Thyroidal influence on the cell surface GM1 of granule cells: Its significance in cell migration during rat brain development

1. No difference was observed in the in vitro growing ability of granule cells isolated from hypothyroid or normal rat brain. When granule cells were taken from hypothyroid rat brain and grown in normal culture medium containing 10% fetal calf serum, they behaved similarly to the granule cells obtained from normal rat brain. 2. In both cases there were progressive losses of in vitro growing ability of the granule cells with the age of the animal and it became impossible to grow them when derived from 21 days or older animals. 3. A marked decrease in cell surface GM1 was observed when the cells were maintained under thyroid hormone-deficient conditions in culture. 4. Anti-GM1 antibody was found to inhibit significantly the migration of granule cells along the astrocyte fibers. 5. These results indicate that GM1 has an important role in thyroid hormone-dependent postnatal brain maturation in rat.     

The dynamics of mitochondrial Ca fluxes

We have investigated the kinetics of mitochondrial Ca²⁺ influx and efflux and their dependence on cytosolic [Ca²⁺] and [Na⁺] using low-Ca²⁺-affinity aequorin. The rate of Ca²⁺ release from mitochondria increased linearly with mitochondrial [Ca²⁺] ([Ca²⁺]_M). Na⁺-dependent Ca²⁺ release was predominant al low [Ca²⁺]_M but saturated at [Ca²⁺]_M around 400 μM, while Na⁺-independent Ca²⁺ release was very slow at [Ca²⁺]_M below 200 μM, and then increased at higher [Ca²⁺]_M, perhaps through the opening of a new pathway. Half-maximal activation of Na⁺-dependent Ca²⁺ release occurred at 5-10 mM [Na⁺], within the physiological range of cytosolic [Na⁺]. Ca²⁺ entry rates were comparable in size to Ca²⁺ exit rates at cytosolic [Ca²⁺] ([Ca²⁺]_c) below 7 μM, but the rate of uptake was dramatically accelerated at higher [Ca²⁺]_c. As a consequence, the presence of [Na⁺] considerably reduced the rate of [Ca²⁺]_M increase at [Ca²⁺]_c below 7 μM, but its effect was hardly appreciable at 10 μM [Ca²⁺]_c. Exit rates were more dependent on the temperature than uptake rates, thus making the [Ca²⁺]_M transients to be much more prolonged at lower temperature. Our kinetic data suggest that mitochondria have little high affinity Ca²⁺ buffering, and comparison of our results with data on total mitochondrial Ca²⁺ fluxes indicate that the mitochondrial Ca²⁺ bound/Ca²⁺ free ratio is around 10- to 100-fold for most of the observed [Ca²⁺]_M range and suggest that massive phosphate precipitation can only occur when [Ca²⁺]_M reaches the millimolar range.     

Role of glycine residues highly conserved in the S2-S3 linkers of domains I and II of voltage-gated calcium channel α1 subunits

The pore-forming component of voltage-gated calcium channels, α₁ subunit, contains four structurally conserved domains (I-IV), each of which contains six transmembrane segments (S1-S6). We have shown previously that a Gly residue in the S2-S3 linker of domain III is completely conserved from yeasts to humans and important for channel activity. The Gly residues in the S2-S3 linkers of domains I and II, which correspond positionally to the Gly in the S2-S3 linker of domain III, are also highly conserved. Here, we investigated the role of the Gly residues in the S2-S3 linkers of domains I and II of Ca_v1.2. Each of the Gly residues was replaced with Glu or Gln to produce mutant Ca_v1.2s; G182E, G182Q, G579E, G579Q, and the resulting mutants were transfected into BHK6 cells. Whole-cell patch-clamp recordings showed that current-voltage relationships of the four mutants were the same as those of wild-type Ca_v1.2. However, G182E and G182Q showed significantly smaller current densities because of mislocalization of the mutant proteins, suggesting that Gly¹⁸² in domain I is involved in the membrane trafficking or surface expression of α₁ subunit. On the other hand, G579E showed a slower voltage-dependent current inactivation (VDI) compared to Ca_v1.2, although G579Q showed a normal VDI, implying that Gly⁵⁷⁹ in domain II is involved in the regulation of VDI and that the incorporation of a negative charge alters the VDI kinetics. Our findings indicate that the two conserved Gly residues are important for α₁ subunit to become functional.     

Kinetic control of multiple forms of Ca(2+) spikes by inositol trisphosphate in pancreatic acinar cells.

