Ketamine inhibition of cytoplasmic calcium signalling in rat pheochromocytoma (PC-12) cells

This study examines the mechanism of action of ketamine, a dissociative anesthetic, with a specific focus on its ability to inhibit changes in the concentration of intracellular free calcium, [Ca²⁺]_i, in PC-12 cells. The resting [Ca²⁺]_i as measured with the fluorescent probe Fura-2 AM in control cells is 184.8±8.6 nM (mean±SEM, n = 15). Changes in [Ca²⁺]_i via influx through voltage-gated calcium channels after membrane depolarization with potassium chloride were monitored in the absence and presence of various concentrations of ketamine. Potassium-depolarization caused a dose-dependent rapid increase in [Ca²⁺]_i, averaging 62±5%, 33±2% and 18±3% (n = 10 each) above control levels for 70 mM, 50 mM and 35 mM KCl, respectively. Ketamine, in the dosage range studied (5 – 500 μM), inhibited the increase in [Ca²⁺]_i stimulated by potassium-depolarization in a dose-dependent manner. The computer-fitted dose-response curve of the pooled data yielded a half maximal suppression concentration, ED₅₀, of 33 μM. In conclusion, this study demonstrates that ketamine inhibits Ca²⁺ influx through voltage-gated Ca²⁺ channels in PC-12 cells at clinically relevant doses, and may play a role in ketamine's action as a general anesthetic agent.     

Effect of Chloride Channel Inhibitors on Cytosolic Ca2+ Levels and Ca2+-Activated K+ (Gardos) Channel Activity in Human Red Blood Cells

DIDS, NPPB, tannic acid (TA) and AO1 are widely used inhibitors of Cl^– channels. Some Cl^– channel inhibitors (NPPB, DIDS, niflumic acid) were shown to affect phosphatidylserine (PS) scrambling and, thus, the life span of human red blood cells (hRBCs). Since a number of publications suggest Ca²⁺ dependence of PS scrambling, we explored whether inhibitors of Cl^– channels (DIDS, NPPB) or of Ca²⁺-activated Cl^? channels (DIDS, NPPB, TA, AO1) modified intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and activity of Ca²⁺-activated K⁺ (Gardos) channel in hRBCs. According to Fluo-3 fluorescence in flow cytometry, a short treatment (15 min, +37 °C) with Cl^? channels inhibitors decreased [Ca²⁺]_i in the following order: TA > AO1 > DIDS > NPPB. According to forward scatter, the decrease of [Ca²⁺]_i was accompanied by a slight but significant increase in cell volume following DIDS, NPPB and AO1 treatments. TA treatment resulted in cell shrinkage. According to whole-cell patch-clamp experiments, TA activated and NPPB and AO1 inhibited Gardos channels. The Cl^– channel blockers further modified the alterations of [Ca²⁺]_i following ATP depletion (glucose deprivation, iodoacetic acid, 6-inosine), oxidative stress (1 mM t-BHP) and treatment with Ca²⁺ ionophore ionomycin (1 μM). The ability of the Cl^? channel inhibitors to modulate PS scrambling did not correlate with their influence on [Ca²⁺]_i as TA and AO1 had a particularly strong decreasing effect on [Ca²⁺]_i but at the same time enhanced PS exposure. In conclusion, Cl^– channel inhibitors affect Gardos channels, influence Ca²⁺ homeostasis and induce PS exposure of hRBCs by Ca²⁺-independent mechanisms.     

Glucose Decouples Intracellular Ca2+ Activity from Glucagon Secretion in Mouse Pancreatic Islet Alpha-Cells

The mechanisms of glucagon secretion and its suppression by glucose are presently unknown. This study investigates the relationship between intracellular calcium levels ([Ca²⁺]_i) and hormone secretion under low and high glucose conditions. We examined the effects of modulating ion channel activities on [Ca²⁺]_i and hormone secretion from ex vivo mouse pancreatic islets. Glucagon-secreting α-cells were unambiguously identified by cell specific expression of fluorescent proteins. We found that activation of L-type voltage-gated calcium channels is critical for α-cell calcium oscillations and glucagon secretion at low glucose levels. Calcium channel activation depends on K_ATP channel activity but not on tetrodotoxin-sensitive Na⁺ channels. The use of glucagon secretagogues reveals a positive correlation between α-cell [Ca²⁺]_i and secretion at low glucose levels. Glucose elevation suppresses glucagon secretion even after treatment with secretagogues. Importantly, this inhibition is not mediated by K_ATP channel activity or reduction in α-cell [Ca²⁺]_i. Our results demonstrate that glucose uncouples the positive relationship between [Ca²⁺]_i and secretory activity. We conclude that glucose suppression of glucagon secretion is not mediated by inactivation of calcium channels, but instead, it requires a calcium-independent inhibitory pathway.     

