Differential effects of anesthetic and nonanesthetic cyclobutanes on neuronal voltage-gated sodium channels

BACKGROUND: Despite their key role in the generation and propagation of action potentials in excitable cells, voltage-gated sodium (Na+) channels have been considered to be insensitive to general anesthetics. The authors tested the sensitivity of neuronal Na+ channels to structurally similar anesthetic (1-chloro-1,2,2-trifluorocyclobutane; F3) and nonanesthetic (1,2-dichlorohexafluorocyclobutane; F6) polyhalogenated cyclobutanes by neurochemical and electrophysiologic methods. METHODS: Synaptosomes (pinched-off nerve terminals) from adult rat cerebral cortex were used to determine the effects of F3 and F6 on 4-aminopyridine- or veratridine-evoked (Na+ channel-dependent) glutamate release (using an enzyme-coupled spectrofluorimetric assay) and increases in intracellular Ca2+ ([Ca2+]i) (using ion-specific spectrofluorimetry). Effects of F3 and F6 on Na+ currents were evaluated directly in rat lumbar dorsal root ganglion neurons by whole-cell patch-clamp recording. RESULTS: F3 inhibited glutamate release evoked by 4-aminopyridine (inhibitory concentration of 50% [IC50] = 0.77 mM [approximately 0.8 minimum alveolar concentration (MAC)] or veratridine (IC50 = 0.42 mM [approximately 0.4 MAC]), and veratridine-evoked increases in [Ca2+]i (IC50 = 0.5 mM [approximately 0.5 MAC]) in synaptosomes; F6 had no significant effects up to 0.05 mM (approximately twice the predicted MAC). F3 caused reversible membrane potential-independent inhibition of peak Na+ currents (70+/-9% block at 0.6 mM [approximately 0.6 MAC]), and a hyperpolarizing shift in the voltage-dependence of steady state inactivation in dorsal root ganglion neurons (-21+/-9.3 mV at 0.6 mM). F6 inhibited peak Na+ currents to a lesser extent (16+/-2% block at 0.018 mM [predicted MAC]) and had minimal effects on steady state inactivation. CONCLUSIONS: The anesthetic cyclobutane F3 significantly inhibited Na+ channel-mediated glutamate release and increases in [Ca2+]i. In contrast, the nonanesthetic cyclobutane F6 had no significant effects at predicted anesthetic concentrations. F3 inhibited dorsal root ganglion neuron Na+ channels with a potency and by mechanisms similar to those of conventional volatile anesthetics; F6 was less effective and did not produce voltage-dependent block. This concordance between anesthetic activity and Na+ channel inhibition supports a role for presynaptic Na+ channels as targets for general anesthetic effects and suggests that shifting the voltage-dependence of Na+ channel inactivation is an important property of volatile anesthetic compounds.     

Inhibition of Presynaptic Sodium Channels by Halothane

Background: Recent electrophysiologic studies indicate that clinical concentrations of volatile general anesthetic agents inhibit central nervous system sodium (Na sup +) channels. In this study, the biochemical effects of halothane on Na sup + channel function were determined using rat brain synaptosomes (pinched-off nerve terminals) to assess the role of presynaptic Na sup + channels in anesthetic effects.

Methods: Synaptosomes from adult rat cerebral cortex were used to determine the effects of halothane on veratridine-evoked Na sup + channel-dependent Na sup + influx (using22 Na sup +), changes in intrasynaptosomal [Na sup +] (using ion-specific spectrofluorometry), and neurotoxin interactions with specific receptor sites of the Na sup + channel (by radioligand binding). The potential physiologic and functional significance of these effects was determined by measuring the effects of halothane on veratridine-evoked Na sup + channel-dependent glutamate release (using enzyme-coupled spectrofluorometry).

Results: Halothane inhibited veratridine-evoked22 Na sup + influx (IC50 = 1.1 mM) and changes in intrasynaptosomal [Na sup +] (concentration for 50% inhibition [IC50] = 0.97 mM), and it specifically antagonized [sup 3 H]batrachotoxinin-A 20-alpha-benzoate binding to receptor site two of the Na sup + channel (IC50 = 0.53 mM). Scatchard and kinetic analysis revealed an allosteric competitive mechanism for inhibition of toxin binding. Halothane inhibited veratridine-evoked glutamate release from synaptosomes with comparable potency (IC50 = 0.67 mM).     