The mechanisms of agonist-induced Ca(2+) spikes have been investigated using a caged inositol 1,4,5-trisphosphate (IP(3)) and a low-affinity Ca(2+) indicator, BTC, in pancreatic acinar cells. Rapid photolysis of caged IP(3) was able to reproduce acetylcholine (ACh)-induced three forms of Ca(2+) spikes: local Ca(2+) spikes and submicromolar (<1 microM) and micromolar (1-15 microM) global Ca(2+) spikes (Ca(2+) waves). These observations indicate that subcellular gradients of IP(3) sensitivity underlie all forms of ACh-induced Ca(2+) spikes, and that the amplitude and extent of Ca(2+) spikes are determined by the concentration of IP(3). IP(3)-induced local Ca(2+) spikes exhibited similar time courses to those generated by ACh, supporting a role for Ca(2+)-induced Ca(2+) release in local Ca(2+) spikes. In contrast, IP(3)- induced global Ca(2+) spikes were consistently faster than those evoked with ACh at all concentrations of IP(3) and ACh, suggesting that production of IP(3) via phospholipase C was slow and limited the spread of the Ca(2+) spikes. Indeed, gradual photolysis of caged IP(3) reproduced ACh-induced slow Ca(2+) spikes. Thus, local and global Ca(2+) spikes involve distinct mechanisms, and the kinetics of global Ca(2+) spikes depends on that of IP(3) production particularly in those cells such as acinar cells where heterogeneity in IP(3) sensitivity plays critical role.     

The effect of fructan on membrane lipid organization and dynamics in the dry state

Fructans are a group of fructose-based oligo- and polysaccharides, which appear to be involved in membrane preservation during dehydration by interacting with the membrane lipids. To get further understanding of the protective mechanism, the consequences of the fructan-membrane lipid interaction for the molecular organization and dynamics in the dry state were studied. POPC and DMPC were investigated in the dry state by (2)H, (31)P NMR, and Fourier transform infrared spectroscopy using two types of fructan and dextran. The order-disorder transition temperature of dry POPC was reduced by 70 degrees C in the presence of fructan. Fructan increased the mobility of the acyl chains, but immobilized the lipid headgroup region. Most likely, fructans insert between the headgroups of lipids, thereby spacing the acyl chains. This results in a much lower phase transition temperature. The headgroup is immobilized by the interaction with fructan. The location of the interaction with the lipid headgroup is different for the inulin-type fructan compared to the levan-type fructan, since inulin shows interaction with the lipid phosphate group, whereas levan does not. Dextran did not influence the phase transition temperature of dry POPC showing that reduction of this temperature is not a general property of polysaccharides.     

A locally-induced increase in intracellular Ca propagates cell-to-cell in the presence of plasma membrane Ca ATPase inhibitors in non-excitable cells

Intercellular Ca²⁺ waves are commonly observed in many cell types. In non-excitable cells, intercellular Ca²⁺ waves are mediated by gap junctional diffusion of a Ca²⁺ mobilizing messenger such as IP₃. Since Ca²⁺ is heavily buffered in the cytosolic environment, it has been hypothesized that the contribution of the diffusion of Ca²⁺ to intercellular Ca²⁺ waves is limited. Here, we report that in the presence of plasma membrane Ca²⁺ ATPase inhibitors, locally-released Ca²⁺ from the flash-photolysis of caged-Ca²⁺ appeared to induce further Ca²⁺ release and were propagated from one cell to another, indicating that Ca²⁺ was self-amplified to mediate intercellular Ca²⁺ waves. Our findings support the notion that non-excitable cells can establish a highly excitable medium to communicate local responses with distant cells.     

The biochemical activation of T-type Ca2+ channels in HEK293 cells stably expressing alpha1G and Kir2.1 subunits

In order to investigate the currently unknown cellular signaling pathways of T-type Ca(2+) channels, we decided to construct a new cell line which would stably express alpha(1G) and Kir2.1 subunits in HEK293 cells (HEK293/alpha(1G)/Kir2.1). Compared to cells which only expressed alpha(1G) (HEK293/alpha(1G)), HEK293/alpha(1G)/Kir2.1 cells produced an enormous inward rectifying current which was blocked by external Ba(2+) and Cs(+) in a concentration-dependent manner. The expression of Kir2.1 channels contributed significantly to the shift of membrane potential from -12.2+/-2.8 to -57.3+/-3.7mV. However, biophysical and pharmacological properties of alpha(1G)-mediated Ca(2+) channels remained unaffected by the expression of Kir2.1 subunits, except for the enlarging of the window current region. Biochemical activation of alpha(1G) channels using 150mM KCl brought about an increase in [Ca(2+)](i), which was blocked by mibefradil, the T-type Ca(2+) channel blocker. These data suggest that the HEK293/alpha(1G)/Kir2.1 cell line would have potential uses in the study of T-type Ca(2)(+) channel-mediated signaling pathways and possibly useful in the development of new therapeutic drugs associated with T-type Ca(2)(+) channels.     