Differential effects of propofol and isoflurane on glucose utilization and insulin secretion

AimsVolatile anesthetics, such as isoflurane, reverse glucose-induced inhibition of pancreatic adenosine triphosphate-sensitive potassium (K_ATP) channel activity, resulting in reduced insulin secretion and impaired glucose tolerance. No previous studies have investigated the effects of intravenous anesthetics, such as propofol, on pancreatic K_ATP channels. We investigated the cellular mechanisms underlying the effects of isoflurane and propofol on pancreatic K_ATP channels and insulin secretion.Main methodsIntravenous glucose tolerance tests (IVGTT) were performed on male rabbits. Pancreatic islets were isolated from male rats and used for a perifusion study, measurement of intracellular ATP concentration ([ATP]_i), and patch clamp experiments.Key findingsGlucose stimulus significantly increased insulin secretion during propofol anesthesia, but not isoflurane anesthesia, in IVGTT study. In perifusion experiments, both islets exposed to propofol and control islets not exposed to anesthetic had a biphasic insulin secretory response to a high dose of glucose. However, isoflurane markedly inhibited glucose-induced insulin secretion. In a patch clamp study, the relationship between ATP concentration and channel activity could be fitted by the Hill equation with a half-maximal inhibition of 22.4, 15.8, and 218.8 μM in the absence of anesthetic, and with propofol, and isoflurane, respectively. [ATP]_i and single K_ATP channel conductance did not differ in islets exposed to isoflurane or propofol.SignificanceOur results indicate that isoflurane, but not propofol, decreases the ATP sensitivity of K_ATP channels and impairs glucose-stimulated insulin release. These differential actions of isoflurane and propofol on ATP sensitivity may explain the differential effects of isoflurane and propofol on insulin release.     

Persistent Na+ influx drives L-type channel resting Ca2+ entry in rat melanotrophs

Rat melanotrophs express several types of voltage-gated and ligand-gated calcium channels, although mechanisms involved in the maintenance of the resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) remain unknown. We analyzed mechanisms regulating resting [Ca²⁺]_i in dissociated rat melanotrophs by Ca²⁺-imaging and patch-clamp techniques. Treatment with antagonists of L-type, but not N- or P/Q-type voltage-gated Ca²⁺ channels (VGCCs) as well as removal of extracellular Ca²⁺ resulted in a rapid and reversible decrease in [Ca²⁺]_i, indicating constitutive Ca²⁺ influx through L-type VGCCs. Reduction of extracellular Na⁺ concentration (replacement with NMDG⁺) similarly decreased resting [Ca²⁺]_i. When cells were champed at –80 mV, decrease in the extracellular Na⁺ resulted in a positive shift of the holding current. In cell-attached voltage-clamp and whole-cell current-clamp configurations, the reduction of extracellular Na⁺ caused hyperpolarisation. The holding current shifted in negative direction when extracellular K⁺ concentration was increased from 5 mM to 50 mM in the presence of K⁺ channel blockers, Ba²⁺ and TEA, indicating cation nature of persistent conductance. RT-PCR analyses of pars intermedia tissues detected mRNAs of TRPV1, TRPV4, TRPC6, and TRPM3-5. The TRPV channel blocker, ruthenium red, shifted the holding current in positive direction, and significantly decreased the resting [Ca²⁺]_i. These results indicate operation of a constitutive cation conductance sensitive to ruthenium red, which regulates resting membrane potential and [Ca²⁺]_i in rat melanotrophs.     

