Depletion Of Extracellular Ca Research Articles

Vibrio vulnificus RtxA1 modulated calcium flux contributes reduced internalization in phagocytes.

Vibrio vulnificusis an opportunistic pathogen that causes primary septicemia and wound infection with high mortality rate. This pathogen produces an RTX toxin (RtxA1) which can cause host cell rounding, cell death and interference with internalization by host phagocytes. However, the mechanism of RtxA1-induced phagocyte paralysis is not clear. Using the murine macrophage cell line RAW264.7, we measured cytotoxicity and phagocytosis of V. vulnificusin normal and calcium-depleted media. To deplete extracellular and cytosolic Ca(2+), cells were exposed to the calcium chelators ethylene glycol tetraacetic acid (EGTA) and 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl esteris (BAPTA-AM), respectively. The cytotoxicity was examined by measuring the activity of lactate dehydrogenase (LDH) released from the damaged cells. The gentamicin protection assay was conducted to determine the number of internalized bacteria, while acridine orange staining was applied to visualize the intracellular bacteria. The fluorescent indicator fura-2-acetoxymethyl ester (fura 2-AM) was used to measure the Ca(2+)signal post-infection. We revealed that extracellular Ca(2+)was essential for phagocytes to internalize V. vulnificus. Meanwhile, cytosolic Ca(2+)flux in RAW264.7 cells induced by an RtxA1 isogenic mutant was repressed by the parent strain. Furthermore, depletion of extracellular Ca(2+)level by EGTA significantly reduced the cytotoxicity but did not affect the antiphagocytic activity of RtxA1 toxin. Our results indicated that RtxA1 may interfere with cytosolic Ca(2+)flux of phagocyte to promote bacteria colonization.

The Ca2+ Channel Iinhibitor NNC 55-0396 Inhibits Voltage-Dependent K+ Channels in Rabbit Coronary Arterial Smooth Muscle Cells

We demonstrated the inhibitory effect of NNC 55-0396, a T-type Ca(2+) channel inhibitor, on voltage-dependent K(+) (K(V)) channels in freshly isolated rabbit coronary arterial smooth muscle cells. NNC 55-0396 decreased the amplitude of K(V) currents in a concentration-dependent manner, with an IC(50) of 0.080 μM and a Hill coefficient of 0.76.NNC 55-0396 did not affect steady-state activation and inactivation curves, indicating that the compound does not affect the voltage sensitivity of K(V) channel gating. Both the K(V) currents and the inhibitory effect of NNC 55-0396 on K(V) channels were not altered by depletion of extracellular Ca(2+) or intracellular ATP, suggesting that the inhibitory effect of NNC 55-0396 is independent of Ca(2+)-channel activity and phosphorylation-dependent signaling cascades. From these results, we concluded that NNC 55-0396 dosedependently inhibits K(V) currents, independently of Ca(2+)-channel activity and intracellular signaling cascades.

Measurement of cell respiration and oxygenation in standard multichannel biochips using phosphorescent O2-sensitive probes

Measurement of cell oxygenation and oxygen consumption is useful for studies of cell bioenergetics, metabolism, mitochondrial function, drug toxicity and common pathophysiological conditions. Here we present a new platform for such applications which uses commercial multichannel biochips (μ-slides, Ibidi) and phosphorescent O2 sensitive probes. This platform was evaluated with both extracellular and intracellular O2 probes, several different cell types and treatments including mitochondrial uncoupling and inhibition, depletion of extracellular Ca(2+) and inhibition of V-ATPase and histone deacetylases. The results show that compared to the standard microwell plates currently used, the μ-slide platform provides facile O2 measurements with both suspension and adherent cells, higher sensitivity and reproducibility, and faster measurement time. It also allows re-perfusion and multiple treatments of cells and multi-parametric analyses in conjunction with other probes. Optical measurements are conducted on standard fluorescence readers and microscopes.