Effects of Propofol on Sodium Channel-dependent Sodium Influx and Glutamate Release in Rat Cerebrocortical Synaptosomes

Background: Previous electrophysiologic studies have implicated voltage-dependent Na+ channels as a molecular site of action for propofol. This study considered the effects of propofol on Na+ channel-mediated Na+ influx and neurotransmitter release in rat brain synaptosomes (isolated presynaptic nerve terminals).

Methods: Purified cerebrocortical synaptosomes from adult rats were used to determine the effects of propofol on Na+ influx through voltage-dependent Na+ channels (measured using22 Na+) and intracellular [Na+] (measured by ion-specific spectrofluorimetry). For comparison, the effects of propofol on synaptosomal glutamate release evoked by 4-aminopyridine (Na+ channel dependent), veratridine (Na (+) channel dependent), and KCl (Na+ channel independent) were studied using enzyme-coupled fluorimetry.

Results: Propofol inhibited veratridine-evoked22 Na+ influx (inhibitory concentration of 50% [IC50] = 46 micro Meter; 8.9 micro Meter free) and changes in intracellular [Na+] (IC50 = 13 micro Meter; 6.3 micro Meter free) in synaptosomes in a dose-dependent manner. Propofol also inhibited 4-aminopyridine-evoked (IC50 = 39 micro Meter; 19 micro Meter free) and veratridine (20 micro Meter)-evoked (IC (50) = 30 micro Meter; 14 micro Meter free), but not KCl-evoked (up to 100 micro Meter) glutamate release from synaptosomes.     

Role of mitochondrial Na+ concentration, measured by CoroNa red, in the protection of metabolically inhibited MDCK cells

In ischemic or hypoxic tissues, elevated cytosolic calcium levels can induce lethal processes. Mitochondria, besides the endoplasmic reticulum, play a key role in clearing excessive cytosolic Ca2+. In a previous study, it was suggested that the clearance of cytosolic Ca2+, after approximately 18 min of metabolic inhibition (MI) in renal epithelial cells, occurs via the reverse action of the mitochondrial Na+/Ca2+ exchanger (NCX). For further investigating the underlying mechanism, changes in the mitochondrial Na+ concentration ([Na+](m)) were monitored in metabolically inhibited MDCK cells. CoroNa Red, a sodium-sensitive fluorescence probe, was used to monitor [Na+]m. In the first 15 min of MI, a twofold increase of [Na+]m was observed reaching 113 +/- 7 mM, whereas the cytosolic Na+ concentration ([Na+]c) elevated threefold, to a level of 65 +/- 6 mM. In the next 45 min of MI, [Na+]m dropped to 91 +/- 7 mM, whereas [Na+]c further increased to 91 +/- 4 mM. The striking rise in [Na+]m is likely sufficient to sustain the driving force for mitochondrial Ca2+ uptake via the NCX. Furthermore, when CGP-37157, a specific inhibitor of the mitochondrial NCX, was applied during MI, the second-phase drop of [Na+]m was completely abolished. The obtained results support the hypothesis that the mitochondrial NCX reverses after approximately 15 min of MI. Moreover, because the cellular homeostasis can recover after MI, the mitochondria likely protect MDCK cells from injury during MI by the reversal of the mitochondrial NCX. This study is the first to report [Na+]m measurements in nonpermeabilized living cells.     

Parathyroid hormone control of free cytosolic Ca2+ in the kidney

We have established a perifusion system to monitor free cytosolic calcium concentrations ([Ca2+]i) in mouse kidney slices, which presumably reflects in vivo status more accurately than renal cells in culture, by means of the fluorescent calcium indicators quin-2 and fura-2. An increase in the extracellular calcium concentrations from 0 (no added Ca2+) to 3.0 mM resulted in an increase in [Ca2+]i from 52 to 239 nM. Replacement of 118 mM of extracellular Na+ with choline, or the addition of ouabain, an inhibitor of Na+,K+-ATPase, at 10(-6) M in the perfusate caused an increase in [Ca2+]i from 161 +/- 13 to 873 +/- 78 nM (n = 10) and 161 +/- 13 to 395 +/- 68 nM (n = 4), respectively, suggesting the possible existence of a Na+,Ca2+ exchange mechanism in the kidney slice. We further examined the effects of PTH on [Ca2+]i mobilization in the kidney. Both human PTH-(1-34) and hPTH-(1-84) increased [Ca2+]i within 60 s at physiologic concentrations of 10(-11)-10(-9) M in a dose-dependent manner. On the other hand, an increase in intracellular cAMP in the slice was also detected above 3 X 10(-9) M hPTH-(1-34) [base 2.1 +/- 0.4 pmol/mg, 3.2 +/- 0.6 pmol/mg (p less than 0.05 versus control values) 5 minutes after the application of 3 X 10(-9) M hPTH-(1-34) and 17.3 +/- 4.3 pmol/mg (p less than 0.05 versus control values) 3 X 10(-8) M hPTH-(1-34), mean +/- SEM, n = 7, p less than 0.05 versus control values].(ABSTRACT TRUNCATED AT 250 WORDS)     