An Analysis of the Leakages of Sodium Ions into and Potassium Ions out of Striated Muscle Cells

Net sodium influx under K-free conditions was independent of the intracellular sodium ion concentration, [Na]_i, and was increased by ouabain. Unidirectional sodium influx was the sum of a component independent of [Na]_i and a component that increased linearly with increasing [Na]_i. Net influx of sodium ions in K-free solutions varied with the external sodium ion concentration, [Na]_o, and a steady-state balance of the sodium ion fluxes occurred at [Na]_o = 40 mM. When solutions were K-free and contained 10^-4 M ouabain, net sodium influx varied linearly with [Na]_o and a steady state for the intracellular sodium was observed at [Na]_o = 13 mM. The steady state observed in the presence of ouabain was the result of a pump-leak balance as the external sodium ion concentration with which the muscle sodium would be in equilibrium, under these conditions, was 0.11 mM. The rate constant for total potassium loss to K-free Ringer solution was independent of [Na]_i but dependent on [Na]_o. Replacing external NaCl with MgCl₂ brought about reductions in net potassium efflux. Ouabain was without effect on net potassium efflux in K-free Ringer solution with [Na]_o = 120 mM, but increased potassium efflux in a medium with NaCl replaced by MgCl₂. When muscles were enriched with sodium ions, potassium efflux into K-free, Mg⁺⁺-substituted Ringer solution fell to around 0.1 pmol/cm²·s and was increased 14-fold by addition of ouabain.     

Dramatic effect of glycolysis inhibition on the cerebellar granule cells bioenergetics

Cultured cerebellar granule cells were co-loaded with Ca²⁺-sensitive dye fura-2FF and rhodamine-123 sensitive to changes in the mitochondrial potential (????_m). A 60-min incubation of cells in glucose-free solution containing 2-deoxy-D-glucose (DG) induced a slow developing mitochondrial depolarization (sMD) without appreciable changes in basal [Ca²⁺]ⁱ. This sMD was insensitive to a removal of external Ca²⁺ or to the NMDA channels blocker memantine but could be readily suppressed by oligomycin due to inhibition of the inward proton current through the Fo channel of mitochondrial ATP synthase. In resting cells glucose deprivation caused a progressive decrease in mitochondrial NADH content ([NADH]), which strikingly enhanced the ability of glutamate to induce a delayed Ca²⁺ deregulation (DCD) associated with a profound mitochondrial depolarization. In glucose-containing medium this DCD appeared in young cells (usually 6?C8 days in vitro) after a prolonged latent period (lag phase). Substitution of glucose by DG led to a dramatic shortening of this lag phase, associated with a critical decrease in [NADH] in most neurons. Addition of pyruvate or lactate to DG-containing solution prevented the sMD and [NADH] decrease in resting cells and greatly diminished the number of cells exhibiting glutamate-induced DCD in glucose-free medium. Measurement of intracellular ATP level ([ATP]) in experiments on sister cells showed that glucose deprivation decreased [ATP] in resting cells and considerably deepened the fall of [ATP] caused by glutamate. This decrease in [ATP] was only slightly attenuated by pyruvate and lactate, despite their ability to prevent the shortening of lag phase preceding the DCD appearance under these conditions. Simultaneous monitoring of cytosolic ATP concentration ([ATP]_c) and ????_m changes in individual CGC expressing fluorescent ATP sensor (AT1.03) revealed that inhibition of either mitochondrial respiration or glycolysis caused a relatively small decrease in [ATP]_c and ????_m. Complete blockade of ATP synthesis in resting CGC with oligomycin in glucose-free DG-containing buffer caused fast ATP depletion and mitochondrial repolarization, indicating that mitochondrial respiratory chain still possess a reserve fuel to support ????_m despite inhibition of glycolysis. The data obtained suggest that the extraordinary enhancement of glutamate-induced deterioration in Ca²⁺ homeostasis caused by glucose deprivation in brain neurons is mainly determined by NADH depletion.     

