Satellite glia cells in dorsal root ganglia express functional NMDA receptors.

Satellite glia cells (SGCs), within the dorsal root ganglia (DRG), surround the somata of most sensory neurons. SGCs have been shown to interact with sensory neurons and appear to be involved in the processing of afferent information. We found that in rat DRG various N-methyl-D-aspartate receptor (NMDAr) subunits were expressed in SGCs in intact ganglia and in vitro. In culture, when SGCs were exposed to brief pulses of NMDA they evoked transient increases in cytoplasmic calcium that were inhibited by specific NMDA blockers (MK-801, AP5) while they were Mg²âº insensitive indicating that SGCs express functional NMDAr. The percentage of NMDA responsive SGCs was similar in mixed- (SGCs plus neurons) and SGC-enriched cultures. The pattern of the magnitude changes of the NMDA-evoked response was similar in SGCs and DRG neurons when they were in close proximity, suggesting that the NMDA response of SGCs and DRG neurons is modulated by their interactions. Treating the cultures with nerve growth factor, and/or prostaglandin E2 did not alter the percentage of SGCs that responded to NMDA. Since glutamate appears to be released within the DRG, the detection of functional NMDAr in SGCs suggests that their NMDAr activity could contribute to the interactions between neurons and SGCs. In summary we demonstrated for the first time that SGCs express functional NMDAr.

The N-methyl-D-aspartate-evoked cytoplasmic calcium increase in adult rat dorsal root ganglion neuronal somata was potentiated by substance P pretreatment in a protein kinase C-dependent manner.

The involvement of substance P (SP) in neuronal sensitization through the activation of the neurokinin-1-receptor (NK1r) in postsynaptic dorsal horn neurons has been well established. In contrast, the role of SP and NK1r in primary sensory dorsal root ganglion (DRG) neurons, in particular in the soma, is not well understood. In this study, we evaluated whether SP modulated the NMDA-evoked transient increase in cytoplasmic Ca2+ ([Ca2+]cyt) in the soma of dissociated adult DRG neurons. Cultures were treated with nerve growth factor (NGF), prostaglandin E2 (PGE2) or both NGF+PGE2. Treatment with NGF+PGE2 increased the percentage of N-methyl-D-aspartate (NMDA) responsive neurons. There was no correlation between the percentage of NMDA responsive neurons and the level of expression of the NR1 and NR2B subunits of the NMDA receptor or of the NK1r. Pretreatment with SP did not alter the percentage of NMDA responsive neurons; while it potentiated the NMDA-evoked [Ca2+]cyt transient by increasing its magnitude and by prolonging the period during which small- and some medium-sized neurons remained NMDA responsive. The SP-mediated potentiation was blocked by the SP-antagonist ([D-Pro4, D-Trp7,9]-SP (4-11)) and by the protein kinase C (PKC) blocker bisindolylmaleimide I (BIM); and correlated with the phosphorylation of PKCε. The Nk1r agonist [Sar9, Met(O2)11]-SP (SarMet-SP) also potentiated the NMDA-evoked [Ca2+]cyt transient. Exposure to SP or SarMet-SP produced a rapid increase in the labeling of phosphorylated-PKCε. In none of the conditions we detected phosphorylation of the NR2B subunit at Ser-1303. Phosphorylation of the NR2B subunit at Tyr1472 was enhanced to a similar extent in cells exposed to NMDA, SP or NMDA+SP, and that enhancement was blocked by BIM. Our findings suggest that NGF and PGE2 may contribute to the injury-evoked sensitization of DRG neurons in part by enhancing their NMDA-evoked [Ca2+]cyt transient in all sized DRG neurons; and that SP may further contribute to the DRG sensitization by enhancing and prolonging the NMDA-evoked increase in [Ca2+]cyt in small- and medium-sized DRG neurons.

Effect of cardiolipin oxidation on solid-phase immunoassay for antiphospholipid antibodies.

