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.     

Regulation of action potential size and excitability in substantia nigra compacta neurons: sensitivity to 4-aminopyridine

Application of the metabotropic glutamate receptor (mGluR) agonist (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) or the selective group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) depolarized both CA3 and CA1 pyramidal cells in guinea pig hippocampal slices. Simultaneous recordings of voltage and intracellular Ca2+ levels revealed that the depolarization was accompanied by a biphasic elevation of intracellular Ca2+ concentration ([Ca2+]i): a transient calcium rise followed by a delayed, sustained elevation. The transient [Ca2+]i rise was independent of the membrane potential and was blocked when caffeine was added to the perfusing solution. The sustained [Ca2+]i rise appeared when membrane depolarization reached threshold for voltage-gated Ca2+ influx and was suppressed by membrane hyperpolarization. The depolarization was associated with an increased input resistance and persisted when either the transient or sustained [Ca2+]i responses was blocked. mGluR-mediated voltage and [Ca2+]i responses were blocked by (+)-alpha-methyl-4-carboxyphenylglycine (MCPG) or (S)-4-carboxy-3-hydroxyphenylglycine (4C3HPG). These data suggest that in both CA3 and CA1 hippocampal cells, activation of group I mGluRs produced a biphasic accumulation of [Ca2+]i via two paths: a transient release from intracellular stores, and subsequently, by influx through voltage-gated Ca2+ channels. The concurrent mGluR-induced membrane depolarization was not caused by the [Ca2+]i rise.     

Mechanisms of GABA and glycine depolarization-induced calcium transients in rat dorsal horn neurons.

1. The mechanisms and effects of GABA- and glycine-evoked depolarization were studied in cultured rat dorsal horn neurons using indo-1 recordings of [Ca2+]i and patch clamp recordings in conventional whole-cell or perforated-patch mode. 2. Application of GABA to unclamped neurons caused [Ca2+]i increases that were dose dependent and exhibited GABAA receptor pharmacology. Calcium entered the neurons via high-threshold voltage-gated calcium channels (conotoxin and nimodipine sensitive). 3. In perforated-patch recordings employing cation-selective ionophores, GABAA receptor activation depolarized 123 of 132 cells to membrane potentials as depolarized as -33 mV (mean -50 mV in all 132 cells, +12 mV above resting potential). The ionic basis of the depolarization was determined by extracellular ion substitution; increased anionic conductance could account fully for the results. 4. Glycine, acting at a strychnine-sensitive receptor, also caused Ca2+ entry into these neurons through voltage-gated Ca2+ channels. Glycine and GABA both evoked [Ca2+]i responses in the same cells and the responses were highly correlated in amplitude. Glycine also depolarized all five cells tested with perforated recording. Each of the five cells was also depolarized by muscimol to a value similar to that obtained for glycine. 5. Both the depolarization and the increases in [Ca2+]i caused by GABA and glycine could potentially play a role in processes of development and differentiation and sensory transmission in the spinal cord dorsal horn.     

Mechanical stimuli induce intracellular calcium response in a subpopulation of cultured rat sensory neurons.

Cultured dorsal root ganglion neurons from newborn rats were mechanically deformed with a fine-tipped glass capillary, and the change in the intracellular Ca2+ concentration ([Ca2+]i) was recorded by Fura-2-based microfluorimetry. The deformation evoked elevation in [Ca2+]i from 18.7 +/- 5.4 nM (mean +/- S.E.M., n = 35) to 137.1 +/- 15.2 nM in some subpopulations of cells, especially those larger than 20 microm in diameter. The largest mechanosensitive cell group was that of cells 20-25 microm in diameter; 56% of the mechanosensitive cells were of this cell size. All of the cells larger than 25 microm in diameter displayed the Ca2+ increase when prodded. The depletion of extracellular Ca2+ diminished the Ca2+ elevation. Verapamil and nickel, blockers of voltage-dependent Ca2+ channels, did not influence the Ca2+ response, whereas gadolinium, a relatively selective blocker of mechanosensitive channels, diminished the response. Na+-free conditions did not influence the response. We concluded that the mechanical stimulation induced a Ca2+ influx in large dorsal root ganglion neurons through mechanosensitive Ca2+-permeable channels.     