CaV1.3 Channels and Intracellular Calcium Mediate Osmotic Stress-induced N-terminal c-Jun Kinase Activation and Disruption of Tight Junctions in Caco-2 Cell Monolayers

We investigated the role of a Ca(2+) channel and intracellular calcium concentration ([Ca(2+)](i)) in osmotic stress-induced JNK activation and tight junction disruption in Caco-2 cell monolayers. Osmotic stress-induced tight junction disruption was attenuated by 1,2-bis(2-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-mediated intracellular Ca(2+) depletion. Depletion of extracellular Ca(2+) at the apical surface, but not basolateral surface, also prevented tight junction disruption. Similarly, thapsigargin-mediated endoplasmic reticulum (ER) Ca(2+) depletion attenuated tight junction disruption. Thapsigargin or extracellular Ca(2+) depletion partially reduced osmotic stress-induced rise in [Ca(2+)](i), whereas thapsigargin and extracellular Ca(2+) depletion together resulted in almost complete loss of rise in [Ca(2+)](i). L-type Ca(2+) channel blockers (isradipine and diltiazem) or knockdown of the Ca(V)1.3 channel abrogated [Ca(2+)](i) rise and disruption of tight junction. Osmotic stress-induced JNK2 activation was abolished by BAPTA and isradipine, and partially reduced by extracellular Ca(2+) depletion, thapsigargin, or Ca(V)1.3 knockdown. Osmotic stress rapidly induced c-Src activation, which was significantly attenuated by BAPTA, isradipine, or extracellular Ca(2+) depletion. Tight junction disruption by osmotic stress was blocked by tyrosine kinase inhibitors (genistein and PP2) or siRNA-mediated knockdown of c-Src. Osmotic stress induced a robust increase in tyrosine phosphorylation of occludin, which was attenuated by BAPTA, SP600125 (JNK inhibitor), or PP2. These results demonstrate that Ca(V)1.3 and rise in [Ca(2+)](i) play a role in the mechanism of osmotic stress-induced tight junction disruption in an intestinal epithelial monolayer. [Ca(2+)](i) mediate osmotic stress-induced JNK activation and subsequent c-Src activation and tyrosine phosphorylation of tight junction proteins. Additionally, inositol 1,4,5-trisphosphate receptor-mediated release of ER Ca(2+) also contributes to osmotic stress-induced tight junction disruption.

Nonsteroidal Anti-Inflammatory Drug Flufenamic Acid Is a Potent Activator of AMP-Activated Protein Kinase

Flufenamic acid (FFA) is a nonsteroidal anti-inflammatory drug (NSAID). It has anti-inflammatory and antipyretic properties. In addition, it modulates multiple channel activities. The mechanisms underlying the pharmacological actions of FFA are presently unclear. Given that AMP-activated protein kinase (AMPK) has both anti-inflammatory and channel-regulating functions, we examined whether FFA induces AMPK activation. 1) Exposure of several different types of cells to FFA resulted in an elevation of AMPKα phosphorylation at Thr172. This effect of FFA was reproduced by functionally and structurally similar mefenamic acid, tolfenamic acid, niflumic acid, and meclofenamic acid. 2) FFA-induced activation of AMPK was largely abolished by the treatment of cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (an intracellular Ca(2+) chelator) or depletion of extracellular Ca(2+), whereas it was mimicked by stimulation of cells with the Ca(2+) ionophore 5-(methylamino)-2-({(2R,3R,6S,8S,9R,11R)-3,9,11-trimethyl-8-[(1S)-1-methyl-2-oxo-2-(1H-pyrrol-2-yl)ethyl]-1,7-dioxaspiro[5.5]undec-2-yl}methyl)-1,3-benzoxazole-4-carboxylic acid (A23187) or ionomycin. 3) FFA triggered a rise in intracellular Ca(2+), which was abolished by cyclosporine, a blocker of mitochondrial permeability transition pore. Cyclosporine also abolished FFA-induced activation of AMPK. 4) Inhibition of Ca(2+)/calmodulin-dependent kinase kinase β (CaMKKβ) with 7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid acetate (STO-609) or down-regulation of CaMKKβ with short interfering RNA largely abrogated FFA-induced activation of AMPK. 5) FFA significantly suppressed nuclear factor-κB activity and inducible nitric-oxide synthase expression triggered by interleukin-1β and tumor necrosis factor α. This suppression was also largely abrogated by STO-609. Taken together, we conclude that FFA induces AMPK activation through the Ca(2+)-CaMKKβ pathway. Activation of AMPK is a presently unrecognized important mechanism underlying the pharmacological effects of FFA.