Diagnostic assays for antiphospholipid antibodies are routinely performed on microtitre plates coated with cardiolipin. Here we show that contact between cardiolipin and NUNC-Immuno plates leads to extensive oxidation, generating a series of peroxy-cardiolipins which were identified by electrospray ionization mass spectrometry. To investigate the impact of oxidation on the antibody assay. cardiolipin was resolved into 12 molecular species, including oxidized species and non-oxidized species with different degrees of unsaturation. All 12 species reacted under anaerobic conditions with serum from patients with primary antiphospholipid syndrome. Immune reactivity was similar for tetralinoleoyl-cardiolipin, trilinoleoyl-oleoyl-cardiolipin, and peroxycardiolipins, but somewhat lower for tristearoyl-oleoyl-cardiolipin. Oxidative treatment of cardiolipin with air, cytochrome c, or Cu2+/tert-butylhydroperoxide, either before or during the assay, did not enhance immune reactivity. Similar results were obtained with a monoclonal IgM from lupus-prone mice, that binds cardiolipin in the absence of protein cofactors. We conclude that the solid-phase assay for antiphospholipid antibodies can be supported by various oxidized and non-oxididized molecular species of cardiolipin.

Isoflurane pretreatment ameliorates postischemic neurologic dysfunction and preserves hippocampal Ca2+/calmodulin-dependent protein kinase in a canine cardiac arrest model.

BACKGROUND: Inhalational anesthetics are neuroprotective in rat models of global ischemia. To determine whether isoflurane at a clinically relevant concentration is neuroprotective in a canine model of cardiac arrest, we measured neurologic function and hippocampal Ca2+/calmodulin-dependent protein kinase II (CaMKII) content 20 h after cardiac arrest. METHODS: We tested the neuroprotective effect of 30 min of 1.5% isoflurane exposure before 8 min of global ischemia induced with ventricular fibrillation. Animals were randomized to four groups: control, isoflurane-control, ischemia, and isoflurane-ischemia. After resuscitation and 20 h of intensive care, each animal's neurologic deficit score was determined by two blinded evaluators. The hippocampal content of CaMKII, determined by immunoblotting, was measured by an individual blinded to the treatment groups. CaMKII activity was measured in samples from the cortex, hippocampus, and striatum of animals in each group. RESULTS: Isoflurane-ischemic animals had a median neurologic deficit score of 22.6% compared with 43.8% for the ischemic animals (P < 0.05). Hippocampal levels of the beta-subunit of CaMKII (CaMKIIbeta) were relatively preserved in isoflurane-ischemic animals (68 +/- 4% of control) compared with ischemic animals (48 +/- 2% of control; P < 0.001), although both groups were statistically significantly lower than control (P < 0. 001 ischemia vs. control and P < 0.05 isoflurane-ischemia vs. control). CONCLUSIONS: Isoflurane is an effective neuroprotective drug in a canine cardiac arrest model in terms of both functional and biochemical criteria.

Advances and strategies for spinal cord regeneration.

Although a cure for spinal cord injuries does not currently exist, advances have been made in the field of spinal cord regeneration. This article discusses the pathophysiology of spinal cord injury, animal models, and strategies for restoration and regeneration of the spinal cord.

Halothane and isoflurane augment depolarization-induced cytosolic CA2+ transients and attenuate carbachol-stimulated CA2+ transients.

BACKGROUND: Neuronal excitability is in part determined by Ca2+ availability that is controlled by regulatory mechanisms of cytosolic Ca2+ ([Ca2+]cyt). Alteration of any of those mechanisms by volatile anesthetics (VAs) may lead to a change in presynaptic transmission and postsynaptic excitability. Using a human neuroblastoma cell line, the effects of halothane and isoflurane on cytosolic Ca2+ concentration ([Ca2+]cyt) in response to K+ and carbachol stimulation were investigated. METHODS: Volatile anesthetic (0.05-1 mm) action on stimulated [Ca2+]cyt transients were monitored in suspensions of SH-SY5Y cells loaded with fura-2. Potassium chloride (KCl; 100 mm) was used to depolarize and activate Ca2+ entry through voltage-dependent calcium channels; 1 mm carbachol was used to activate muscarinic receptor-mediated inositol triphosphate (IP3)-dependent intracellular Ca2+ release. Sequential stimulations, KCl followed by carbachol and vice versa, were used to investigate interactions between intracellular Ca2+ stores. RESULTS: Halothane and isoflurane in clinically relevant concentrations enhanced the K+-evoked [Ca2+]cyt transient whether intracellular Ca2+ stores were full or partially depleted. In contrast, halothane and isoflurane reduced the carbachol-evoked [Ca2+]cyt transient when the intracellular Ca2+ stores were full but had no effect when the Ca2+ stores were partially depleted by KCl stimulation. CONCLUSIONS: Volatile anesthetics acted on sites that differently affect the K+- and carbachol-evoked [Ca2+]cyt transients. These data suggest the involvement of an intracellular Ca2+ translocation from the caffeine-sensitive Ca2+ store to the inositol triphosphate-sensitive Ca2+ store that was altered by halothane and isoflurane.

