Cytosolic free calcium in lymphoblasts from young, aged and Alzheimer subjects.

Altered cellular calcium homeostasis may be important in the pathophysiology of aging and Alzheimer's disease. Calcium transport by freshly isolated lymphocytes declines with Alzheimer's disease compared with age-matched controls. To determine if these changes occur in the absence of complications due to drugs, diet or any of the other variables that are dependent upon the state of the patients, cytosolic free calcium ([Ca2+]i) was determined in cultured lymphoblasts from young and aged control subjects, as well as from Alzheimer patients. Lymphoblast [Ca2+]i was determined with the fluorescent probe fura-2 in either the presence or absence of serum. In cells that were grown in serum free medium, neither growth rates nor [Ca2+]i varied between groups. Growing cells in serum containing medium doubled growth rates and [Ca2+]i. However, [Ca2+]i from young, aged and Alzheimer groups were still similar. Thus, an age- or Alzheimer-related alteration in [Ca2+]i does not occur in cultured lymphoblasts.     

Mycoplasma hyopneumoniae increases intracellular calcium release in porcine ciliated tracheal cells

We investigated the effects of intact pathogenic Mycoplasma hyopneumoniae, nonpathogenic M. hyopneumoniae, and Mycoplasma flocculare on intracellular free Ca2+ concentrations ([Ca2+]i) in porcine ciliated tracheal epithelial cells. The ciliated epithelial cells had basal [Ca2+]i of 103 +/- 3 nM (n = 217 cells). The [Ca2+]i increased by 250 +/- 19 nM (n = 47 cells) from the basal level within 100 s of the addition of pathogenic M. hyopneumoniae strain 91-3 (300 microg/ml), and this increase lasted approximately 60 s. In contrast, nonpathogenic M. hyopneumoniae and M. flocculare at concentrations of 300 microg/ml failed to increase [Ca2+]i. In Ca2+-free medium, pathogenic M. hyopneumoniae still increased [Ca2+]i in tracheal cells. Pretreatment with thapsigargin (1 microM for 30 min), which depleted the Ca2+ store in the endoplasmic reticulum, abolished the effect of M. hyoneumoniae. Pretreatment with pertussis toxin (100 ng/ml for 3 h) or U-73122 (2 microM for 100 s), an inhibitor of phospholipase C, also abolished the effect of M. hyopneumoniae. The administration of mastoparan 7, an activator of pertussis toxin-sensitive proteins G(i) and G(o), increased [Ca2+]i in ciliated tracheal cells. These results suggest that pathogenic M. hyopneumoniae activates receptors that are coupled to G(i) or G(o), which in turn activates a phospholipase C pathway, thereby releasing Ca2+ from the endoplasmic reticulum. Thus, an increase in Ca2+ may serve as a signal for the pathogenesis of M. hyopneumoniae.     

Endometrial connexin expression in the mare and pig: Evidence for the suppression of cell-cell communication in uterine luminal epithelium

This investigation examines the relationship between implantation strategy and gap junction protein expression in uterine endometrium. The pattern of gap junction and connexin protein expression was analyzed in porcine and equine endometrium from cycling and pregnant animals using electron microscopy and immunocytochemistry. Functional analysis of cell-cell communication was also monitored by laser cytometry in primary cultures of endometrial epithelial cells. Gap junctions were detected in endometrial stroma of cycling and pregnant animals, which was correlated with immunoreactive Cx43 within stromal fibroblasts and vascular elements. No Cx26, Cx32, or Cx43 immunostaining was detected in luminal endometrial epithelium in either the mare or the pig at any stage of the estrous cycle or pregnancy. In contrast, endometrial glands of the mare exhibited a spatiotemporal pattern of Cx43 expression in the apicolateral plasma membrane which, when present, colocalized with the tight junction–associated protein, ZO-1. Uterine glandular Cx43 expression in mares was present from day 3 postovulation through day 14 of diestrus and until day 23 of pregnancy, whereas Cx43 was absent within uterine glands during seasonal anestrus, estrus, and after day 30 of pregnancy. Primary cultures of equine endometrial epithelial cells expressed both immunoreactive Cx43 and significant gap junction–mediated intercellular communication (GJIC) which was rapidly upregulated by 1.0 mM 8-bromo-cAMP or blocked with 1.0 mM octanol. No GJIC or connexin protein was detected in cultured porcine epithelial cells despite incubation with a variety of agents, including 8-bromo-cAMP, steroid hormones, retinoic acid, and/or prolactin. Junctional communication in endometrial epithelium of domestic farm animals is different than that reported for species exhibiting invasive implantation. The absence of GJIC in uterine luminal epithelium of the gilt and mare may be involved in limiting trophoblast invasiveness. Anat. Rec. 251:277–285, 1998. © 1998 Wiley-Liss, Inc.     