Extracellular calcium depletion transiently elevates oxygen consumption in neurosecretory PC12 cells through activation of mitochondrial Na +/Ca 2+ exchange

Fluctuating extracellular Ca 2+ regulates many aspects of neuronal (patho)physiology including cell metabolism and respiration. Using fluorescence-based intracellular oxygen sensing technique, we demonstrate that depletion of extracellular Ca 2+ from 1.8 to ≤ 0.6 mM by chelation with EGTA induces a marked spike in O 2 consumption in differentiated PC12 cells. This respiratory response is associated with the reduction in cytosolic and mitochondrial Ca 2+, minor depolarization on the mitochondrial membrane, moderate depolarization of plasma membrane, and no changes in NAD(P)H and ATP. The response is linked to the influx of extracellular Na + and the subsequent activation of mitochondrial Na +/Ca 2+ and Na +/H + exchange. The mitochondrial Na +/Ca 2+ exchanger ( mNCX) activated by Na + influx reduces Ca 2+ and increases Na + levels in the mitochondrial matrix. The excess of Na + activates the mitochondrial Na +/H + exchanger (NHE) increasing the outward pumping of protons, electron transport and O 2 consumption. Reduction in extracellular Na + and inhibition of Na + influx through the receptor operated calcium channels and plasmalemmal NHE reduce the respiratory response. Inhibition of the mNCX, L-type voltage gated Ca 2+ channels or the release of Ca 2+ from the endoplasmic reticulum also reduces the respiratory spike, indicating that unimpaired intercompartmental Ca 2+ exchange is critical for response development.

Evidence for the possible involvement of the P2Y6 receptor in Ca2+ mobilization and insulin secretion in mouse pancreatic islets

Subtypes of purinergic receptors involved in modulation of cytoplasmic calcium ion concentration ([Ca2+]i) and insulin release in mouse pancreatic β-cells were examined in two systems, pancreatic islets in primary culture and beta-TC6 insulinoma cells. Both systems exhibited some physiological responses such as acetylcholine-stimulated [Ca2+]i rise via cytoplasmic Ca2+ mobilization. Addition of ATP, ADP, and 2-MeSADP (each 100 µM) transiently increased [Ca2+]i in single islets cultured in the presence of 5.5 mM (normal) glucose. The potent P2Y1 receptor agonist 2-MeSADP reduced insulin secretion significantly in islets cultured in the presence of high glucose (16.7 mM), whereas a slight stimulation occurred at 5.5 mM glucose. The selective P2Y6 receptor agonist UDP (200 µM) transiently increased [Ca2+]i and reduced insulin secretion at high glucose, whereas the P2Y2/4 receptor agonist UTP and adenosine receptor agonist NECA were inactive. [Ca2+]i transients induced by 2-MeSADP and UDP were antagonized by suramin (100 µM), U73122 (2 µM, PLC inhibitor), and 2-APB (10 or 30 µM, IP3 receptor antagonist), but neither by staurosporine (1 µM, PKC inhibitor) nor depletion of extracellular Ca2+. The effect of 2-MeSADP on [Ca2+]i was also significantly inhibited by MRS2500, a P2Y1 receptor antagonist. These results suggested that P2Y1 and P2Y6 receptor subtypes are involved in Ca2+ mobilization from intracellular stores and insulin release in mouse islets. In beta-TC6 cells, ATP, ADP, 2-MeSADP, and UDP transiently elevated [Ca2+]i and slightly decreased insulin secretion at normal glucose, while UTP and NECA were inactive. RT-PCR analysis detected mRNAs of P2Y1 and P2Y6, but not P2Y2 and P2Y4 receptors.