Calcium signaling in cold cells studied in cultured dorsal root ganglion neurons.

Thermosensitive cold cells were identified in cultured dorsal root ganglion neurons from newborn rats. The neurons were loaded with a calcium indicator, Fura-PE3, and the change in intracellular Ca2+ concentration ([Ca2+]i) of the neurons was measured with microfluorimetry. Thirteen per cent of the cells responded to the cold stimulation. The diameter of the responder cells was 16.3+/-3.2 microm (mean+/-S.D., n = 25). The lowering of the temperature from 35 degrees C to 20 degrees C increased [Ca2+]i from 59.6+/-10.6 nM to 203.4+/-14.8 nM (n = 25). The [Ca2+]i response was dependent on the intensity of the cold stimulation. The depletion of extracellular Ca2+ diminished the Ca2+ elevation. However, a Na(+)-free condition did not influence the response. We concluded that the cold stimulation opens Ca2(+)-permeable channels in putative cold cells from dorsal root ganglion neurons.     

Role of TRP channels and NCX in mediating hypoxia-induced [Ca(2+)](i) elevation in PC12 cells

Mammalian cells require a constant O2 supply to produce adequate energy, and sustained hypoxia can kill cells. Mammals therefore have evolved sophisticated mechanisms to allow their cells to adapt to hypoxia. In this study, we investigated the role of TRP channels and the Na+-Ca2+ exchanger (NCX) in mediating hypoxia-induced [Ca2+]i elevation in a model of the O2-sensing rat pheochromocytoma (PC12) cell line by using Ca2+ imaging and molecular biological approaches. Non-selective cation channels, such as TRPC1, 3 and 6, were found to be functionally expressed in PC12 cells. They mediated Ca2+ entry when cells were exposed to acute hypoxia (PO2 of 15 mmHg), in addition to Ca2+ entry via VGCCs. Blockage of TRPCs by 2APB and SKF96365 could significantly reduce hypoxia-mediated [Ca2+]i elevation. Suramin and U73122 attenuated the hypoxia-induced [Ca2+]i elevation, implying the involvement of the G-protein and PLC pathways in the hypoxic response. In addition to TRPCs and VGCCs, NCX also contributed to the hypoxia-induced [Ca2+]i elevation, and blockade of NCX by KBR7943 could significantly decrease the hypoxia-induced [Ca2+]i elevation. Our results suggest that the activation of TRP by hypoxia could lead to NCX reversal; furthermore, membrane depolarization and TRPCs may play a primary role in mediating the hypoxic response in PC12 cells.     

Relationships between intracellular calcium and afterhyperpolarizations in neocortical pyramidal neurons

We examined the effects of recent discharge activity on [Ca2+]i in neocortical pyramidal cells. Our data confirm and extend the observation that there is a linear relationship between plateau [Ca2+]i and firing frequency in soma and proximal apical dendrites. The rise in [Ca2+] activates K+ channels underlying the afterhyperpolarization (AHP), which consists of 2 Ca(2+)-dependent components: the medium AHP (mAHP) and the slow AHP (sAHP). The mAHP is blocked by apamin, indicating involvement of SK-type Ca(2+)-dependent K+ channels. The identity of the apamin-insensitive sAHP channel is unknown. We compared the sAHP and the mAHP with regard to: 1) number and frequency of spikes versus AHP amplitude; 2) number and frequency of spikes versus [Ca2+]i; 3) IAHP versus [Ca2+]i. Our data suggest that sAHP channels require an elevation of [Ca2+]i in the cytoplasm, rather than at the membrane, consistent with a role for a cytoplasmic intermediate between Ca2+ and the K+ channels. The mAHP channels appear to respond to a restricted Ca2+ domain.     