Mitochondria adjust Ca signaling regime to a pattern of stimulation in salivary acinar cells

The salivary acinar cells have unique Ca²⁺ signaling machinery that ensures an extensive secretion. The agonist-induced secretion is governed by Ca²⁺ signals originated from the endoplasmic reticulum (ER) followed by a store-operated Ca²⁺ entry (SOCE). During tasting and chewing food a frequency of parasympathetic stimulation increases up to ten fold, entailing cells to adapt its Ca²⁺ machinery to promote ER refilling and ensure sustained SOCE by yet unknown mechanism. By employing a combination of fluorescent Ca²⁺ imaging in the cytoplasm and inside cellular organelles (ER and mitochondria) we described the role of mitochondria in adjustment of Ca²⁺ signaling regime and ER refilling according to a pattern of agonist stimulation. Under the sustained stimulation, SOCE is increased proportionally to the degree of ER depletion. Cell adapts its Ca²⁺ handling system directing more Ca²⁺ into mitochondria via microdomains of high [Ca²⁺] providing positive feedback on SOCE while intra-mitochondrial tunneling provides adequate ER refilling. In the absence of an agonist, the bulk of ER refilling occurs through Ca²⁺-ATPase-mediated Ca²⁺ uptake within subplasmalemmal space. In conclusion, mitochondria play a key role in the maintenance of sustained SOCE and adequate ER refilling by regulating Ca²⁺ fluxes within the cell that may represent an intrinsic adaptation mechanism to ensure a long-lasting secretion.     

Mechanisms of signal transduction in photo-stimulated secretion in Phaeocystis globosa

Phaeocystis globosa, a leading agent in marine carbon cycling, releases its photosynthesized biopolymers via regulated exocytosis. Release is elicited by blue light and relayed by a characteristic cytosolic Ca(2+) signal. However, the source of Ca(2+) in these cells has not been established. The present studies indicate that Phaeocystis' secretory granules work as an intracellular Ca(2+) oscillator. Optical tomography reveals that photo-stimulation induces InsP(3)-triggered periodic lumenal [Ca(2+)] oscillations in the granule and corresponding out-of-phase cytosolic oscillations of [Ca(2+)] that trigger exocytosis. This Ca(2+) dynamics results from an interplay between the intragranular polyanionic matrix, and two Ca(2+)-sensitive ion channels located on the granule membrane: an InsP(3)-receptor-Ca(2+) channel, and an apamin-sensitive K(+) channel.     

Anoxic depolarization of hippocampal astrocytes: Possible modulation by P2X7 receptors

Current responses from CA1 neurons and stratum oriens astrocytes were recorded from hippocampal brain slices by means of the whole-cell patch-clamp technique. Anoxic depolarization (AD) was induced by an oxygen/glucose-deprived (OGD) medium also containing sodium iodoacetate and antimycin, in order to block glycolysis and oxidative phosphorylation, respectively. Anoxic depolarization has been reported to be due to the sudden increase of the extracellular K⁺ concentration and the accompanying explosive rise in glutamate concentration. We asked ourselves whether the release of ATP activating P2X7 receptors is also involved in the AD. Although, the AD was evoked in absolute synchrony in neurons and astrocytes, and the NMDA receptor antagonistic AP-5 depressed these responses, neither the non-selective P2 receptor antagonist PPADS, nor the highly selective P2X7 receptor antagonist A438079 interfered with the AD or its delay time in neurons/astrocytes after inducing chemical hypoxia. However, A438079, but not PPADS increased in astrocytes the slow inward current observed in a hypoxic medium. It is concluded that ATP co-released with glutamate by hypoxic stimulation has only a minor function in the present brain slice system.     