Intracellular Calcium Dynamics and Cellular Energetics in Ischemic NG108-15 Cells Studied by Concurrent 31P/19F and 23Na Double-Quantum Filtered NMR Spectroscopy

Abstract: The role of voltage-sensitive Ca²⁺ channels in mediating Ca²⁺ influx during ischemia was investigated in NG108-15 cells, a neuronal cell line that does not express glutamate-sensitive receptor-mediated Ca²⁺ channels. Concurrent ³¹P/¹⁹F and ²³Na double-quantum filtered (DQF) NMR spectra were used to monitor cellular energy status, intracellular [Ca²⁺] ([Ca²⁺]_i), and intracellular Na⁺ content in cells loaded with the calcium indicator 1,2-bis-(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5FBAPTA) during ischemia and reperfusion. Cells loaded with 5FBAPTA were indistinguishable from unloaded cells except for small immediate decreases in levels of phosphocreatine (PCr) and ATP. Ischemia induced a steady decrease in intracellular pH and PCr and ATP levels, and a steady increase in intracellular Na⁺ content; however, a substantial increase in [Ca²⁺]_i (about threefold) was seen only following marked impairment of cellular energy status, when PCr was undetectable and ATP content was reduced to 55% of control levels. A depolarization-induced increase in [Ca²⁺]_i could be completely blocked by 1 µM nifedipine, whereas up to 20 µM nifedipine had no effect on the increase in [Ca²⁺]_i seen during ischemia. These data demonstrate that voltage-gated Ca²⁺ channels do not mediate significant Ca²⁺ flux during ischemia in this cell line and suggest an important role for Ca²⁺_i stores, the Na⁺/Ca²⁺ antiporter, or other processes linked to cellular energy status in the increase in cytosolic Ca²⁺ level during ischemia.     

Suppression of programmed neuronal death by a thapsigargin-induced Ca2+ influx

Rat sympathetic neurons undergo programmed cell death (PCD) in vitro and in vivo when they are deprived of nerve growth factor (NGF). Chronic depolarization of these neurons in cell culture with elevated concentrations of extracellular potassium ([K⁺]_o) prevents this death. The effect of prolonged depolarization on neuronal survival is thought to be mediated by a rise of intracellular calcium concentration ([Ca²⁺]_i) caused by Ca²⁺ influx through voltage-gated channels. In this report we investigate the effects of chronic treatment of rat sympathetic neurons with thapsigargin, an inhibitor of intracellular Ca²⁺ sequestration. In medium containing a normal concentration of extracellular Ca²⁺ ([Ca²⁺]_o), thapsigargin caused a sustained rise of intracellular Ca²⁺ concentration and partially blocked death of NGF-deprived cells. Elevating [Ca²⁺]_o in the presence of thapsigargin further increased [Ca²⁺]_i, suggesting that the sustained rise of [Ca²⁺]_i was caused by a thapsigargin-induced Ca²⁺ influx. This treatment potentiated the effect of thapsigargin on survival. The dihydropyridine Ca²⁺ channel antagonist, nifedipine, blocked both a sustained elevation of [Ca²⁺]_i and enhanced survival caused by depolarization with elevated [K⁺]_o, suggesting that these effects are mediated by Ca²⁺ influx through L-type channels. Nifedipine did not block the sustained rise of [Ca²⁺]_i or enhanced survival caused by thapsigargin treatment, indicating that these effects were not mediated by influx of Ca²⁺ through L-type channels. These results provide additional evidence that increased [Ca²⁺]_i can suppress neuronal PCD and identify a novel method for chronically raising neuronal [Ca²⁺]_i for investigation of this and other Ca²⁺-dependent phenomena. © 1995 John Wiley & Sons, Inc.     

Differential sensitivity to nickel and SK&F96365 of second messengerm operated and receptormoperated calcium channels in rat submandibular ductal cells

The intracellular concentration of calcium ([Ca²⁺]_i) of rat submandibular ductal cells was measured with the intracellular fluorescent dye Fura-2. Carbachol (100 μM) and ATP (1 mM) both increased the [Ca²⁺]_j. The late response to ATP was blocked by 0.5 mM Ni²⁺. This concentration of Ni²⁺ also blocked the increase of the [Ca²⁺]_i and the uptake of manganese and calcium in response to 2′- and 3′-O-(4-benzoylbenzoyl) adenosine 5′-triphosphate (BzATP, 100 μM), a specific agonist of P2X receptors from salivary glands. The increase of the [Ca²⁺]_i in response to 2-methylthioadenosine 5′-triphosphate (2-McSATP, 100 μM) a specific P2Y agonist in salivary glands or to a muscarinic agonist (carbachol) was not affected by 0.5 mM Ni²⁺. Only higher concentrations of Ni²⁺ (in the millimolar range) inhibited the uptake of extracellular calcium in response to carbachol. SK&F 96365, a blocker of store-operated calcium channels, inhibited the uptake of extracellular calcium in response to carbachol without affecting the response to BzATP. It is concluded that at low concentrations (below 0.5 mM), Ni²⁺ inhibits the non-specific cation channel coupled to P2X receptors. The uptake of extracellular calcium by store-operated calcium channels is inhibited by higher concentrations of Ni²⁺ and by SK&F96365.     