Calcium regulates L-Type Ca2+ channel expression in rat skeletal muscle cells

Primary skeletal muscle cells were cultured in a normal- (1.8 mM) or high- (4.8 mM) Ca2+ culture medium to determine whether Ca2+ modulates the number of L-type Ca2+ channels. Skeletal myoballs cultured in a normal medium showed, when exposed to a high extracellular [Ca2+], ([Ca2+]e) a transient increase in intracellular [Ca2+] ([Ca2+]i) from a resting concentration of 60 to 160 nM. By day 3, however, when the experiments were made, [Ca2+]i no longer differed from control (pre-exposure to high Ca2+). The maximum charge movements in myoballs incubated in 1.8 and 4.8 mM were 16.4+/-1.05 (n=56) and 24.1+/-1.18 nC/microF (n=58; P<0.01), respectively, and peak Ca2+ currents at 20 mV were -10.8+/-1.09 (n=46) and -12.8+/-0.75 nA/microF (n=82), respectively (P>0.05). The tail current amplitudes in 1.8 and 4.8 mM Ca2+-treated cells were -9.3+/-1.23 and -14.2+/-1.37 nA/microF (P<0.05), respectively, at 10 mV and -15.3+/-1.76 and -23.6+/-2.02 nA/microF (P<0.05), respectively at 60 mV. The maximum binding of [3H]PN200-110 (a radioligand specific for L-type Ca2+ channel alpha1 subunits) in myoballs cultured in 1.8 and 4.8 mM [Ca2+]e was 1.34+/-0.23 and 3.2+/-0.63 pmol/mg protein (n=8; P<0.02), respectively. The increase in [Ca2+]i associated with the increases in charge movements, tail currents and the number of L-type Ca2+ channel alpha1 subunits in skeletal muscle cells cultured in high [Ca2+]e support the concept that extracellular Ca2+ influx modulates the expression of L-type Ca2+ channels in skeletal muscle cells.     

Functional expression of a subtype of the Ca2+ channels in the embryonic rat hippocampus as detected by dual-fluorescence flow cytometric analysis

We have analyzed the expression of the Ca2+ channels in hippocampal cell suspensions from the 18- to 20-day-old rat embryo using dual-fluorescence flow cytometry. A high concentration of K+ induced elevation of the cytoplasmic free Ca2+ concentration ([Ca2+]i) as well as membrane depolarization. The high K+-evoked [Ca2+]i increase was inhibited by phenytoin, but not by either nifedipine or nicardipine. These agents had no effect on the high K+-induced membrane depolarization. These findings suggest that a subtype corresponding to the low voltage-activated Ca2+ channel is expressed in the embryonic rat hippocampal cells.     

Characterisation of explanted endothelial cells from mouse aorta: electrophysiology and Ca2+ signalling

We describe here the isolation and primary culture of endothelial cells from mouse aorta ("primary explant technique"). These cells provide an excellent model for functional studies in transgenic mice. The primary explant method delivers cells that grow out from small pieces of mouse aorta placed on Matrigel enriched with endothelial growth factors. Cells can be studied on the Matrigel after removing the pieces of aorta or after passages by using dispase and reseeding the cells on gelatine-coated cover-slips. Cells on Matrigel or from the first and second passages were characterised using the combined patch-clamp and fura-2 fluorescence methods. Cells had a mean membrane resting potential of -19+/-3 mV (n=21), a membrane capacitance of 49+/-5 pF (n=37) and a resting cytosolic free [Ca2+] ([Ca2+]i) of 103+/-8 nM (n=30). Adenosine 5'-triphosphate (ATP), acetylcholine and bradykinin, but not histamine, induced fast release of intracellular Ca2+ followed by a sustained rise in [Ca2+]i. Oscillations in [Ca2+]i were observed at lower agonist concentrations. In nearly all cells (93%, n=30), these agonists activated charybdotoxin-sensitive, Ca2+-activated K+ channels and induced hyperpolarisation. In 84% of the cells (n=32), an increase in [Ca2+]i also activated strongly outwards-rectifying Cl- channels. These activated slowly at positive potentials and inactivated rapidly at negative potentials. Increasing [Ca2+]i to 1 microM activated a non-selective cation channel in 86% of the cells (n=28). Each tested cell responded to a challenge with hypotonic solution by activating a Cl- current that was modestly outwards rectifying and inactivated at positive potentials. This current is similar to the well-described swelling-activated current through volume-regulated anion channels (VRAC) in endothelial cells. However, its activation is slower, its inactivation faster and the current density lower than in cultured endothelial cells. It is concluded that the primary explant technique provides a reliable cell model for studying mouse vascular endothelial cell function.     