Oscillations of cytosolic free calcium in bombesin-stimulated HIT-T15 cells

The mechanism underlying the generation of cytosolic free Ca²⁺ ([Ca²⁺_i) oscillations by bombesin, a receptor agonist activating phospholipase C, in insulin secreting HIT-T15 cells was investigated. At 25 μM, 61% of cells displayed [Ca²⁺]_i oscillations with variable patterns. The bombesin-induced [Ca²⁺]_i oscillations could last more than 1 h and glucose was required for maintaining these [Ca²⁺ fluctuations. Bombesin-evoked [Ca²⁺]_i oscillations were dependent on extracellular Ca²⁺ entry and were attenuated by membrane hype rpolarization or by L-type Ca²⁺ channel blockers. These [Ca²⁺]_i oscillations were apparently not associated with fluctuations in plasma membrane Ca²⁺ permeability as monitored by the Mn²⁺ quenching technique. 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) and 4-chloro-m-cresol, which interfere with intracellular Ca²⁺ stores, respectively, by inhibiting Ca²⁺-ATPase of endoplasmic reticulum and by affecting Ca²⁺-induced Ca²⁺ release, disrupted bombesin-induced [Ca²⁺]_i oscillations. 4-chloro-m-resol raised [Ca²⁺]_i by mobilizing an intracellular Ca²⁺ pool, an effect not altered by ryanodine. Caffeine exerted complex actions on [Ca²⁺]_i It raised [Ca²⁺]_i by promoting Ca²⁺ entry while inhibiting bombesin-elicited [Ca²⁺]_i oscillations. Our results suggest that in bombesin-elicited [Ca²⁺]_i oscillations in HIT-T15 cells: (i) the oscillations originate primarily from intracellular Ca²⁺ stores; and (ii) the Ca²⁺ influx required for maintaining the oscillations is in part membrane potential-sensitive and not coordinated with [Ca²⁺]_i oscillations. The interplay between intracellular Ca²⁺ stores and voltage-sensitive and voltage-insensitive extracellular Ca²⁺ entry determines the [Ca²⁺]_i oscillations evoked by bombesin.     

Temporary membrane distortion of vascular smooth muscle cells is responsible for their apoptosis induced by platelet-activating factor-like oxidized phospholipids and their degradation product, lysophosphatidylcholine

To obtain information about the mechanism of apoptosis induced by oxidized low density lipoproteins (oxLDL) in atherosclerotic plaques, we examined the effects of lysophosphatidylcholine (LPC) and platelet-activating factor (PAF)-like lipids (PAF-LL), which can be derived from oxLDL, on rat vascular smooth muscle cells (VSMC). All the lipids with different structures examined induced apoptosis of VSMC, so we studied the mechanism of induction of apoptosis by LPC. LPC-induced apoptosis was inhibited by alpha-tocopherol (alpha-T) and cholesterol (Chol), but not by other antioxidants such as palmitoyl ascorbic acid and PAF receptor antagonist. The cells temporarily became spherical and highly permeable before induction of apoptosis, and their change in shape was prevented by alpha-T and Chol. From these results, we suggest that the apoptosis induced by oxLDL-derived phospholipids in VSMC is caused by temporary membrane distortion, not through specific receptors.     

Bcl-2 acts subsequent to and independent of Ca fluxes to inhibit apoptosis in thapsigargin- and glucocorticoidmtreated mouse lymphoma cells

The mechanism by which Bcl-2 inhibits apoptosis is unknown. One proposal is that Bcl-2 regulates intracellular Ca²⁺ fluxes thought to mediate apoptosis. In the present study, we investigated Bcl-2's mechanism of action by determining the effect of Bcl-2 on intracellular Ca²⁺ fluxes in the WEH17.2 mouse lymphoma cell line, which does not express Bcl-2, and its stable transfectant, which expresses a high level of Bcl-2. Treatment with the endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin produced marked alterations in intracellular Ca²⁺ homeostasis in both WEH17.2 and W.Hb12 cells, including elevation of free cytosolic Ca²⁺, endoplasmic reticulum Ca²⁺ pool depletion, capacitative entry of extracellular Ca²⁺, and increased loading of Ca²⁺ into mitochondria. Similar changes in intracellular Ca²⁺ occurred spontaneously in both cell lines following exponential growth. In both situations, W.Hb12 cells maintained optimal viability despite marked alterations in intracellular Ca ²⁺' whereas WEH17.2 cells underwent apoptosis. Treatment with the glucocorticoid hormone, dexamethasone, induced apoptosis in WEH17.2 cells, but not in W.HB12 cells, even though dexamethasone treatment did not alter intracellular Ca²⁺ homeostasis in either cell line. These findings indicate that Bcl-2 acts downstream from intracellular Ca ²⁺ fluxes in a pathway where Ca²⁺-dependent and Ca²⁺-independent death signals converge.     