CNS voltage-dependent Na(+) channel expression and distribution in an undifferentiated and differentiated CNS cell line.

Upon serum removal, CAD-R1 cells undergo neurite outgrowth and an increase in voltage-dependent Na(+) current (VDNaC) density without changing their activation and inactivation properties. Insulin and endothelial cell growth supplement inhibited the increase in VDNaC density but not the neurite outgrowth. RI, RII, RIII Na(+) channel proteins were expressed in CAD-R1 cells. These proteins exhibited both similar and different distribution and clustering patterns which suggested the channel's structural differences play a role in channel distribution.

Microanalysis of cardiolipin in small biopsies including skeletal muscle from patients with mitochondrial disease.

Cardiolipin is a specific mitochondrial phospholipid that is present in mammalian tissues in low concentration. To measure cardiolipin in small biopsies from patients with mitochondrial disease, we developed a new technique that can detect subnanomolar levels of well-resolved molecular species, the most abundant of which are tetralinoleoyl-cardiolipin (L(4)) and trilinoleoyl-oleoyl-cardiolipin (L(3)O). To this end, a fluorescence-labeled derivative of cardiolipin (2-[naphthyl-1'-acetyl]-cardiolipin dimethyl ester) was formed and analyzed by high performance liquid chromatography. Cardiolipin was measured in skeletal muscle biopsies from 8 patients with mitochondrial disease and in 17 control subjects. In 5 patients with mitochondrial disease, cardiolipin content was higher than normal (2. 4;-7.0 vs. 0.4;-2.2 nmol/mg protein). In 3 patients with mitochondrial disease, the L(4)/L(3)O ratio was lower than normal (2;-4 vs. 4;-6). Cardiolipin was also measured in various rat and dog muscle tissues. The L(4)/L(3)O ratio was higher in condensed "muscle" type mitochondria (heart ventricle, skeletal muscle, ratios 4;-7) than in orthodox "liver" type mitochondria (liver, smooth muscle, heart auricular appendage, H9c2 myoblasts, ratios 0.4;-3), suggesting that the L(4)/L(3)O proportion is important for cristae membrane structure. We concluded that the L(4)/L(3)O ratio is a tissue-specific variable that may change in the presence of mitochondrial disease. The new method is suitable to measure cardiolipin in muscle biopsies in order to estimate concentration of mitochondria.

Volatile anesthetic action on muscle Ca2+ homeostasis.

It is proposed that volatile anesthetics act through the modification of Ca2+ homeostasis in excitable cells. To test this hypothesis, cardiac and skeletal muscles were used as models to examine Ca2+ response, and Ca2+ regulatory and delivery mechanisms. I found that halothane did not alter Ca2+ binding to cardiac troponin C. However, halothane and isoflurane reversibly decreased the Ca2+ affinity of calmodulin at low anesthetic concentration, and irreversibly increased the Ca2+ affinity of calmodulin at high anesthetic concentration. The volatile anesthetics also increased the permeability of light fraction of sarcoplasmic reticulum (SR) to Ca2+. I conclude that volatile anesthetics alter calcium homeostasis in cardiac and skeletal muscles. This work was in part performed in collaboration with Giovanni Salviati and the author benefited from Salviati's work in similar areas.

Permeability of rat heart myocytes to cytochrome c.

Rat heart myocytes undergoing progressive damage demonstrate morphological changes of shortening and swelling followed by the formation of intracellular vacuoles and plasma membrane blebbing. The damaged myocytes displayed impaired N,N'-tetramethyl-p-phenyldiamine (TMPD) ascorbate-stimulated respiratory activity which was restored by the addition of reduced cytochrome c to the cell culture medium. To clarify the role played by cytochrome c in the impairment of cell respiration, polarographic, spectrophotometric and fluorescence as well as electron microscopy imaging experiments were performed. TMPD/ascorbate-stimulated respiratory activity returned to control levels, at approximately 20 microM cytochrome c, establishing the threshold below which the turnover rate by cytochrome c oxidase in the cell depends on cytochrome concentration. Mildly damaged cardiac myocytes, as indicated by cell shortening, retention of visible striations and free-fluorescein exclusion, together with the absence of lactate dehydrogenase leakage and exclusion of trypan blue, were able to oxidize exogenous cytochrome c and were permeable to fluorescein-conjugated cytochrome c. The results, while consistent with an early cytochrome c release observed at the beginning of cell death, elucidate the role played by cytochrome c in the kinetic control of mitochondrial electron transfer under pathological conditions, particularly those involving the terminal part of the respiratory chain. These data are the first to demonstrate that the sarcolemma of cardiac myocytes, damaged but still viable, is permeable to cytochrome c.