Gαq-TRPC6-mediated Ca2+ Entry Induces RhoA Activation and Resultant Endothelial Cell Shape Change in Response to Thrombin

RhoA activation and increased intracellular Ca(2+) concentration mediated by the activation of transient receptor potential channels (TRPC) both contribute to the thrombin-induced increase in endothelial cell contraction, cell shape change, and consequently to the mechanism of increased endothelial permeability. Herein, we addressed the possibility that TRPC signals RhoA activation and thereby contributes in actinomyosin-mediated endothelial cell contraction and increased endothelial permeability. Transduction of a constitutively active Galphaq mutant in human pulmonary arterial endothelial cells induced RhoA activity. Preventing the increase in intracellular Ca2+ concentration by the inhibitor of Galphaq or phospholipase C and the Ca2+ chelator, BAPTA-AM, abrogated thrombin-induced RhoA activation. Depletion of extracellular Ca2+ also inhibited RhoA activation, indicating the requirement of Ca2+ entry in the response. RhoA activation could not be ascribed to storeoperated Ca2+ (SOC) entry because SOC entry induced with thapsigargin or small interfering RNA-mediated inhibition of TRPC1 expression, the predominant SOC channel in these endothelial cells, failed to alter RhoA activity. However, activation of receptor-operated Ca2+ entry by oleoyl-2-acetyl-sn-glycerol, the membrane permeable analogue of the Galphaq-phospholipase C product diacylglycerol, induced RhoA activity. Receptor-operated Ca2+ activation was mediated by TRPC6 because small interfering RNA-induced TRPC6 knockdown significantly reduced Ca2+ entry. TRPC6 knockdown also prevented RhoA activation, myosin light chain phosphorylation, and actin stress fiber formation as well as inter-endothelial junctional gap formation in response to either oleoyl-2-acetyl-sn-glycerol or thrombin. TRPC6-mediated RhoA activity was shown to be dependent on PKCalpha activation. Our results demonstrate that Galphaq activation of TRPC6 signals the activation of PKCalpha, and thereby induces RhoA activity and endothelial cell contraction.

A novel neurotrophic role of secretory phospholipases A 2 for cerebellar granule neurons

Cultured cerebellar granule neurons (CGNs) require membrane depolarization or neurotrophic factors for their survival in vitro and undergo apoptosis when deprived of these survival-promoting stimuli. Here, we show that secretory phospholipases A 2s (sPLA 2s) rescue CGNs from apoptosis after potassium deprivation. The neurotrophic effect required the enzymatic activity of sPLA 2s, since catalytically inactive mutants of sPLA 2s failed to protect CGNs from apoptosis. Consistently, the ability of sPLA 2s to protect CGNs from apoptosis correlated with the extent of sPLA 2-induced arachidonic acid release from live CGNs. The survival-promoting effect of sPLA 2 was inhibited by depletion of extracellular Ca 2+ or by the presence of L-type Ca 2+ channel blocker nicardipine, suggesting that Ca 2+ influx occurs upon sPLA 2 treatment. Among the mammalian sPLA 2s tested, only group X sPLA 2, but not group IB nor IIA sPLA 2s, displayed neurotrophic activity. These results suggest a novel, unexpected neurotrophin-like role of sPLA 2 in the nervous system.