Intracellular free sodium concentrations in GH4C1 cells

In the present investigation, intracellular sodium ([Na⁺]_i) levels were determined in GH₄C₁ cells using the fluorescent probe SBFI. Fluorescence was determined by excitation at 340 nm and 385 nm, and emission was measured at 500 nm. Intracellular free sodium ([Na⁺]_i) was determined by comparing the ratio 340/385 to a calibration curve. The ratio was linear between 10 and 60 mM Na⁺. Resting [Na⁺]_i in GH₄C₁ cells was 26 ± 6.2 mM (mean ± SD). In cells incubated in Na⁺-buffer [Na⁺]_i decreased to 3 ± 3.6 mM. If Na⁺/K⁺ ATPase was inhibited by incubating the cells with 1 mM ouabain, [Na⁺]_i increased to 47 ± 12.8 mM in 15 min. Stimulating the cells with TRH, phorbol myristyl acetete, or thapsigargin had no effect on [Na⁺]_i. Incubating the cells in Ca²⁺-buffer rapidly increased [Na⁺]_i. The increase was not inhibited by tetrodotoxin. Addition of extracellular Ca²⁺, nimodipine, or Ni²⁺ to these cells immediately decreased [Na⁺]_i, whereas Bay K 8644 enhanced the influx of Na⁺. In cells where [Na⁺]_i was increased the TRH-induced increase in intracellular free calcium ([Ca²⁺]_i) was decreased compared with control cells. Our results suggest that Na⁺ enters the cells via Ca²⁺ channels, and [Na⁺]_i may attenuate TRH-induced changes in [Ca²⁺]_i in GH₄C₁ cells. © 1993 Wiley-Liss, Inc.     

Ionic control of renal gluconeogenesis IV. Effect of extracellular phosphate concentration

Isolated rat renal tubules from glucose from pyruvate, malate, glycerol and α-ketoglutarate. The rate of glucose formation from all but glycerol is enhanced by an increase in Ca²⁺ concentration. Because changes in inorganic phosphate concentrations influence the uptake and retention of calcium by isolated cells, the effect of changes in phosphate concentration upon renal gluconeogenesis was examined. It was found that changing phosphate concentration altered the metabolism of isolated rat renal tubules in three ways which dependend upon the Ca²⁺ concentration. In the absence of Ca²⁺, increasing phosphate concentration from 0.07 to 1.2 mM led to a stimulation of the decarboxylation of [U-¹⁴C]malate, [1-14C]pyruvate, [2-¹⁴C]-pyruvate, α-keto[5-¹⁴C]glutarate and [1,3-¹⁴C₂]glycerol, and to an increase in ATP concentration but had no effect upon the rate of glucose formation from malate, pyruvate, α-ketoglutarate but a slight stimulation of glucose production from glycerol. A further increase in phosphate above 1.2 mM had no effect on any of these parameters. In the presence of either low (0.2 mM) or high (2.0 mM) Ca²⁺, changing phosphate concentration had no effect upon the decarboxylation of any of these substrates except glycerol whose decarboxylation was stimulated by increasing medium phosphate concentration. In the presence of calcium, increasing phosphate concentration led to an inhibition of glucose formation from malate, pyruvate and α-ketoglutarate but not from glycerol. Also in the presence of calcium both parathyroid hormone and cyclic AMP stimulated glucose formation, and under these conditions increasing phosphate concentration led to an inhibition of glucose formation. In tubules treated with parathyroid hormone an increase in phosphate concentration from 0.07 to 6.0 mM led to a significant increase in cyclic AMP concentration even though the rate of glucose formation decreased.Analysis of metabolite concentrations and rates of substrates decarboxylations, under a variety of conditions, revealed that P_i altered renal gluconeogenesis at a site different from those controlled by changes in Ca²⁺ concentration. The P_i-control site was tentatively identified as the glyceraldehyde phosphate dehydrogenase-glycerate kinase reaction sequence. However, the effect of changing P_i concentration upon parathyroid hormone-induced alterations in cyclic AMP concentration could not be explained by this action of P_i, and was probably due to an effect of P_i upon cellular calcium distribution. Thus, changes in P_i concentration appear to have two cellular effects, only one of which is related to a change in cellular calcium metabolism.     