Junctional complexes of the blood-testis barrier in the Japanese quail (Coturnix Coturnix japonica)

This study investigated the developmental changes in the adherens junctions, gap junctions, as well as tight junctions forming the blood-testis barrier (BTB) in Japanese quail (Coturnix Coturnix japonica) testis. Testicular tissue from pre-pubertal, pubertal, adult, and aged Japanese quail were examined by immunohistochemistry and transmission electron microscopy (TEM). The tight junction proteins claudin-3, claudin-11, occludin and zonula occludens-1 (ZO-1), were generally localised in the cytoplasm of Sertoli cells, spermatogonia, and spermatocytes of pre-pubertal, pubertal, some adult birds. The adherens junction protein E-cadherin had a similar distribution pattern. During pre-pubertal development, the gap junction protein connexin-43 (Cx43) was only localised between Leydig cells in the testicular interstitium. However, TEM revealed the presence of gap junctions between cells of the seminiferous epithelium as early as the pre-pubertal stage. Furthermore, TEM confirmed the presence of tight and adherens junctions in the seminiferous epithelia of all age groups. The findings of this study document age-related differences in the immunolocalisation and intensity of the junctional proteins and the ultrastructure of the junctional complexes forming the BTB in quail testes. Additionally, the junctional complexes forming the BTB in the Japanese quail are well established prior to puberty. This study provides baseline information for the future evaluation of pathological changes in the BTB of avian species at different developmental stages.     

Role of Cl- co-transporters in the excitation produced by GABAA receptors in juvenile bovine adrenal chromaffin cells

GABA is the primary inhibitory neurotransmitter in the adult mammalian brain. However, in neonatal animals, activation of Cl(-)-permeable GABA receptors is excitatory and appears to depend on the expression of a Na(+)-K(+)-2Cl- cotransporter (NKCC) that elevates intracellular Cl- levels, leading to a depolarized Cl- equilibrium potential (ECl). The change from excitation to inhibition appears to involve the expression of the K+/Cl- co-transporter, KCC2, which lowers intracellular Cl- levels resulting in a hyperpolarized ECl. In this study, we show that bovine chromaffin cells from 4- to 5-mo-old animals are excited by GABA. Activation of GABAA receptors depolarizes the cells, opens voltage-dependent Ca2+ channels, elevates [Ca2+]i, and promotes the release of catecholamines. Blockade of voltage-dependent Ca2+ channels prevents the elevation of [Ca2+]i by GABA. The extrapolated anion reversal potential in these cells is approximately -28 mV, indicating a resting intracellular anion concentration of approximately 50 mM. Expression of KCC2 protein was not detected in the juvenile chromaffin cells. In contrast, clear expression of NKCC1 was observed. Blockade of NKCC1 should reduce the intracellular Cl- concentration and hyperpolarize ECl. Bumetanide, an NKCC1 blocker, reduced the elevation of [Ca2+]i by GABA. In some cells, activation of GABAA receptors inhibits responses to excitatory neurotransmitters, even though GABA itself is depolarizing. Co-activation of cholinergic and GABAA receptors in chromaffin cells produced elevations in [Ca2+]i that were comparable to those produced by cholinergic receptors alone. Our data showing the selective expression of chloride co-transporters and the resulting strongly depolarized anion reversal potential may help explain how activation of GABAA receptors causes sufficient excitation to elicit catecholamine release from chromaffin cells.     