Halothane decreases calcium sensitivity of rat aortic smooth muscle

PURPOSE: To examine the effect of halothane on the cytosolic Ca2+ concentration ([Ca2+]i)-tension relationship of rat aortic smooth muscle. METHODS: Rat aortic rings without endothelia were loaded with the fluorescent Ca2+ indicator, Fura PE3-AM, and then mounted in organ baths. The changes in isometric tension and [Ca2+]i were measured simultaneously. In one series ionomycin (10 nM-3 microM) was added to normal Krebs' solution cumulatively in the absence and presence of halothane (1.5%, 3%). In the other series, CaCl2 (0.3-3 mM) was added to Ca2+-free Krebs' solution including high KCl (50 mM), phenylephrine (100 nM) or prostaglandin F2alpha (PGF2alpha, 1-3 microM) in the absence and presence of halothane (1.5%, 3%). The linear part of [Ca2+]i-tension relationship was analyzed by a linear regression. RESULTS: Halothane, 1.5%, had no effect on the normal [Ca2+]i-tension relationship obtained with the calcium ionophore, ionomycin (10 nM-3 microM), but halothane 3% decreased the slope of the relationship (0.239 +/- 0.037 for control and 0.110 +/- 0.010 for halothane 3%, P < 0.05). Halothane, 1.5% and 3%, did not change the [Ca2+]i-tension relationship obtained with CaCl2 (0.3-3 mM) in the presence of high KCl (50 mM) or phenylephrine (100 nM). In contrast, halothane, 3%, inhibited the intercept of [Ca2+]i-tension relationship obtained with CaCl2 (0.3-3 mM) in the presence of prostaglandin F2alpha (PGF2alpha, 1-3 microM) (45.708 +/- 4.233 for control and 26.997 +/- 2.522 for halothane 3%, P < 0.01). CONCLUSION: Halothane decreases the Ca2+ sensitivity and that in the presence of PGF2.     

Ca2+ signaling mediated by IP3-dependent Ca2+ releasing and store-operated Ca2+ channels in rat odontoblasts.

In the phospholipase-C (PLC) signaling system, Ca2+ is mobilized from intracellular Ca2+ stores by an action of inositol 1,4,5-trisphosphate (IP3). The depletion of IP3-sensitive Ca2+ stores activates a store-operated Ca2+ entry (SOCE). However, no direct evidence has been obtained about these signaling pathways in odontoblasts. In this study, we investigate the characteristics of the SOCE and IP3-mediated Ca2+ mobilizations in rat odontoblasts using fura-2 microfluorometry and a nystatin-perforated patch-clamp technique. In the absence of extracellular Ca2+ ([Ca2+]o), thapsigargin (TG) evoked a transient rise in intracellular Ca2+ concentration ([Ca2+]i). After TG treatment to deplete the store, the subsequent application of Ca2+ resulted in a rapid rise in [Ca2+]i caused by SOCE. In the absence of TG treatment, no SOCE was evoked. The Ca2+ influx was dependent on [Ca2+]o (KD = 1.29 mM) and was blocked by an IP3 receptor inhibitor, 2-aminoethoxydiphenyl borate (2-APB), as well as La3+ in a concentration-dependent manner (IC50 = 26 microM). In TG-treated cells, an elevation of [Ca2+]o from 0 to 2.5 mM elicited an inwardly rectifying current at hyperpolarizing potentials with a positive reversal potential. The currents were selective for Ca2+ over the other divalent cations (Ca2+ > Ba2+ > Sr2+ > Mn2+). In the absence of [Ca2+]o, carbachol, bradykinin, and 2-methylthioadenosine 5'triphosphate activated Ca2+ release from the store; these were inhibited by 2-APB. These results indicate that odontoblasts possessed Ca2+ signaling pathways through the activation of store-operated Ca2+ channels by the depletion of intracellular Ca2+ stores and through the IP3-induced Ca2+ release activated by PLC-coupled receptors.     