Cold stress increases reactive oxygen species formation via TRPA1 activation in A549 cells

Reactive oxygen species (ROS) are responsible for lung damage during inhalation of cold air. However, the mechanism of the ROS production induced by cold stress in the lung is still unclear. In this work, we measured the changes of ROS and the cytosolic Ca²⁺ concentration ([Ca²⁺]_c) in A549 cell. We observed that cold stress (from 20 to 5 °C) exposure of A549 cell resulted in an increase of ROS and [Ca²⁺]_c, which was completely attenuated by removing Ca²⁺ from medium. Further experiments showed that cold-sensing transient receptor potential subfamily member 1 (TRPA1) agonist (allyl isothiocyanate, AITC) increased the production of ROS and the level of [Ca²⁺]_c in A549 cell. Moreover, HC-030031, a TRPA1 selective antagonist, significantly inhibited the enhanced ROS and [Ca²⁺]_c induced by AITC or cold stimulation, respectively. Taken together, these data demonstrated that TRPA1 activation played an important role in the enhanced production of ROS induced by cold stress in A549 cell.     

Allosteric voltage gating of potassium channels I. Mslo ionic currents in the absence of Ca(2+).

Activation of large conductance Ca(2+)-activated K(+) channels is controlled by both cytoplasmic Ca(2+) and membrane potential. To study the mechanism of voltage-dependent gating, we examined mSlo Ca(2+)-activated K(+) currents in excised macropatches from Xenopus oocytes in the virtual absence of Ca(2+) (<1 nM). In response to a voltage step, I(K) activates with an exponential time course, following a brief delay. The delay suggests that rapid transitions precede channel opening. The later exponential time course suggests that activation also involves a slower rate-limiting step. However, the time constant of I(K) relaxation [tau(I(K))] exhibits a complex voltage dependence that is inconsistent with models that contain a single rate limiting step. tau(I(K)) increases weakly with voltage from -500 to -20 mV, with an equivalent charge (z) of only 0.14 e, and displays a stronger voltage dependence from +30 to +140 mV (z = 0.49 e), which then decreases from +180 to +240 mV (z = -0.29 e). Similarly, the steady state G(K)-V relationship exhibits a maximum voltage dependence (z = 2 e) from 0 to +100 mV, and is weakly voltage dependent (z congruent with 0.4 e) at more negative voltages, where P(o) = 10(-5)-10(-6). These results can be understood in terms of a gating scheme where a central transition between a closed and an open conformation is allosterically regulated by the state of four independent and identical voltage sensors. In the absence of Ca(2+), this allosteric mechanism results in a gating scheme with five closed (C) and five open (O) states, where the majority of the channel's voltage dependence results from rapid C-C and O-O transitions, whereas the C-O transitions are rate limiting and weakly voltage dependent. These conclusions not only provide a framework for interpreting studies of large conductance Ca(2+)-activated K(+) channel voltage gating, but also have important implications for understanding the mechanism of Ca(2+) sensitivity.     

Regulation of K+ channels in the basolateral membrane ofNecturus oxyntic cells

Summary Patch-clamp methods were used to study single-channel events in isolated oxyntic cells and gastric glands fromNecturus maculosa. Cell-attached, excised inside-out and outside-out patches from the basolateral membrane frequently contained channels which had conductances of 67±21 pS in 24% of the patches and channels of smaller conductance, 33±6 pS in 56% of the patches. Channels in both classes were highly selective for K⁺ over Na⁺ and Cl^–, and shared linear current-voltage relations. The 67-pS channel was activated by membrane depolarization, whereas the activity of the 33-pS channel was relatively voltage independent. The larger conductance channels were activated by intracellular Ca²⁺ in the range between 5 and 500nm, but unaffected by cAMP. The smaller conductance channels were activated by cAMP, but not Ca²⁺. The presence of K⁺ channels in the basolateral membrane which are regulated by these known second messengers can account for the increase in conductance and the hyperpolarization of the membrane observed upon secretagogue stimulation.     

Sphingosine 1-phosphate receptors modulate intracellular Ca2+ homeostasis

Ligation of sphingosine 1-phosphate (S1P) to a set of specific receptors named S1P receptors (S1PRs) regulates important biological processes. Although the ability of S1P to increase cytosolic Ca2+ in various cell types is well known, the role of the individual S1PRs has not been fully characterized. Here, we provide a complete analysis of S1P-dependent intracellular Ca2+ homeostasis in HeLa cells. Overexpression of S1P2, or S1P3, but not S1P1, leads to a significant increase in cytosolic and mitochondrial [Ca2+] in response to S1P challenge. Moreover, cells ectopically expressing S1P2, or S1P3 exhibited an appreciable decrease of the free Ca2+ concentration in the endoplasmic reticulum, dependent on stimulation of receptors by S1P endogenously present in the culture medium which was accompanied by a reduced susceptibility to C2-ceramide-induced cell death. These results demonstrate a differential contribution of individual S1PRs to Ca2+ homeostasis and its possible implication in the regulation of cell survival.