ATP-induced [Ca2+]i changes in the human corneal epithelium

ATP, when leaked from damaged cells, is capable of eliciting responses in neighboring cells. A better understanding of the mechanism of this response is essential for designing therapeutic strategies for disease, there have been only a limited number of studies on the effect of ATP on the human cornea. We examined ATP-induced intracellular Ca2+ ([Ca2+]i) changes in the human corneal epithelia, cultured to near confluence. Cells were loaded with the Ca2+ indicators, Indo-1 or Fluo-4, and [Ca2+]i was monitored. ATP was found to induce an increase in [Ca2+]i, which was initiated from the perinuclear region and the nuclear envelope per se, and then propagated gradually towards the periphery. Intranuclear Ca2+ was momentarily increased. UTP elicited an identical response, but adenosine and alpha, beta-methylene ATP had no effect. Pretreatment with U73122 or thapsigargin inhibited the ATP-induced increase in [Ca2+]i. When a cell was topically stimulated with ATP, the [Ca2+]i increase spread beyond the cell boundary. The intercellular communications that accompanied the [Ca2+]i changes were inhibited by octanol. We conclude that extracellular ATP in the human cornea caused the mobilization of Ca2+ from intracellular Ca2+ stores (e.g. the endoplasmic reticulum and nuclear envelope) via P2Y purinoceptors of the epithelial cell. The response to ATP appears to spread to neighboring regions through gap junctions in the epithelium.     

Modeling the calcium gate of cardiac gap junction channel

We addressed the question how Ca2+ transients affect gap junction conductance (Gj) during action potential (AP) propagation by constructing a dynamic gap junction model coupled with a cardiac cell model. The kinetics of the Ca2+ gate was determined based on published experimental findings that the Hill coefficient for the [Ca2+]i-Gj relationship ranges from 3 to 4, indicating multiple ion bindings. It is also suggested that the closure of the Ca2+ gate follows a single exponential time course. After adjusting the model parameters, a two-state (open-closed) model, assuming simultaneous ion bindings, well described both the single exponential decay and the [Ca2+]i-Gj relationship. Using this gap junction model, 30 cardiac cell models were electrically connected in a one-dimensional cable. However, Gj decreased in a cumulative manner by the repetitive Ca2+ transients, and a conduction block was observed. We found that a reopening of the Ca2+ gate is possible only by assuming a sequential ion binding with one rate limiting step in a multistate model. In this model, the gating time constant (T) has a bell-shaped dependence on [Ca2+]i, with a peak around the half-maximal concentration of [Ca2+]i. Here we propose a five-state model including four open states and one closed state, which allows normal AP propagation; namely, the Gj is decreased -15% by a single Ca2+ transient, but well recovers to the control level during diastole. Under the Ca(2+)-overload condition, however, the conduction velocity is indeed decreased as demonstrated experimentally. This new gap junction model may also be useful in simulations of the ventricular arrhythmia.     

Effects of ATP and its analogues on [Ca2+]i dynamics in the rabbit corneal epithelium.