Study on ATP concentration changes in cytosol of individual cultured neurons during glutamate-induced deregulation of calcium homeostasis

For the first time, simultaneous monitoring of changes in the concentration of cytosolic ATP ([ATP]_c), pH (pH_c), and intracellular free Ca²⁺ concentration ([Ca²⁺]_i) of the individual neurons challenged with toxic glutamate (Glu) concentrations was performed. To this end, the ATP-sensor AT1.03, which binds to ATP and therefore enhances the efficiency of resonance energy transfer between blue fluorescent protein (energy donor) and yellow-green fluorescent protein (energy acceptor), was expressed in cultured hippocampal neurons isolated from 1–2-day-old rat pups. Excitation of fluorescence in the acceptor protein allowed monitoring changes in pH_c. Cells were loaded with fluorescent low-affinity Ca²⁺ indicators Fura-FF or X-rhod-FF to register [Ca²⁺]_i. It was shown that Glu (20 μM, glycine 10 μM, Mg²⁺-free) produced a rapid acidification of the cytosol and decrease in [ATP]_c. An approximately linear relationship (r ² = 0.56) between the rate of [ATP]_c decline and latency of glutamate-induced delayed calcium deregulation (DCD) was observed: higher rate of [ATP]_c decrease corresponded to shorter DCD latency period. DCD began with a decrease in [ATP]_c of as much as 15.9%. In the phase of high [Ca²⁺]_i, the plateau of [ATP]_c dropped to 10.4% compared to [ATP]_c in resting neurons (100%). In the presence of the Na⁺/K⁺-ATPase inhibitor ouabain (0.5 mM), glutamate-induced reduction in [ATP]_c in the phase of the high [Ca²⁺]_i plateau was only 36.6%. Changes in [ATP]_c, [Ca²⁺]_i, mitochondrial potential, and pH_c in calcium-free or sodium-free buffers, as well as in the presence of the inhibitor of Na⁺/K⁺-ATPase ouabain, led us to suggest that in addition to increase in proton conductivity and decline in [ATP]_c, one of the triggering factors of DCD might be a reversion of the neuronal plasma membrane Na⁺/Ca²⁺ exchange.     

Effects of intracellular magnesium on Kv1.5 and Kv2.1 potassium channels

We characterized the effects of intracellular Mg²⁺ (Mg²⁺_i) on potassium currents mediated by the Kv1.5 and Kv2.1 channels expressed in Xenopus oocytes. Increase in Mg²⁺_i caused a voltage-dependent block of the current amplitude, apparent acceleration of the current kinetics (explained by a corresponding shift in the steady-state activation) and leftward shifts in activation and inactivation dependencies for both channels. The voltage-dependent block was more potent for Kv2.1 [dissociation constant at 0 mV, K_d(0), was ~70 mM and the electric distance of the Mg²⁺ binding site, , was 0.2] than for the Kv1.5 channel [K_d(0)~40 mM and =0.1]. Similar shifts in the voltage-dependent parameters for both channels were described by the Gouy-Chapman formalism with the negative charge density of 1 e^–/100 Ų. Additionally, Mg²⁺_i selectively reduced a non-inactivating current and increased the accumulation of inactivation of the Kv1.5, but not the Kv2.1 channel. A potential functional role of the differential effects of Mg²⁺_i on the Kv channels is discussed.     