Control of chondrocyte regulatory volume decrease (RVD) by [Ca2+]i and cell shape

OBJECTIVES: Optimal matrix metabolism by articular chondrocytes is controlled by the 'set-point' volume which is determined mainly by membrane transporters. The signal transduction pathway(s) for the key membrane transporter which responds to cell swelling ('osmolyte channel') and mediates regulatory volume decrease (RVD) is poorly understood, so here the role of Ca2+ and the effects of 2D culture have been clarified. METHODS: Changes to the volume and intracellular calcium levels ([Ca2+]i) of freshly isolated and 2D cultured bovine articular chondrocytes subjected to hypotonic challenge using a 43% reduction in medium osmolarity were studied by single-cell fluorescence microscopy. The effects of ethylene glycol tetraacetic acid (EGTA), REV5901 and Gd(3+) were studied and the role of Ca2+ influx determined by Mn2+ quench. RESULTS: In freshly isolated cells, approximately 50% of chondrocytes exhibited 'robust RVD' (6[120]). RVD was inhibited by REV 5901 (4+/-2% responding) (3[23]) and 2 mM EGTA (18+/-5% responding) (4[166]) whereas Gd3+ had no effect (3[89]). The hypotonic challenge resulted in a Gd3+-insensitive rise in [Ca2+]i that did not correlate with RVD in all cells. Following 2D culture, chondrocytes also demonstrated Gd3+-insensitive RVD, but in contrast, the [Ca2+]i rise was blocked by this agent. CONCLUSIONS: The data suggested that in freshly isolated and 2D cultured chondrocytes, the rise in [Ca2+]i occurring during hypotonic challenge could be related to RVD, but only in some cells. However, with 2D culture, the Ca2+ response switched to being Gd3+-sensitive, suggesting that as a result of changes to chondrocyte shape, stretch-activated cation channels although present, do not appear to play a role in volume regulation.     

Thiopental Alters Contraction, Intracellular Ca2+, and pH in Rat Ventricular Myocytes

Background: Myocardial contractility is regulated by intracellular concentration of free Ca2+ ([Ca2+]i) and myofilament Ca2+ sensitivity. The objective of this study was to elucidate the direct effects of thiopental on cardiac excitation-contraction coupling using individual, field-stimulated ventricular myocytes.

Methods: Freshly isolated rat ventricular myocytes were loaded with the Ca2+ indicator, fura-2, and placed on the stage of an inverted fluorescence microscope in a temperature-regulated bath. [Ca2+]i (340/380 ratio) and myocyte shortening (video-edge detection) were monitored simultaneously in individual cells field-stimulated at 0.3 Hz. Amplitude and timing of myocyte shortening and [Ca2+]i were compared before and after addition of thiopental. Intracellular pH was measured with the pH indicator, BCECF (500/440 ratio). Real-time uptake of Ca2+ into isolated sarcoplasmic reticulum vesicles was measured using fura-2 free acid in the extravesicular compartment. One hundred thirty-two cells were studied.

Results: Field stimulation increased [Ca2+]i from 85 +/- 10 nM to 355 +/- 22 nM (mean +/- SEM). Myocytes shortened by 10% of resting cell length (127 +/- 5 [micro sign]m). Times to peak [Ca2+]i and shortening were 139 +/- 6 and 173 +/- 7 msec, respectively. Times to 50% recovery for [Ca2+]i and shortening were 296 +/- 6 and 290 +/- 6 ms, respectively. Addition of thiopental (30-1,000 [micro sign]M) resulted in dose-dependent decreases in peak [Ca2+]i and myocyte shortening. Thiopental altered time to peak and time to 50% recovery for [Ca2+]i and myocyte shortening and inhibited the rate of uptake of Ca2+ into isolated sarcoplasmic reticulum vesicles. Thiopental did not, however, alter the amount of Ca2+ released in response to caffeine in sarcoplasmic reticulum vesicles or intact cells. Thiopental (100 [micro sign]M) increased intracellular pH and caused an upward shift in the dose-response curve to extracellular Ca2+ for shortening, with no concomitant effect on peak [Ca2+]i. These effects were abolished by ethylisopropyl amiloride, an inhibitor of Na+ -H+ exchange.     