Adenosine-5'-triphosphate (ATP) plays a pivotal role in various tissues as an extracellular transmitter. ATP released from nerve endings and/or damaged cells may elicit reactions in adjacent cells. To identify such reactions, we investigated the dynamics of the intracellular calcium ion concentrations ([Ca2+]i) in the rabbit corneal epithelium during ATP-stimulation. Intact epithelial sheets isolated from corneal tissue were loaded with Fura-2, and [Ca2+]i dynamics in each cell layer were analyzed using a digital imaging system (Argus 50/CA). Normal architecture was preserved, suggesting that functional integrity remained intact. Perfusion with HEPES-buffered Ringer's solution containing ATP (10 microM) and uridine-5'-triphosphate (UTP; 10 microM) caused a biphasic [Ca2+]i increase in the superficial layer that manifested itself as a rapid initial spike followed by a long-lasting plateau phase. Adenosine-5'-diphate (10 microM) elevated the [Ca2+]i level, but induced only the initial spike, which was smaller than those induced by ATP and UTP. Adenosine (10 microM) did not elicit any [Ca2+]i changes in the epithelial cells. Suramin (10 microM; a P2 receptor antagonist) blocked the ATP-induced [Ca2+]i increase, whereas the P2X receptor agonists, alpha, beta-methylene ATP (10 microM), 2-methyl-thio ATP (10 microM) and Benzoylbenzoyl ATP (10 microM), did not elicit any increases in [Ca2+]i. In the basal cell layer, ATP-induced [Ca2+]i dynamics were biphasic, while oscillatory fluctuations of [Ca2+]i were induced in the wing cells of the mid layer of the corneal epithelium by ATP stimulation. Ca2+ oscillations were sometimes synchronized among adjacent wing cells, but these waves did not propagate to other cell layers. These results suggest that extracellular ATP elicits a [Ca2+]i increase mainly via P2Y receptors. In addition, synchronized Ca2+ oscillation in the wing cell layer indicates that intracellular events may spread to neighboring cells within the layer.     

Effects of gender on intracellular [Ca2+] in rat cardiac myocytes

The present study investigated the effects of gender on intracellular [Ca2+] ([Ca2+]i) in freshly isolated rat cardiac myocytes. Changes in [Ca2+]i in response to varied extracellular [Ca2+], different stimulus frequencies and addition of caffeine and isoprenaline were monitored using fura-2 in both male and female cardiac myocytes. Increasing extracellular [Ca2+] and stimulus frequency resulted in significant increases in peak [Ca2+] and the amplitude of the Ca2+ transient in both male and female cardiac myocytes. However, as extracellular [Ca2+] was raised, peak [Ca2+] and the amplitude of the Ca2+ transient increased significantly more in male than female cardiac myocytes. In addition a significant difference between male and female cells at each stimulus frequency was apparent. The time course of decay of the Ca2+ transient was significantly slower in female cardiac myocytes when compared with male cardiac myocytes, along with significantly slowed times to peak shortening and 50% relaxation, and a reduced extent of shortening. There was no significant difference in the amplitude of caffeine-induced [Ca2+]i responses between male and female cells, however, [Ca2+]i increased more readily in male cells than in female cells when isoprenaline was added. The data demonstrate that, under a variety of conditions, intracellular [Ca2+] rises to higher levels in cardiac myocytes from male as compared to female rats.     

Connexin expression and functional analysis of gap junctional communication in mouse embryonic stem cells

Gap junctional intercellular communication (GJIC) has been suggested to be necessary for cellular proliferation and differentiation. We wanted to investigate the function of GJIC in mouse embryonic stem (ES) cells using pharmacological inhibitors or a genetic approach to inhibit the expression of connexins, that is, the subunit proteins of gap junction channels. For this purpose, we have analyzed all known connexin genes in mouse ES cells but found only three of them, Cx31, Cx43, and Cx45, to be expressed as proteins. We have demonstrated by coimmunoprecipitation that Cx31 and Cx43, as well as Cx43 and Cx45, probably form heteromeric gap junction channels, whereas Cx31 and Cx45 do not. The pharmacological inhibitors reduced GJIC between ES cells to approximately 3% and initiated apoptosis, suggesting an antiapoptotic effect of GJIC. In contrast to these results, reduction of GJIC to approximately 5% by decreased expression of Cx31 or Cx45 via RNA interference in homozygous Cx43-deficient ES cells did not lead to apoptosis. Additional studies suggested that apoptotic death of ES cells and adult stem cells reported in the literature is likely due to a cytotoxic side effect of the inhibitors and not due to a decrease of GJIC. Using the connexin expression pattern in mouse ES cells, as determined in this study, multiple connexin-deficient ES cells can now be genetically engineered in which the level of GJIC is further decreased, to clarify whether the differentiation of ES cells is qualitatively or quantitatively compromised.     

