_i peak was always reached before Mn²⁺ influx started. Every agonist showed this delayed stimulation of the Ca²⁺ influx with a lag time of 23±1.5 s (n=15) indicating a similar mechanism in each case. Verapamil (10^–6–10^–4 mol/l) blocked dose dependently both phases (peak and plateau) of the CCH-induced [Ca²⁺]_i increase. Short pre-incubation with verapamil augmented the effect on the [Ca²⁺]_i peak, whereas no further influence on the plateau was observed. Ni²⁺ (10^–3 mol/l) reduced the plateau value by 70%.     

Ca2+ release in cultured rat epididymal cells during hypoosmotic swelling

Microfluorimetric studies were carried out to investigate the effects of hypoosmotic swelling on intracellular Ca²⁺ concentration ([Ca²⁺]_i) in single rat epididymal cells. In Ca²⁺-free solution containing 50 mol/l ethylenebis(oxonitrilo)tetraacetate (EGTA) hypoosmotic swelling (–160 mosmol/l) induced a transient rise in [Ca²⁺]_i which was either monophasic, biphasic or oscillatory. The [Ca²⁺]_i responses to repeated hypoosmotic stimulations followed a decremental pattern. However, if 2.5 mmol/l Ca²⁺ was admitted during the recovery period between successive stimulations, the second and the third [Ca²⁺]_i responses were slightly greater than the first. Increasing the change in osmolarity from –14±1.0 to –154±1.5 mosmol/l increased the rise in [Ca²⁺]_i but reduced the [Ca²⁺]_i response to subsequent ionomycin stimulation (4 mol/l). The swelling- and the ionomycin-induced rises in [Ca²⁺]_i followed a reciprocal pattern. It was suggested that intracellular Ca²⁺ release in response to cell swelling in the epididymal epithelium might play a role in cell volume regulation and the control of epididymal fluid osmolarity.