Identification and characteristics of a Na+/Ca2+ exchanger in cultured human mesangial cells

Excitable cells express Na+/Ca2+ exchange activity among other mechanisms modulating rapid fluctuations of cytosolic free Ca2+ ([Ca2+]i). We studied functions and regulation of a Na+/Ca2+ exchanger in cultured human glomerular mesangial cells. Fura-2-loaded confluent monolayers reacted to removal of ambient Na+ with an immediate, transient elevation of [Ca2+]i, assessed by single/dual wavelength fluorometry. Peak [Ca2+]i was inversely correlated with the extracellular Na+ concentration. Ca2+ influx was the sole mechanism implicated, as the [Ca2+]i rise was prevented by EGTA. The process was inhibited by 1 mM amiloride, but not by blockers of voltage-operated Ca2+ channels. Re-addition of Na+ resulted in a rapid decrease of [Ca2+]i, indicating bimodal operation of the exchanger. Na(+)-loading the cells with monensin and ouabain enhanced Ca2+ uptake. Prior stimulation of [Ca2+]i with the thromboxane A2 mimetic, U-46619, or angiotensin II also increased Ca2+ uptake upon subsequent Na+ removal, suggesting induction of the exchanger by vasoconstrictors. Moreover, the magnitude of agonist-induced [Ca2+]i transients was amplified by Na+ removal, indicating that the exchanger modulates the effects of vasoconstrictors. These results demonstrate that an inducible Na+/Ca2+ antiporter is operative in resting and stimulated human mesangial cells, further confirming their smooth muscle origin and potential regulatory role on glomerular hemodynamics.     

Effects of Halothane on Sarcoplasmic Reticulum Calcium Release Channels in Porcine Airway Smooth Muscle Cells

Background: Volatile anesthetics relax airway smooth muscle (ASM) by altering intracellular Ca2+ concentration ([Ca2+]i). The authors hypothesized that relaxation is produced by decreasing sarcoplasmic reticulum Ca2+ content via increased Ca2+ "leak" through both inositol trisphosphate (IP3) and ryanodine receptor channels.

Methods: Enzymatically dissociated porcine ASM cells were exposed to acetylcholine in the presence or absence of 2 minimum alveolar concentration (MAC) halothane, and IP3 levels were measured using radioimmunoreceptor assay. Other cells were loaded with the Ca2+ indicator fluo-3 and imaged using real-time confocal microscopy.

Results: Halothane increased IP3 concentrations in the presence and absence of acetylcholine. Inhibition of phospholipase C blunted the IP3 response to halothane. Exposure to 2 MAC halothane induced a transient [Ca2+]i response, suggesting depletion of sarcoplasmic reticulum Ca2+. Exposure to 20 [mu]m Xestospongin D, a cell-permeant IP3 receptor antagonist, resulted in a 45 +/- 13% decrease in the [Ca2+]i response to halothane compared with halothane exposure alone. In permeabilized cells, Xestospongin D or 0.5 mg/ml heparin decreased the [Ca2+]i response to halothane by 65 +/- 13% and 68 +/- 22%, respectively, compared with halothane alone. In both intact and permeabilized cells, 20 [mu]m ryanodine blunted the [Ca2+]i response to halothane by 32 +/- 13% and 39 +/- 21%, respectively, compared with halothane alone. Simultaneous exposure to Xestospongin D and ryanodine completely inhibited the [Ca2+]i response to halothane.     

Widespread inhibition of sodium channel-dependent glutamate release from isolated nerve terminals by isoflurane and propofol.

BACKGROUND: Controversy persists concerning the mechanisms and role of general anesthetic inhibition of glutamate release from nerve endings. To determine the generality of this effect and to control for methodologic differences between previous studies, the authors analyzed the presynaptic effects of isoflurane and propofol on glutamate release from nerve terminals isolated from several species and brain regions. METHODS: Synaptosomes were prepared from rat, mouse, or guinea pig cerebral cortex and also from rat striatum and hippocampus. Release of endogenous glutamate evoked by depolarization with 20 microm veratridine (which opens voltage-dependent Na+ channels by preventing inactivation) or by 30 mm KCl (which activates voltage-gated Ca2+ channels by membrane depolarization) was monitored using an on-line enzyme-linked fluorometric assay. RESULTS: Glutamate release evoked by depolarization with increased extracellular KCl was not significantly inhibited by isoflurane up to 0.7 mM ( approximately 2 minimum alveolar concentration; drug concentration for half-maximal inhibition [IC50] > 1.5 mM) [corrected] or propofol up to 40 microm in synaptosomes prepared from rat, mouse, or guinea pig cerebral cortex, rat hippocampus, or rat striatum. Lower concentrations of isoflurane or propofol significantly inhibited veratridine-evoked glutamate release in all three species (isoflurane IC50 = 0.41-0.50 mm; propofol IC50 = 11-18 microm) and rat brain regions. Glutamate release was evoked by veratridine or increased KCl (from 5 to 35 mM) to assess the involvement of presynaptic ion channels as targets for drug actions [corrected]. CONCLUSIONS: Isoflurane and propofol inhibited Na+ channel-mediated glutamate release evoked by veratridine with greater potency than release evoked by increased KCl in synaptosomes prepared from three mammalian species and three rat brain regions. These findings are consistent with a greater sensitivity to anesthetics of presynaptic Na+ channels than of Ca2+ channels coupled to glutamate release. This widespread presynaptic action of general anesthetics is not mediated by potentiation of gamma-aminobutyric acid type A receptors, though additional mechanisms may be involved.     