人鼻咽上皮组织中间隙连接蛋白基因表达谱的研究

目的:研究细胞间隙连接蛋白基因(Cx)在正常人鼻咽上皮组织中的表达谱,在此基础上探索细胞间隙连接蛋白在鼻咽癌组织中的表达,为研究鼻咽组织癌变的原因和机理提供新思路。方法:根据基因库中人间隙连接蛋白基因的mRNA序列,合成间隙连接蛋白基因家族中13个基因的PCR引物,用RT-PCR的方法检测这些基因在鼻咽组织中的表达。结果:18例正常人鼻咽组织中,检出Cx30、31.1、37、40、43分别有16、16、17、15、17例表达,Cx26、31、32、45分别有10、11、9、9例表达,Cx36、46、46.6、50分别有1、0、1、3例表达。结论:正常人鼻咽组织中,主要表达的间隙连接蛋白为Cx30、31.1、37、40、43。     

Response to ATP is accompanied by a Ca2+ influx via P2X purinoceptors in the coronary arterioles of golden hamsters

In the vascular wall, adenosine-5'-triphosphate (ATP) released along with noradrenaline from sympathetic nerve terminals is considered to play an important role in controlling intracellular calcium ion ([Ca2+]i) levels in arteries. The present study examined how vascular smooth muscle cells in coronary arterioles respond to ATP in relation to [Ca2+]i dynamics. For this purpose, the dynamics of [Ca2+]i in the coronary arterioles of golden hamsters was examined by real-time laser scanning confocal microscopy. This technique enabled the visualization of [Ca2+]i changes in response to ATP in the intact coronary arterioles, the ultrastructure of which was well preserved. It was shown that an increase in [Ca2+]i in the arteriole smooth muscle cells was elicited by ATP. While P1 purinoceptor agonists have no effect on this process, P2 purinoceptor agonists were found to induce a [Ca2+]i increase in the smooth muscle cells. Suramin (an antagonist of P2X and P2Y receptors) completely inhibited ATP-induced [Ca2+]i dynamics, but reactive blue 2 (a P2Y receptor antagonist) did not. Uridine-5'-triphosphate (a P2Y receptor agonist) had no effect on [Ca2+]i, but alpha,beta-methylene ATP (a P2X receptor agonist) caused a strong increase in [Ca2+]i. We conclude that smooth muscle cells of the hamster coronary arterioles possess P2X, but not P1 or P2Y purinoceptors. The smooth muscle cells probably respond to extracellular ATP via P2X purinoceptors, resulting in the contraction of the coronary arterioles.     

Upregulation of GluR2 decreases intracellular Ca2+ following ischemia in developing gerbils

Developing animals are known to be resistant to cerebral ischemia. To investigate the mechanisms by which developing animals exhibit ischemic resistance, we examined the changes in intracellular calcium ([Ca2+]i) after oxygen-glucose deprivation (OGD) using hippocampal slices from gerbils. We found that increases of [Ca2+]i in hippocampal CA1 neurons is significantly less after OGD in developing gerbils than in adults. Western blot analysis of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid) receptors (AMPARs) showed that GluR2 expression, but not that of the other AMPARs is significantly higher in developing gerbils than in adults. Expression of the anti-apoptotic proteins such as HSP70, Bcl-XL, and plasma membrane Ca2+-ATPase type1 (PMCA1) are not higher in the developing gerbils than in adults. These results suggest that the higher expression of GluR2 is important for the smaller increases in [Ca2+]i and enhanced resistance to ischemia-induced neuronal damage in developing animals.     

Roles of Ca2+ and F-actin in intracellular aggregation of Chlamydia trachomatis in eucaryotic cells.