The ATP-sensitivity of K+ channels in rat pancreatic B-cells is modulated by ADP

ATP-sensitive K+ channels in inside-out membrane patches from dispersed rat pancreatic B-cells were studied using patch-clamp methods. The dose-response curve for ATP-induced channel inhibition was shifted to higher concentrations in the presence of ADP (2 mM). In glucose-free solution, the total intracellular concentration of ATP was 3.8 mM and of ADP 1.5 mM; glucose (20 mM) increased ATP and decreased ADP by approx. 40%. These results suggest that both ADP and ATP may be involved in regulating the activity of the glucose-sensitive K+ channel in intact B-cells.     

Low affinity purinergic receptor modulates the response of rat submandibular glands to carbachol and substance P

The effect of extracellular ATP on the intracellular calcium concentration ([Ca²⁺]_i) in rat submandibular glands was tested. The dose-response curve for ATP was biphasic with a first increase in the 1–30 μM concentration range and a further increase at concentrations higher than 100 μM. Among ATP analogs, only benzoyl-ATP stimulated the low affinity component. ATPτS blocked this response. All the other analogs tested reproduced the high-affinity low capacity response. Magnesium and Coomassie blue selectively blocked the low affinity component. High concentrations of ATP blocked the increase of the intracellular calcium concentration [Ca²⁺]_i in response to 100 μM carbachol. By itself, substance P (100 pM-1 μM) increased the [Ca²⁺]_i. One mM ATP potentiated the response to concentrations of substance P higher than 10 nM. This potentiation was reversed by extracellular magnesium. Carbachol 100 μM and substance P (100 pM-1 μM) increased the release of inositol trisphosphate (IP₃) from polyphosphoinositides (polyPI). Activation of the low affinity ATP receptors did not activate the polyPI-specific phospholipase C but inhibited its activation by 100 μM carbachol (−50%) and by 100 nM substance P (−60% at 1 nM substance P and −40% at 100 nM substance P). Substance P induced a strong homologous desensitization: a preincubation with 1 nM substance P nearly completely abolished the response to 1 μM substance P. When the cells were exposed to ATP before the second addition of substance P, the purinergic agonist partially restored the response to the tachykinin without totally reversing the desensitization. It is concluded that two types of purinergic receptors coexist in rat submandibular glands; a high-affinity, low capacity receptor which remains pharmacologically and functionally undefined and a low affinity site, high capacity receptor of the P_2Z type coupled to a non-selective cation channel. The occupancy of these low affinity sites blocks the increase of the [Ca²⁺]_i in response to a muscarinic agonist and the activation of polyPI-specific phospholipase C by carbachol and substance P. It potentiates the effect of high concentrations of substance P on the [Ca²⁺]_i. © 1996 Wiley-Liss, Inc.     

A P-NMR saturation transfer study of the regulation of creatine kinase in the rat heart

(1) ³¹P nuclear magnetic resonance was used to measure the creatine kinase-catalysed fluxes in Langendorff-perfused rat hearts consuming oxygen at different rates and using either of two exogenous substrates (11 mM glucose or 5 mM acetate). (2) Fluxes in the direction of ATP synthesis were between 3.5–12-times the steady-state rates of ATP utilization (estimated from rates of O₂-consumption), demonstrating that the reaction is sufficiently rapid to maintain the cytosolic reactants near their equilibrium concentrations. (3) Under all conditions studied, the cytosolic free [ADP] was primarily responsible for regulating the creatine kinase fluxes. The enzyme displayed a K_m for cytosolic ADP of 35 μM and an apparent V_max of 5.5 mM/s in the intact tissue. (4) Although the reaction is maintained in an overall steady-state, the measured ratio of the forward flux (ATP synthesis) to the reverse flux (phosphocreatine synthesis) was significantly greater than unity under some conditions. It is proposed that this discrepancy may be a consequence of participation of ATP in reactions other than the PCr /ag ATP or ATP /ag ADP + P_i interconversions specifically considered in the analysis. (5) The results support the view that creatine kinase functions primarily to maintain low cytosolic concentrations of ADP during transient periods in which energy utilization exceeds production.     