Optimal concentration of cellular free calcium for AVP-induced cAMP in collecting tubules

The role of Ca2+ in the cellular action of arginine vasopressin (AVP) has been demonstrated in renal papillary collecting tubule. We further examined whether an optimal concentration of cellular free Ca2+ [( Ca2+]i) exists for AVP-induced cAMP production in rat renal papillary collecting tubule cells in culture. [Ca2+]i was measured using fura-2. When cells were exposed for one hour to the media supplemented with 0 mM Ca2+ (containing 1 mM EGTA), 1, 2, 3, 4, 6.4, or 8 mM Ca2+, basal [Ca2+]i ranged as below: 24.9 +/- 5.6, 90.7 +/- 7.4, 107.4 +/- 9.8, 146.1 +/- 13.7, 162.0 +/- 14.6, 241.5 +/- 32.8, and 234.9 +/- 29.6 nM, respectively. When medium Ca2+ was 1 mM, 1 x 10(-7) M AVP increased [Ca2+]i to 181.5 +/- 13.2 nM from 90.7 +/- 7.4 nM (P less than 0.01). AVP-induced increases in [Ca2+]i were obtained with the varying Ca2+ media described above, though the increases in [Ca2+]i were quantitatively variable. AVP-induced cellular cAMP production was examined during three minute incubation period in the presence of 5 x 10(-4) M 3-isobutyl-1-methylxanthine. Basal level of cellular cAMP was 87.6 +/- 7.9 fmol/micrograms protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anesthetic Preconditioning Enhances Ca2+ Handling and Mechanical and Metabolic Function Elicited by Na+-Ca2+ Exchange Inhibition in Isolated Hearts

Background: Anesthetic preconditioning (APC) is well known to protect against myocardial ischemia-reperfusion injury. Studies also show the benefit of Na+-Ca2+ exchange inhibition on ischemia-reperfusion injury. The authors tested whether APC plus Na+-Ca2+ exchange inhibitors given just on reperfusion affords additive protection in intact hearts.

Methods: Cytosolic [Ca2+] was measured by fluorescence at the left ventricular wall of guinea pig isolated hearts using indo-1 dye. Sarcoplasmic reticular Ca2+-cycling proteins, i.e., Ca2+ release channel (ryanodine receptor [RyR2]), sarcoplasmic reticular Ca2+-pump adenosine triphosphatase (SERCA2a), and phospholamban were measured by Western blots. Hearts were assigned to seven groups (n = 8 each): (1) time control; (2) ischemia; (3, 4) 10 [mu]m Na+-Ca2+ exchange inhibitor KB-R7943 (KBR) or 1 [mu]m SEA0400 (SEA), given during the first 10 min of reperfusion; (5) APC initiated by sevoflurane (2.2%, 0.41 +/- 0.03 mm) given for 15 min and washed out for 15 min before ischemia-reperfusion; (6, 7) APC plus KBR or SEA.

Results: The authors found that APC reduced the increase in systolic [Ca2+], whereas KBR and SEA both reduced the increase in diastolic [Ca2+] on reperfusion. Each intervention improved recovery of left ventricular function. Moreover, APC plus KBR or SEA afforded better functional recovery than APC, KBR, or SEA alone (P < 0.05). Ischemia-reperfusion-induced degradation of major sarcoplasmic reticular Ca2+-cycling proteins was attenuated by APC, but not by KBR or SEA.     

Effects of advanced glycation end products on cytosolic Ca2+ signaling of cultured human mesangial cells.