The effect of intracellular free Ca2+ ([Ca2+]i) on the intracellular aggregation of Chlamydia trachomatis serovars L2 and E in McCoy and HeLa cells is investigated. Loading the cells with the Ca2+ chelator MAPT/AM (1,2-bis-5-methyl-amino-phenoxylethane-N,N-n'-tetra-acetoxymethyl acetate), thereby decreasing the [Ca2+]i from 67 to 19 nM, decreased the number of cells with a local aggregation of chlamydiae in a dose-dependent manner. Neither the attachment nor the uptake of elementary bodies (EBs) was, however, affected after depletion of Ca2+ from the cells. There was no significant difference in the level of measured [Ca2+]i between infected and uninfected cells. Reducing the [Ca2+]i also significantly inhibited chlamydial inclusion formation. Differences in the organization of the actin filament network were observed in response to [Ca2+]i depletion. In Ca(2+)-depleted cells, where few EB aggregates were formed, few local accumulations of F-actin were observed in the cytosol. These results suggest that the aggregation of EBs in eucaryotic cells requires a normal homeostasis of intracellular Ca2+. By affecting F-actin reorganization and putatively certain Ca(2+)-binding proteins, [Ca2+]i plays a vital role in the infectious process of chlamydiae.     

Osteoclasts from medullary bone of egg-laying Japanese quail do not express the putative calcium 'receptor'.

The present study reports the contrasting effects of extracellular calcium ([Ca2+]e) elevation on cytosolic free calcium levels ([Ca2+]i) of osteoclasts, freshly isolated either from medullary bone of the egg-laying Japanese quail or from rat cortical bone. [Ca2+]i was measured in single osteoclasts using the Ca(2+)-sensitive fluorochrome, Indo-1. We found that elevation of [Ca2+]e failed to induce a rise of [Ca2+]i in quail osteoclasts, whilst causing an elevation of [Ca2+]i in rat osteoclasts. The calcium ionophore, ionomycin, led to a sustained elevation of [Ca2+]i in both cell types. These findings suggest that osteoclasts isolated from egg-laying quail do not possess the calcium sensor or 'receptor' that appears to be vital for the survival and function of rat osteoclasts.     

Cell-to-cell communication in the ovarian follicle: developmental and hormonal regulation of the expression of connexin43

The extensively developed network of cell-to-cell communication in the ovarian follicle is generated by gap junctions. In addition to the transmission of nutrients from the follicular cells to the oocyte, junctional communication in the ovarian follicle mediates the transfer of cAMP, the regulatory signal that maintains the oocyte in meiotic arrest. Luteinizing hormone (LH) interrupts cell-to-cell communication within the ovarian follicle, leading to a decrease in intra-oocyte concentrations of cAMP followed by resumption of meiosis. The developmental and hormonal regulation of the ovarian gap junction protein connexin43 (Cx43) and gene expression throughout folliculogenesis is reviewed in this article. An age-dependent increase in the amount of the Cx43 protein that was accompanied by its phosphorylation in preovulatory follicles has been observed. This protein disappeared after ovulation. The changes in both the amount and phosphorylation state of Cx43 were mimicked by exogenous administration of hormones as follows. Pregnant mare serum gonadotrophin increased Cx43 protein expression with a concurrent induction of its phosphorylation while a further human chorionic gonadotrophin injection resulted in a significant decrease of the protein. Cx43 mRNA showed a similar pattern of expression. In-vitro analysis of isolated ovarian follicles revealed that short time exposure (10 min) to LH stimulates phosphorylation of Cx43 followed by its immediate dephosphorylation, while longer incubations (8 and 24 h) with this hormone result in elimination of the protein. A significant decrease in Cx43 mRNA concentration at 24 h of incubation with LH was observed in these follicles. These results suggest that: (i) the presence of the gap junction protein in the ovary is developmentally regulated; (ii) after sexual maturation, both the amount of the Cx43 ovarian gap junction protein and its phosphorylation state are subjected to regulation by gonadotrophins; (iii) the LH-induced gating mechanism of the gap junctions in rat ovarian follicles is comprised of two steps: the immediate response is represented by a change in the phosphorylation state of the Cx43 protein, and the later response is manifested by a reduction of Cx43 protein concentration, due to attenuation of its gene expression.     