N-methyl-D-aspartate receptors enhance mechanical responses and voltage-dependent Ca2+ channels in rat dorsal root ganglia neurons through protein kinase C

N-methyl-D-aspartate (NMDA)receptors (NMDARs) located on peripheral terminals of primary afferents are involved in the transduction of noxious mechanical stimuli. Exploiting the fact that both NMDARs and stretch-activated channels are retained in short-term culture and expressed on the soma of dorsal root ganglia (DRG) neurons, we examined the effect of NMDA on mechanically mediated changes in intracellular calcium concentration ([Ca2+]i). Our aims were to determine whether NMDARs modulate the mechanosensitivity of DRG neurons. Primary cultures of adult rat lumbosacral DRG cells were cultured for 1-3 days. [Ca2+]i responses were determined by Fura-2 ratio fluorescence. Somas were mechanically stimulated with fire-polished glass pipettes that depressed the cell membrane for 0.5 s. Voltage-activated inward Ca2+ currents were measured by the whole cell patch clamp. Stimulation of neurons with 100 microM NMDA in the presence, but not the absence, of co-agonist (10 microM D-serine) caused transient [Ca2+]i responses (101+/-9 nM) and potentiated [Ca2+]i peak responses to subsequent mechanical stimulation more than two-fold (P < 0.001). NMDA-mediated potentiation of mechanically induced [Ca2+]i responses was inhibited by the selective protein kinase C (PKC) inhibitor GF109203X (GFX; 10 microM), which had no independent effects on NMDA- or mechanically induced responses. Short-term treatment with the PKC activator phorbol dibutyrate (1 microM PDBu for 1-2 min) also potentiated mechanically induced [Ca2+]i responses nearly two-fold (P < 0.001), while longer exposure (>10 min) inhibited the [Ca2+]i transients by 44% (P < 0.001). Both effects of PDBu were prevented by prior treatment with GFX. Inhibition of voltage-dependent Ca2+ channels with 25 microM La3+ had no effect on mechanically induced [Ca2+]i transients prior to NMDA, but prevented enhancement of the transients by NMDA and PDBu. NMDA pretreatment transiently enhanced nifedipine-sensitive, voltage-activated Ca2+ currents by a process that was sensitive to GFX. In conclusion, activation of NMDARs on cultured DRG neurons sensitize voltage-dependent L-type Ca2+ channels which contribute to mechanically induced [Ca2+]i transients through a PKC-mediated process.     