Advanced glycation end product (AGE) accumulation in a high glucose (HG) environment is thought to mediate some of the vascular complications of diabetes. Transmembrane signaling of contractile cells is generally inhibited by HG, with implications for systemic and target organ hemodynamics. In the kidney, glomerular mesangial cells grown in HG media are hyporesponsive to the effects of vasoconstrictor agents, possibly explaining the hyperfiltration and increased capillary pressure that eventually lead to diabetic glomerulopathy. To verify whether AGE binding to specific mesangial receptors could mediate these effects of HG, cultured human mesangial cells (HMC) were exposed to in vitro glycated bovine serum albumin (BSA) for 60 min at 37 degrees C before measurement of cytosolic Ca2+ ([Ca2+]i) by microfluorometric techniques in monolayers or single cells. AGE-BSA (2 mg/ml) reduced Ca2+ release from intracellular stores by 1 microM angiotensin II from peak [Ca2+]i levels of 843+/-117 to 390+/-50 nM in monolayers and from 689+/-68 to 291+/-36 nM in individual cells (P < 0.05). Nonglycated BSA and BSA exposed to 250 mM glucose-6-phosphate for 30 d in the presence of 250 mM aminoguanidine (AMGD), an inhibitor of nonenzymatic glycation, had no effect on the angiotensin II-induced [Ca2+]i spike (peak 766+/-104 and 647+/-87 nM, monolayers/ single cells, respectively, P = NS). AGE also inhibited store-operated Ca2+ influx through plasma membrane channels, assessed by addition of 1 to 10 mM extracellular Ca2+ to cells previously held in Ca2(+)-free media (control 339+/- 46/593 +/- 51, +AGE-BSA 236 +/- 25/390 +/- 56, +AMGD 483+/-55/ 374+/-64 nM [Ca2+]i, monolayers/single cells at 10 mM Ca2+, respectively; +AGE-BSA, P < 0.05 versus control). Contrary to HG, AGE-BSA did not translocate protein kinase C isoforms alpha, zeta, and delta to the plasma membrane. Culture of HMC in HG supplemented with 1 mM AMGD prevented downregulation of [Ca2+]i signaling. These data suggest that glycated macromolecules or matrix components may inhibit transmembrane Ca2+ signaling of glomerular cells through binding to a specific AGE receptor, thus mediating some of the known functional effects of HG on the kidney.     

Effects of halothane on sarcoplasmic reticulum calcium release channels in porcine airway smooth muscle cells

BACKGROUND: Volatile anesthetics relax airway smooth muscle (ASM) by altering intracellular Ca2+ concentration ([Ca2+]i). The authors hypothesized that relaxation is produced by decreasing sarcoplasmic reticulum Ca2+ content via increased Ca2+ "leak" through both inositol trisphosphate (IP3) and ryanodine receptor channels. METHODS: Enzymatically dissociated porcine ASM cells were exposed to acetylcholine in the presence or absence of 2 minimum alveolar concentration (MAC) halothane, and IP3 levels were measured using radioimmunoreceptor assay. Other cells were loaded with the Ca2+ indicator fluo-3 and imaged using real-time confocal microscopy. RESULTS: Halothane increased IP3 concentrations in the presence and absence of acetylcholine. Inhibition of phospholipase C blunted the IP3 response to halothane. Exposure to 2 MAC halothane induced a transient [Ca2+]i response, suggesting depletion of sarcoplasmic reticulum Ca2+. Exposure to 20 microM Xestospongin D, a cell-permeant IP3 receptor antagonist, resulted in a 45+/-13% decrease in the [Ca2+]i response to halothane compared with halothane exposure alone. In permeabilized cells, Xestospongin D or 0.5 mg/ml heparin decreased the [Ca2+]i response to halothane by 65+/-13% and 68+/-22%, respectively, compared with halothane alone. In both intact and permeabilized cells, 20 microM ryanodine blunted the [Ca2+]i response to halothane by 32+/-13% and 39+/-21%, respectively, compared with halothane alone. Simultaneous exposure to Xestospongin D and ryanodine completely inhibited the [Ca2+]i response to halothane. CONCLUSIONS: The authors conclude that halothane reduces sarcoplasmic reticulum Ca2+ content in ASM cells via increased Ca2+ leak through both IP3 receptor and ryanodine receptor channels. Effects on IP3 receptor channels are both direct and indirect via elevation of IP3 levels.     

Anesthetic preconditioning enhances Ca2+ handling and mechanical and metabolic function elicited by Na+-Ca2+ exchange inhibition in isolated hearts