The role of protease-activated receptors on the intracellular calcium ion dynamics of vascular smooth muscles, with special reference to cerebral arterioles

Protease-activated receptors (PARs) mediate cellular responses to various proteases in numerous cell types, including smooth muscles and the endothelium of blood vessels. To clarify whether the stimulation of PARs induces responses in smooth muscle cells of cerebral arterioles, intracellular Ca2+([Ca2+]i) dynamics and nitric oxide (NO) production during PARs stimulation were investigated in the rat cerebral arterioles by real-time confocal microscopy, since [Ca2+]i and NO are both key factors in the maintenance of strain in blood vessels. Testicular arterioles were also investigated for comparison. In smooth muscle cells of small cerebral arterioles (< 50 microm in diameter), thrombin and PAR1-activating peptide (AP) induced an increase in [Ca2+]i and contraction. The response to PAR1 activation was caused by Ca2+ mobilization from intracellular Ca2+ stores. Trypsin and PAR2-AP induced a decrease in [Ca2+]i in the cells which was considered to be mediated by endothelium-derived NO and/or by promoting a Ca2+ sequestration mechanism. PAR3- and 4-AP had little effect. In contrast to small cerebral arterioles, [Ca2+]i dynamics in smooth muscle cells of large cerebral arterioles (< 150 microm in diameter) or testicular arterioles remained unchanged during PARs activation. The effects of PARs activation on the [Ca2+]i dynamics and the contraction/relaxation of cerebral arterioles are also discussed in relation to the role of proteases in the regional tissue circulation of the brain.     

Calcium homeostasis in dissociated embryonic neurons: a flow cytometric analysis.

1. Ca2+ homeostasis in freshly dissociated neurons from embryonic rat hypothalamus, cortex, and brain stem was investigated with flow cytometry. Cells were dissociated from embryonic brain by enzymatic and mechanical means and were incubated with the acetoxymethylester derivative of the Ca(2+)-sensitive dye indo-1. Neurons hydrolyzed and retained the dye as determined by the intensity of fluorescence emission, whereas similarly treated cultured astrocytes gave very low-level fluorescence. 2. The fluorescence of the indo-1 dye was measured at two wavelengths (405 and 485 nm) for each cell. Data were collected only from those cells (presumptive neurons) with high levels of fluorescence. Methods were developed to calibrate the level of intracellular free calcium ([Ca2+]i) as the ratio of fluorescence at 410 and 485 nm. The level of intracellular free Ca2+ was then calculated for each neuron. 3. A wide distribution of resting [Ca2+]i was found, with a median of approximately 90 nM. After addition of ionomycin to cells in Ca(2+)-free medium, there was a transient increase in [Ca2+]i, suggesting that all embryonic neurons had internal Ca2+ stores. The presence of active calcium extrusion mechanisms was demonstrated with the use of ionomycin in Ca(2+)-containing medium and with metabolic inhibitors. Furthermore, incubation in sodium-free medium resulted in a transient increase in [Ca2+]i and a reduced ability to eliminate elevated [Ca2+]i from the cytoplasm, suggesting that calcium homeostasis was dependent on the activity of the Na(+)-Ca2+ exchange mechanism. 4. Depolarization with K+ or veratrine increased [Ca2+]i in approximately 20% of the cells. This increase was blocked by eliminating extracellular free Ca2+ or adding Co2+, nifedipine, or verapamil, suggesting mediation by voltage-sensitive calcium channels. 5. Neurons were sorted on the basis of high [Ca2+]i and placed into dissociated culture. After 24 h, neurons in culture retained indo-1 fluorescence, suggesting that populations of neurons can be collected on the basis of their levels of [Ca2+]i. 6. These results demonstrate that flow cytometric analysis allows the characterization of a variety of Ca(2+)-regulatory mechanisms in populations of freshly dissociated embryonic neurons. Although only a proportion of embryonic day 17 neurons exhibit voltage-sensitive calcium channels, all neurons have developed the ability to sequester and extrude Ca2+.     