Human chymase stimulates Ca 2+ signaling in human polymorphonuclear cells

Human chymase is known to function as a chemoattractant for human leukocytes. To investigate the mechanism of the chymase-induced cell migration, change in intracellular calcium concentration ([Ca 2+]i) was examined in human polymorphonuclear (PMN) cells using Fluo-3 as a fluorescent Ca 2+ indicator. Treatment of PMN cells with human chymase caused [Ca 2+]i elevation in a concentration-dependent manner. Depletion of extracellular Ca 2+ from the medium partially attenuated the chymase-induced [Ca 2+]i increase, showing that both Ca 2+ influx and Ca 2+ release from internal stores might be involved in the [Ca 2+]i response. Pretreatment of the cells with pertussis toxin completely blocked the chymase-induced [Ca 2+]i signal, suggesting an involvement of G protein in the chymase-mediated [Ca 2+]i elevation. The data in the present study raise the possibility that the chymase-induced cell migration is mediated by the [Ca 2+]i elevation, which might be caused by stimulation of a G-protein-coupled receptor such as protease-activated receptors (PARs).

Extracellular Ca 2+ Depletion Contributes to Fast Activity-Dependent Modulation of Synaptic Transmission in the Brain

Synaptic activation is associated with rapid changes in intracellular Ca 2+, while the extracellular Ca 2+ level is generally assumed to be constant. Here, using a novel optical method to measure changes in extracellular Ca 2+ at high spatial and temporal resolution, we find that brief trains of synaptic transmission in hippocampal area CA1 induce transient depletion of extracellular Ca 2+. We show that this depletion, which depends on postsynaptic NMDA receptor activation, decreases the Ca 2+ available to enter individual presynaptic boutons of CA3 pyramidal cells. This in turn reduces the probability of consecutive synaptic releases at CA3−CA1 synapses and therefore contributes to short-term paired-pulse depression of minimal responses. This activity-dependent depletion of extracellular Ca 2+ represents a novel form of fast retrograde synaptic signaling that can modulate glutamatergic information transfer in the brain.

Physiology of sleep and wakefulness as it relates to the physiology of epilepsy.

This paper reviews the present knowledge about the cellular origins of vigilance states (wakefulness and slow-wave sleep) from the perspective of their involvement in the triggering of epileptic seizures. The data stem from intracellular recordings (most of them dual impalements of pairs of neurons and glia), extracellular ionic concentrations (mainly K and Ca ) and simultaneous intracortical field potentials from the cortex of cats. These data were corroborated with recordings from naturally sleeping animals and humans. It is shown that sleep is dominated by a cortically generated slow (<1 Hz) oscillation resulting from the complex interplay within networks of neurons and glia, which are modulated by the more diffuse action of extracellular currents of ions. Wakefulness is produced through the activation of brainstem and basal forebrain structures, which disrupt sleep oscillations and elicit a global change of the extraneuronal milieu, with profound modifications of glial and cerebral blood flow parameters. Paroxysmal events arising during quiet sleep evolve within the cortex from normal slow sleep oscillations. The synchronization of large cortical and eventually subcortical territories relies on the propagation of increased currents of K through the glial syncytium, which compensate for the reduced synaptic efficacy due to the depletion of extracellular Ca.

Attenuation of negatively regulated ANP secretion by calcium in hypertrophied atria.