BACKGROUND: Anesthetic preconditioning (APC) is well known to protect against myocardial ischemia-reperfusion injury. Studies also show the benefit of Na+-Ca2+ exchange inhibition on ischemia-reperfusion injury. The authors tested whether APC plus Na+-Ca2+ exchange inhibitors given just on reperfusion affords additive protection in intact hearts. METHODS: Cytosolic [Ca2+] was measured by fluorescence at the left ventricular wall of guinea pig isolated hearts using indo-1 dye. Sarcoplasmic reticular Ca2+-cycling proteins, i.e., Ca2+ release channel (ryanodine receptor [RyR2]), sarcoplasmic reticular Ca2+-pump adenosine triphosphatase (SERCA2a), and phospholamban were measured by Western blots. Hearts were assigned to seven groups (n = 8 each): (1) time control; (2) ischemia; (3, 4) 10 microM Na+-Ca2+ exchange inhibitor KB-R7943 (KBR) or 1 microM SEA0400 (SEA), given during the first 10 min of reperfusion; (5) APC initiated by sevoflurane (2.2%, 0.41 +/- 0.03 mm) given for 15 min and washed out for 15 min before ischemia-reperfusion; (6, 7) APC plus KBR or SEA. RESULTS: The authors found that APC reduced the increase in systolic [Ca2+], whereas KBR and SEA both reduced the increase in diastolic [Ca2+] on reperfusion. Each intervention improved recovery of left ventricular function. Moreover, APC plus KBR or SEA afforded better functional recovery than APC, KBR, or SEA alone (P < 0.05). Ischemia-reperfusion-induced degradation of major sarcoplasmic reticular Ca2+-cycling proteins was attenuated by APC, but not by KBR or SEA. CONCLUSIONS: APC plus Na+-Ca2+ exchange inhibition exerts additive protection in part by reducing systolic and diastolic Ca2+ overload, respectively, during ischemia-reperfusion. Less degradation of sarcoplasmic reticular Ca2+-cycling proteins may also contribute to cardiac protection.     

Widespread Inhibition of Sodium Channel-dependent Glutamate Release from Isolated Nerve Terminals by Isoflurane and Propofol

Background : Controversy persists concerning the mechanisms and role of general anesthetic inhibition of glutamate release from nerve endings. To determine the generality of this effect and to control for methodologic differences between previous studies, the authors analyzed the presynaptic effects of isoflurane and propofol on glutamate release from nerve terminals isolated from several species and brain regions.

Methods : Synaptosomes were prepared from rat, mouse, or guinea pig cerebral cortex and also from rat striatum and hippocampus. Release of endogenous glutamate evoked by depolarization with 20 [mu]m veratridine (which opens voltage-dependent Na+ channels by preventing inactivation) or by 30 mm KCl (which activates voltage-gated Ca2+ channels by membrane depolarization) was monitored using an on-line enzyme-linked fluorometric assay.

Results : Glutamate release evoked by depolarization with increased extracellular KCl was not significantly inhibited by isoflurane up to 0.7 mm (~2 minimum alveolar concentration; drug concentration for half-maximal inhibition > 1.5 mm) or propofol up to 40 [mu]m in synaptosomes prepared from rat, mouse, or guinea pig cerebral cortex, rat hippocampus, or rat striatum. Lower concentrations of isoflurane or propofol significantly inhibited veratridine-evoked glutamate release in all three species (isoflurane IC50 = 0.41-0.50 mm; propofol IC50 = 11-18 [mu]m) and rat brain regions. Inhibition of veratridine-evoked release was insensitive to the [gamma]-aminobutyric acid receptor type A antagonist bicuculline (100 [mu]m) in rat cortical synaptosomes.     

Differential Effects of Sevoflurane, Isoflurane, and Halothane on Ca (2+) Release from the Sarcoplasmic Reticulum of Skeletal Muscle

Background: Although malignant hyperthermia after application of sevoflurane has been reported, little is known about its action on intracellular calcium homeostasis of skeletal muscle. The authors compared the effect of sevoflurane with that of isoflurane and halothane on Ca2+ release of mammalian sarcoplasmic reticulum and applied a novel method to quantify Ca2+ turnover in permeabilized skeletal muscle fibers.

Methods: Liquid sevoflurane, isoflurane, and halothane at 0.6 mM, 3.5 mM, and 7.6 mM were diluted either in weakly calcium buffered solutions with no added Ca2+ (to monitor Ca2+ release) or in strongly Ca2+ buffered solutions with [Ca2+] values between 3 nM and 24.9 [micro sign]M for [Ca2+]-force relations. Measurements were taken on single saponin skinned muscle fiber preparations of BALB/c mice. Individual [Ca2+]-force relations were characterized by the Ca2+ concentration at half-maximal force that indicates the sensitivity of the contractile proteins and by the steepness. Each force transient was transformed directly into a Ca (2+) transient with respect to the individual [Ca2+]-force relation of the fiber.

Results: At 0.6 mM, single force transients induced by sevoflurane were lower compared with equimolar concentrations of isoflurane and halothane (P < 0.05). Similarly, calculated peak Ca2+ transients of sevoflurane were lower than those induced by equimolar halothane (P < 0.05). The Ca2+ concentrations at half maximal force were decreased after the addition of sevoflurane, isoflurane, and halothane in a concentration-dependent manner (P < 0.05).