Reciprocal interactions between calcium and chloride in rod photoreceptors

This study used imaging and electrophysiological techniques in salamander retinal slices to correlate Ca2+ and Cl- levels in rods and thus test the hypothesis of a feedback interaction between Ca2+- and Ca2+-activated Cl- channels whereby Cl- efflux through Cl- channels can inhibit Ca2+ channels. Increasing [K+]o levels produced a concentration-dependent depolarization of rods accompanied by increases in [Ca2+]i measured with Fura-2. The voltage dependence of increases in [Ca2+]i was compared with the voltage dependence of the calcium current (ICa). [Cl-]i was measured with the dye, MEQ. Depolarization with high K+ to membrane potentials below -20 mV reduced [Cl-]i; larger depolarizations increased [Cl-]i. The Na/K/Cl cotransport inhibitor, bumetanide, shifted the apparent Cl- equilibrium potential (ECl) to more negative potentials, suggesting that this cotransporter helps establish a relatively depolarized ECl. MEQ fluorescence changes evoked by high K+ were inhibited by niflumic acid (0.1 mM), NPPB (2 microM), or replacement of Ca2+ with Ba2+, suggesting that depolarization-evoked Cl- changes result partly from stimulation of Ca2+-activated Cl- channels. Replacing >/=12 mM [Cl-]o with CH3SO4- produced a significant reduction in [Cl-]i. [Ca2+]i increases evoked by 20 or 50 mM K+ were also significantly inhibited by replacing >/=12 mM [Cl-]o with CH3SO4-. Thus modest depolarization can evoke increases in [Ca2+]i that lead to reductions in [Cl-]i, and conversely, reductions in [Cl-]i inhibit depolarization-evoked [Ca2+]i increases. These findings support the hypothesis that feedback interactions between Ca2+- and Ca2+-activated Cl- channels may contribute to the regulation of presynaptic Ca2+ currents involved in synaptic transmission from rod photoreceptors.     

Voltage-gated calcium channels mediate intracellular calcium increase in weaver dopaminergic neurons during stimulation of D2 and GABAB receptors

The weaver (wv) mutation affects the pore-forming region of the inwardly rectifying potassium channel (GIRK) leading to degeneration of cerebellar granule and midbrain dopaminergic neurons. The mutated channel (wvGIRK) loses its potassium selectivity, allowing sodium (Na+) and possibly calcium ions (Ca2+) to enter the cell. Here we performed whole cell patch-clamp recordings combined with microfluorometry to investigate possible differences in calcium ([Ca2+]i) dynamics in native dopaminergic neurons (expressing the wvGIRK2 subunits) in the midbrain slice preparation from homozygous weaver (wv/wv) and control (+/+) mice. Under resting conditions, [Ca2+]i was similar in wv/wv compared with +/+ neurons. Activation of wvGIRK2 channels by D2 and GABAB receptors increased [Ca2+]i in wv/wv neurons, whereas activation of wild-type channels decreased [Ca2+]i in +/+ neurons. The calcium rise in wv/wv neurons was abolished by antagonists of the voltage-gated calcium channels (VGCC); voltage clamp of the neuron at -60 mV; and hyperpolarization of the neuron to -80 mV or more, in current clamp, and was unaffected by TTX. Therefore we propose that wvGIRK2 channels in native dopamine neurons are not permeable to Ca2+, and when activated by D2 and GABAB receptors they mediate membrane depolarization and an indirect Ca2+ influx through VGCC rather than via wvGIRK2 channels. Such calcium influx may be the trigger for calcium-mediated excitotoxicity, responsible for selective neuronal death in weaver mice.     

Role of Na+/H+ exchangers, excitatory amino acid receptors and voltage-operated Ca2+ channels in human immunodeficiency virus type 1 gp120-mediated increases in intracellular Ca2+ in human neurons and astrocytes.