Abnormal intracellular Ca(2+)-handling has been described in various heart diseases associated with cardiac hypertrophy. The crucial role of Ca(2+) in the excitation-secretion coupling in atrial cardiomyocytes is not well established. To investigate modulation of atrial natriuretic peptide (ANP) secretion regulated by Ca(2+) in hypertrophied atria, responsiveness of stretch-induced ANP to Ca(2+) was studied using isolated perfused quiescent hypertrophied rat atria. Male Sprague-Dawley rats were given a single subcutaneous injection of 50 mg/kg monocrotaline (MCT) and were sacrificed at 5-6 weeks. In isolated perfused hypertrophied right atria from MCT rats, changes in atrial volume induced by increased atrial pressure caused proportional increases in mechanically stimulated extracellular fluid (ECF) translocation and stretch-induced ANP secretion. Stretch-induced ANP secretion was markedly increased by the depletion of extracellular Ca(2+). However, an accentuation of stretch-induced ANP secretion by Ca(2+) depletion was markedly attenuated in hypertrophied right atria, as compared to control right atria. Therefore, stretch-induced ANP secretion in terms of ECF translocation by Ca(2+) depletion in hypertrophied atria was significantly lower than in control right atria. However, no significant differences were observed between nonhypertrophied and control left atria. Depletion of extracellular Ca(2+) caused a decrease in intracellular calcium in single beating atrial myocytes, which was significantly attenuated in hypertrophied atrial myocytes. The results suggest that attenuation of Ca(2+)-induced negative regulation of ANP secretion in hypertrophied atria may be due to the disturbance of intracellular Ca(2+) regulation.

Calcium transient and its propagation by poking a single human microvascular endothelial cell with a heat-blunt-ended micropipette

In order to elucidate signal transduction mechanism on how cells sense mechanical deformation, cytosolic Ca transients, by poking with a heat-blunted glass micropipette, were directly observed in human microvascular endothelial cell (HMEC). Cytosolic Ca transients were observed in almost all the HMECs poked with a micropipette. The transients started at the point of poking, and propagated at the peripheral regions of the poked cell. Those transients were also observed to propagate to the adjacent cells. Neither transient of poked cells nor propagation to adjacent cells was detected under the condition of depletion of extracellular Ca or intracellular Ca stores. Either stretch-activated channel or voltage-sensitive ion channel blockage inhibited significantly Ca transients. Phospholipase C inhibition, resulting in inhibition of inositol 1,4,5-triphosphate (IP 3) production, caused no Ca transient by poking. The results suggested that Ca influx might be a trigger for Ca release from intracellular Ca stores by IP 3 in the case of HMEC poking experiments.

The Role of Perisynaptic Glial Sheaths in Glutamate Spillover and Extracellular Ca 2+ Depletion

Recent findings suggest that rapid activation of extrasynaptic receptors and transient depletion of extracellular Ca 2+ may represent an important component of glutamatergic synaptic transmission. These phenomena imply a previously unrecognized role for synaptic glial sheaths: to retard extracellular diffusion in the synaptic vicinity. The present study is an attempt to assess the extent and physiological implications of this retardation using a detailed compartmental model of the typical synaptic environment. The model allows reconstruction of a partial (asymmetric) glial sheath covered with transporter molecules, which gives a more realistic representation of the vicinity of central synapses. Simulations show to what extent, in conditions compatible with physiology, the occupancy of synaptic receptors and the depletion of Ca 2+ in the cleft increase with increased glial coverage. The impact of glial sheaths on synaptic transmission is shown to become greater with smaller synapses and with slower kinetics of perisynaptic ion transients. At a calyceal synapse, a profound temporal filtering of fast Ca 2+ influx is found, and similar phenomena are predicted to occur following simultaneous activation of multiple synapses in the neuropil. The results provide a quantitative guidance for interpretation of physiological experiments that address fast transients of neurotransmitters and small ions in the brain tissue.

Characterization of a novel fungal protein, p15, which induces neuronal differentiation of PC12 cells.

In our previous paper, we reported that a 15 kDa protein (p15) produced by a fungus, genus Helicosporium, enhanced NGF-induced neurite outgrowth from PC12 cells. Here we further characterized the actions of p15. The complete amino acid sequence of p15 was determined and it was shown to be a hydrophilic protein composed of 118 amino acid residues with two intramolecular disulfide bridges. p15-induced neurite outgrowth was blocked by the depletion of extracellular Ca(2+) in the culture medium and was significantly inhibited by L-type Ca(2+) channel inhibitor nicardipine. p15 stimulated Src kinase and MAPK activities, and neurite outgrowth was not observed in srcDN2, a dominant negative c-src(K295R)-expressing cell line, and was significantly reduced in RasN17-expressing cells. These results suggest that p15 stimulates neurite outgrowth through the potentiation of L-type Ca(2+) channels, thereby activating the Src-Ras-MAPK cascade.