Increased Cation Conductance in Human Erythrocytes Artificially Aged by Glycation

Excessive glucose concentrations foster glycation and thus premature aging of erythrocytes. The present study explored whether glycation-induced erythrocyte aging is paralleled by features of suicidal erythrocyte death or eryptosis, which is characterized by cell membrane scrambling with subsequent phosphatidylserine exposure at the cell surface and cell shrinkage. Both are triggered by increases of cytosolic Ca²⁺ concentration ([Ca²⁺]_i), which may result from activation of Ca²⁺ permeable cation channels. Glycation was accomplished by exposure to high glucose concentrations (40 and 100 mM), phosphatidylserine exposure estimated from annexin binding, cell shrinkage from decrease of forward scatter, and [Ca²⁺]_i from Fluo3-fluorescence in analysis via fluorescence-activated cell sorter. Cation channel activity was determined by means of whole-cell patch clamp. Glycation of total membrane proteins, immunoprecipitated TRPC3/6/7, and immunoprecipitated L-type Ca²⁺ channel proteins was estimated by Western blot testing with polyclonal antibodies used against advanced glycation end products. A 30–48-h exposure of the cells to 40 or 100 mM glucose in Ringer solution (at 37°C) significantly increased glycation of membrane proteins, hemoglobin (HbA_1c), TRPC3/6/7, and L-type Ca²⁺ channel proteins, enhanced amiloride-sensitive, voltage-independent cation conductance, [Ca²⁺]_i, and phosphatidylserine exposure, and led to significant cell shrinkage. Ca²⁺ removal and addition of Ca²⁺ chelator EGTA prevented the glycation-induced phosphatidylserine exposure and cell shrinkage after glycation. Glycation-induced erythrocyte aging leads to eryptosis, an effect requiring Ca²⁺ entry from extracellular space.     

A 31P-NMR saturation transfer study of the regulation of creatine kinase in the rat heart

(1) ³¹P nuclear magnetic resonance was used to measure the creatine kinase-catalysed fluxes in Langendorff-perfused rat hearts consuming oxygen at different rates and using either of two exogenous substrates (11 mM glucose or 5 mM acetate). (2) Fluxes in the direction of ATP synthesis were between 3.5–12-times the steady-state rates of ATP utilization (estimated from rates of O₂-consumption), demonstrating that the reaction is sufficiently rapid to maintain the cytosolic reactants near their equilibrium concentrations. (3) Under all conditions studied, the cytosolic free [ADP] was primarily responsible for regulating the creatine kinase fluxes. The enzyme displayed a K_m for cytosolic ADP of 35 μM and an apparent V_max of 5.5 mM/s in the intact tissue. (4) Although the reaction is maintained in an overall steady-state, the measured ratio of the forward flux (ATP synthesis) to the reverse flux (phosphocreatine synthesis) was significantly greater than unity under some conditions. It is proposed that this discrepancy may be a consequence of participation of ATP in reactions other than the PCr /ag ATP or ATP /ag ADP + P_i interconversions specifically considered in the analysis. (5) The results support the view that creatine kinase functions primarily to maintain low cytosolic concentrations of ADP during transient periods in which energy utilization exceeds production.     

Interaction between thapsigargin and ATP4− in the regulation of the intracellular calcium in rat submandibular glands

Rat submandibular glands were digested with crude collagenase, and the intracellular calcium concentration of the cellular suspension was measured using fura-2. In the absence of extracellular magnesium and calcium ([Ca²⁺]_o), ATP had no effect; the response to ATP peaked at 1–2.5 mM [Ca²⁺]_o and was inhibited at 5 mM. One millimolar (mM) extracellular ATP did not increase the leak of LDH or fura-2; 10 m?M Coomassie brilliant blue G specifically inhibited the effect of ATP on [Ca²⁺]_in. Depleting intracellular calcium pools with thapsigargin did not affect the response to ATP. Using a Ca²⁺-free/Ca²⁺ reintroduction protocol, it was shown that ATP and thapsigargin increase the uptake of extracellular calcium. The effect of the two agonists was synergistic. Removal of extracellular sodium inhibited the effect of carbachol on [Ca²⁺]_in and the calcium uptake but potentiated the response to ATP. These results suggest that, after binding to purinergic receptors, extracellular ATP^4- increases [Ca²⁺]_in. ATP^4- does not mobilize thapsigargin-sensitive intracellular calcium pools (among which is the IP₃-sensitive calcium pool) but stimulates the uptake of extracellular calcium by a mechanism inhibited by extracellular sodium, probably by opening a nonselective cation channel. © 1994 Wiley-Liss, Inc.