Human immunodeficiency virus type 1 (HIV-1) dementia is the commonest form of dementia in North American people less than 60 years of age. HIV-1 envelope glycoprotein gp120 has been implicated in the neurotoxicity observed in, and the pathogenesis of, HIV-1 dementia. Recombinant gp120 (gp120) was pressure-applied on to cultured human fetal neurons and astrocytes and, by using single-cell calcium imaging, we determined the mechanisms responsible for gp120-induced increases in the levels of intracellular calcium ([Ca2+]i). Significant dose-related increases in [Ca2+]i were observed in neurons and astrocytes. In neurons, 5 pM gp120 increased [Ca2+]i by 290+/-13 nM and increases of 2210+/-211 nM were found at 209 nM, the highest concentration of gp120 tested. The apparent EC50 value for gp120 of 223+/-40 pM in neurons was not significantly different from that in astrocytes. Immunoelution of gp120 with polyclonal anti-gp120 and Ca2+-free conditions blocked increases in [Ca2+]i by gp120. Increases in [Ca2+]i were significantly (P < 0.005) attenuated by the Na+/H+ exchange blocker 5-(N-methyl-N-isobutyl)-amiloride in neurons and astrocytes. The L-type calcium channel blockers nimodipine, diltiazem and CdCl2 + NiCl2 significantly (P < 0.005) reduced increases in [Ca2+]i in neurons, but not astrocytes. Increases in [Ca2+]i by gp120 were not significantly affected by blockers of N-, P- and Q-type calcium channels. The N-methyl-D-aspartate receptor antagonists (+/-)-2-amino-5-phosphonopentanoic acid (AP5), memantine and dizocilpine significantly (P < 0.01) lowered gp120-induced increases in [Ca2+]i in neurons. AP5 and memantine, but not dizocilpine, significantly (P < 0.01) reduced increases in [Ca2+]i by gp120 in astrocytes. Gp120 appears to activate astrocyte Na+/H+ exchangers to release glutamate and potassium and, subsequent to this, increases in [Ca2+]i in neurons and astrocytes result from activation of excitatory amino acid receptors on astrocytes and neurons, and voltage-operated calcium channels on neurons. Drugs that block gp120-induced changes in [Ca2+]i in neurons and astrocytes may help in the treatment of HIV-1 dementia.     

Somatostatin inhibits prolactin secretion by multiple mechanisms involving a site of action distal to increased cyclic adenosine 3',5'-monophosphate and elevated cytosolic Ca2+ in rat lactotrophs

The release of prolactin (PRL) from a clonal cell-line of anterior pituitary cells (GH4C1) was inhibited by somatostatin (SRIH) in a dose-dependent manner (ED50 nM). The inhibition (20% of control levels) was detectable within 50 s and maximal within 90 s. Thyroliberin (TRH) enhancement of PRL secretion was biphasic. SRIH inhibited both phases equally. Ionomycin in combination with the phorbol ester, TPA, mimics the TRH-elicited PRL release, and SRIH partly inhibited this effect. SRIH had no effect on TRH-stimulated formation of inositol trisphosphate, and only small effects on TRH-activated adenylate cyclase. Vasoactive intestinal peptide (VIP) and forskolin stimulated cAMP formation and PRL release potently. SRIH inhibited both effects of VIP and forskolin, and there was a close correlation between the inhibition of PRL secretion and cAMP accumulation. 8-Bromo-cAMP enhanced PRL release, an effect that was also partly reduced by SRIH. The Ca2+ channel activator, BAY-K-8644 and high extracellular K+ increased PRL release, and SRIH caused a partial reduction in the release response to both secretagogues. SRIH lowered [Ca2+]i, and markedly reduced the rise in [Ca2+]i elicited by TRH, VIP and K+. SRIH did not influence the Ca2+ spikes recorded in Na+-free solution, and had no effect on the TRH-induced membrane potential changes. Our results demonstrate that SRIH may inhibit PRL release from GH4C1 cells by (1) inhibiting hormone-sensitive adenylate cyclase, (2) blocking the effect of cAMP and (3) lowering [Ca2+]i. None of these effects is, however, sufficient to explain all the effects of SRIH, suggesting that SRIH also exerts a major action at a step subsequent to cAMP accumulation and [Ca2+]i elevation. Since the GH4C1 cells possess one single class of binding sites, this implies that the same SRIH receptor is coupled to several cellular signalling systems.     