Calmodulin antagonists suppress gap junction coupling in isolated Hensen cells of the guinea pig cochlea

The effect of calmodulin (CaM) antagonists W7, trifluoperazine (TFP) and a calmodulin inhibitory peptide on gap junction coupling in isolated Hensen cells of the organ of Corti was analysed by the double whole-cell patch-clamp technique. Addition of the conventional antagonists W7 and TFP in the micromolar range caused a rapid decrease of gap junction conductance after a delay of a few minutes in a dose-dependent manner. Fluorescence spectroscopy of cytoplasmic free calcium concentration ([Ca(2+)](i)) by Fura-2 showed no significant change of [Ca(2+)](i) by W7. Chelation of [Ca(2+)](i) by 10 mM BAPTA or use of nominally Ca(2+)-free external bath did not suppress the W7-induced gap junction uncoupling. The results suggest that W7 and TFP induce gap junction uncoupling at unchanged global [Ca(2+)](i) in Hensen cells. To obtain additional evidence for an involvement of CaM in regulating gap junction conductance a calmodulin inhibitory peptide, the MLCK peptide (250 nM), was added to the standard pipette solution. Again gap junction uncoupling was observed, but on a significantly slower time scale. This is the first study of an effect of calmodulin antagonists on gap junction coupling in isolated Hensen cells. The question whether the effect of calmodulin inhibitors is specific and involves CaM-dependent gating of gap junction coupling in Hensen cells is discussed.     

Action potential-dependent calcium transients in myenteric S neurons of the guinea-pig ileum.

Simultaneous intracellular microelectrode recording and Fura-2 imaging was used to investigate the relationship between intracellular calcium ion concentration ([Ca2+]i) and excitability of tonic S neurons in intact myenteric plexus of the guinea-pig ileum. S neurons were impaled in myenteric ganglia, at locations near connections with internodal strands. The calcium indicator Fura-2 was loaded via the recording microelectrode. The estimated [Ca2+]i of these neurons was approximately 95 nM (n = 25). Intracellular current injection (200 ms pulses, 0.2 nA, delivered at 0.05 Hz) resulted in action potential firing throughout the stimulus pulse, accompanied by transient increases in [Ca2+]i (to approximately 240 nM, n = 12). Increasing the number of evoked action potentials by increasing stimulus duration (100-500 ms) or intensity (0.05-0.3 nA) produced correspondingly larger [Ca2+]i transients. Single action potentials rarely produced resolvable [Ca2+]i events, while short bursts of action potentials (three to five events) invariably produced resolvable [Ca2+]i increases. Some neurons demonstrated spontaneous action potential firing, which was accompanied by sustained [Ca2+]i increases. Action potential firing and [Ca2+]i increases were also observed by activation of slow synaptic input to these neurons, in cases where the slow depolarization initiated action potential firing. Action potentials (evoked or spontaneous) and associated [Ca2+]i transients were abolished by tetrodotoxin (1 microM). Omega-conotoxin GVIA (100 nM) reduced [Ca2+]i transients by approximately 67%, suggesting that calcium influx through N-type calcium channels contributes to evoked [Ca2+]i increases. The S neurons in this study showed prominent afterhyperpolarizations following bursts of action potential firing. The time-course of afterhyperpolarizations was correlated with the time-course of evoked [Ca2+]i transients. Afterhyperpolarizations were blocked by tetrodotoxin and reduced by omega-conotoxin GVIA, suggesting that calcium influx through N-type channels contributes to these events. The electrical properties of Fura-2-loaded neurons were not significantly different from properties of neurons recorded without Fura-2 injection, suggesting that Fura-2 injection alone does not significantly influence the electrical properties of these cells. These data indicate that myenteric S neurons in situ show prominent, activity-dependent increases in [Ca2+]i. These events can be generated spontaneously, or be evoked by intracellular current injection or synaptic activation. [Ca2+]i transients in these neurons appear to involve action potential-dependent opening of N-type calcium channels, and the elevation in [Ca2+]i increase may underlie afterhyperpolarizations and regulate excitability of these enteric neurons.