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 Ca 2+ concentration ([Ca 2] i) was recorded by Fura-2-based microfluorimetry. The deformation evoked elevation in [Ca 2+] 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 μm in diameter. The largest mechanosensitive cell group was that of cells 20–25 μm in diameter; 56% of the mechanosensitive cells were of this cell size. All of the cells larger than 25 μm in diameter displayed the Ca 2+ increase when prodded. The depletion of extracellular Ca 2+ diminished the Ca 2+ elevation. Verapamil and nickel, blockers of voltage-dependent Ca 2+ channels, did not influence the Ca 2+ 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 Ca 2+ influx in large dorsal root ganglion neurons through mechanosensitive Ca 2+-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 Ca 2+ concentration ([Ca 2+] 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 μm (mean±S.D., n=25). The lowering of the temperature from 35°C to 20°C increased [Ca 2+] i from 59.6±10.6 nM to 203.4±14.8 nM ( n=25). The [Ca 2+] i response was dependent on the intensity of the cold stimulation. The depletion of extracellular Ca 2+ diminished the Ca 2+ elevation. However, a Na +-free condition did not influence the response. We concluded that the cold stimulation opens Ca 2+-permeable channels in putative cold cells from dorsal root ganglion neurons.

Glucose and non-glucidic nutrients exert permissive effects on 2-keto acid regulation of pancreatic β-cell function

Insulin-releasing effects of straight and branched chain 2-keto acids were assessed using clonal glucose-responsive β-cells. Pyruvic acid (PA), 2-ketovaleric acid (KV), 2-ketoisovaleric acid (KIV) or 2-keto-3-methylvaleric acid (KMV) dose-dependently promoted the stimulatory effects of D-glucose, whereas 2-ketobutyric acid (KB) did not affect insulin release. The stimulatory 2-keto acids also promoted the stimulatory activity of D-glyceraldehyde, L-leucine or L-arginine. Responses to PA, KV, KIV or KMV were significantly reduced by transport inhibition with 2-cyano-3 hydroxycinnamate, glucokinase inhibition with mannoheptulose or metabolic inhibition with sodium azide or sodium cyanide. Membrane hyperpolarisation with K + depletion or diazoxide reduced insulin output, but failed to abolish secretory responses to KV, KIV and KMV. Secretory effects of these 2-keto acids also persisted in β-cells depolarised with high KCl and glucose. Voltage-dependent Ca 2+ channel blockade, with verapamil, or depletion of extracellular Ca 2+ abolished the secretory activity of 2-keto acids. Collectively, these results indicate that glucose and metabolisable nutrients exert permissive effects on 2-keto acid-induced insulin release. In addition, KV, KIV and KMV can regulate β-cell function at least partially independently of K +-ATP channel activity, both through their mitochondrial metabolism and regulation of Ca 2+ influx.

Warm cells revealed by microfluorimetry of Ca 2+ in cultured dorsal root ganglion neurons

Warm cells were identified by Fura-PE3-based microfluorimetry of Ca 2+ in cultured dorsal root ganglion (DRG) neurons. In response to a physiologically relevant stimulus temperature (43°C), a subpopulation of small DRG neurons from new born rats increased the intracellular Ca 2+ concentration ([Ca 2+] i). Seven percent of the cells responded to the warm stimulus. The stimulus evoked elevation in [Ca 2+] i from 52.5±9.5 nM (mean±S.D., n=18) to 171.0±15.6 nM in cells between 15 and 25 μm in diameter. The depletion of extracellular Ca 2+ diminished the Ca 2+ elevation. The Na +-free condition also diminished the response. We concluded that the heat stimulation opens nonselective cation channels in putative warm cells from DRG neurons.