GABA-induced intracellular Ca2+ elevation in the embryonic chick brainstem slice

We examined the intracellular Ca2+ ([Ca2+]i) elevation evoked by GABA in an 8-day embryonic chick brainstem slice using a Ca imaging technique with Ca green-1 AM. When small quantities of GABA were pressure-ejected on the surface of the slice, the [Ca2+]i elevation was clearly detected. The GABA-induced [Ca2+]i elevation was eliminated in a Ca2+-free solution, whereas the previously reported GABA-induced light-scattering change was independent of extracellular Ca2+. Although, micro-application of glycine or glutamate also induced [Ca2+]i elevation, these changes were smaller than that by GABA. These results suggest that the GABA-induced [Ca2+]i elevation is due to Ca2+ entry resulting from membrane depolarization and may play an important role in the development of the central nervous system (CNS).     

Effect of streptozotocin-induced diabetes on the activity of calcium channels in rat dorsal horn neurons

We have previously found that spinal dorsal horn neurons from streptozotocin-diabetic rats, an animal model for diabetes mellitus, show the prominent changes in the mechanisms responsible for [Ca2+]i regulation. The present study aimed to further characterize the effects of streptozotocin-induced diabetes on neuronal calcium homeostasis. The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured in Fura-2AM-loaded dorsal horn neurons from acutely isolated spinal cord slices using fluorescence technique. We studied Ca2+ entry through plasmalemmal Ca2+ channels during potassium (50 mM KCl)-induced depolarization. The K+-induced [Ca2+]i elevation was inhibited to a different extent by nickel ions, nifedipine and omega-conotoxin suggesting the co-expression of different subtypes of plasmalemmal voltage-gated Ca2+ channels. The suppression of [Ca2+]i transients by Ni2+ (50 microM) was the same in control and diabetic neurons. On the other hand, inhibition of [Ca2+]i transients by nifedipine (50 microM) and omega-conotoxin (1 microM) was much greater in diabetic neurons compared with normal animals. These data suggest that under diabetic conditions the activity of N- and L- but not T-type voltage-gated Ca2+ channels substantially increased in dorsal horn neurons.     

Inhibition of Ca2+ influx by surfactant in NR8383 alveolar macrophages

OBJECTIVE: Pulmonary surfactants reduce alveolar surface tension and alter inflammatory cell function. We studied the effects of surfactant preparations on Ca2+ influx regulated by protein kinase C (PKC) and mitogen-activated protein kinases (MAPK) and cytokine secretion in the alveolar macrophage (AM) cell line NR8383.METHODS: Fura-2-loaded AMs were stimulated with zymosan (200 microg/ml), 1,2-dioctanoyl-sn-glycerol (DOG, 20 microM) or C6-ceramide (C6C, 10 microM) in the presence of exogenous surfactants (beractant, calfactant or colfosceril) or surfactant phospholipid (dipalmitoyl phosphatidylcholine, DPPC), at 250 microg/ml phospholipid and changes in cytosolic free Ca2+ (Delta[Ca2+]i) and cytokines were measured. RESULTS: Zymosan-induced Delta[Ca2+]i (117 +/- 5 nM) at 3 min was reduced (p <0.001) by beractant (50 +/- 6 nM), colfosceril (61 +/- 2 nM), calfactant (46 +/- 5 nM), and DPPC (52 +/- 5 nM). Beractant inhibited the Delta[Ca2+]i by PKC stimulation with DOG and all preparations reduced the MAPK-induced Ca2+ influx by C6C. Beractant and Ca2+ channel blocker SKF 96365 (10 microM) together abolished the zymosan-stimulated Delta[Ca2+]i. Zymosan-stimulated TNF-alpha and IL-1beta secretion was also inhibited by surfactant pretreatment. CONCLUSIONS: These results indicate that exogenous surfactant inhibits Ca2+ influx and cytokine secretion in zymosan-stimulated AMs. This anti-inflammatory activity may be through an interaction with downstream signaling elements or Ca2+ channels.