Halothane and isoflurane alter calcium dynamics in rat cerebrocortical synaptosomes.

UNLABELLED: An increase in synaptosomal Ca2+ triggers neurotransmitter release and volatile anesthetics have been shown to inhibit neurotransmitter release by inhibition of Ca2+ entry. We have examined the effect of isoflurane and halothane on the kinetics of increase and decrease of Ca2+ in rat cerebrocortical synaptosomes ([Ca2+]in). We have also used specific Ca2+ antagonists to examine the role of L-, N-, and P-type Ca2+ channels. Synaptosomal [Ca2+]in was measured spectrofluorometrically using fura-2 as a Ca2+ reporter; Ca2+ transients were initiated by depolarization with 40 mM KCl. We found that < or = 1 minimum alveolar anesthetic concentration halothane and isoflurane decreased peak [Ca2+]in by approximately 40%, that both anesthetics decreased the rate of [Ca2+]in increase and decrease, that specific voltage-dependent calcium channel antagonists had little effect on peak or plateau [Ca2+]in, and that the volatile anesthetics increased the permeability of synaptosomal membranes to Ca2+. These results suggest that the volatile anesthetics, at clinically relevant concentrations, can alter Ca2+ homeostasis in the synapse. IMPLICATIONS: Clinically relevant concentrations of halothane and isoflurane markedly depress K+-evoked increases in rat cerebrocortical synaptosomal calcium (Ca2+) unrelated to L-, N-, and P-type voltage-dependent calcium channels and increase the Ca2+ permeability of the synaptosomal membrane. These changes in Ca2+ dynamics could have profound effects on Ca2+ signaling in the synapse.

The effects of halothane on single human neuronal L-type calcium channels.

UNLABELLED: We investigated halothane's effects on the function of L-type Ca2+ channels in a human neuronal cell line, SH-SY5Y, by using the cell-attached patch voltage clamp configuration and Ba2+ as the charge carrier. In multiple-channel patches, halothane decreased the peak and persistent Ba2+ currents, accelerated the rate of inactivation, and slowed the rate of activation. Single-channel analysis showed that halothane (0.14-1.26 mM) increased the latency time for the first channel opening, increased the lifetime of nonconducting events, increased the proportion of short-lived open events, decreased the lifetime of the two open populations, and increased the percentage of current traces without channel activity. All of the observed halothane effects contribute to the halothane-induced decrease in macroscopic Ba2+ currents. The halothane concentration producing 50% reduction (IC50) of the peak Ba2+ current was 0.80 mM (approximately 1.9 hypothetical minimum alveolar anesthetic concentration [H-MAC] at 28 degrees C) and of the persistent Ba2+ current was 0.69 mM (approximately 1.7 H-MAC). The halothane effects did not always occur together, and the Hill slope of 1.6 suggested the presence of more than one interaction site or of more than one population of L-type Ca2+ channels. Halothane reduces L-type Ca2+ channel currents in human neuronal cells primarily through the stabilization of nonconducting states such as closed (before and after channel opening) and inactivated states. IMPLICATIONS: Calcium is a signaling molecule in neurons. We measured the effect of halothane on Ba2+ (a Ca2+ surrogate) movement into a human neuron-like cell electronically. Ba2+ entry through the L-type channel was depressed. Halothane decreased the likelihood of the channel opening and enhanced the rate at which the channel closed and inactivated. These actions of halothane are probably related to its anesthetic action.

Global ischemia increases the density of voltage-dependent calcium channels in porcine cardiac sarcolemma.

The objective of this work was to determine whether normothermic global cardiac ischemia in a porcine model was associated with a change in the density (Bmax) of voltage-dependent calcium channels in myocardial sarcolemmal membranes. Pigs were anesthetized, a thoracotomy was performed, and samples were taken of the left and right ventricles from control and ischemic hearts. Dihydropyridine-binding sites were quantified using [3H]isradipine, and 5'-nucleotidase activity was measured by the liberation of inorganic phosphate from adenosine monophosphate. Bmax and dissociation constants and 5'-nucleotidase activity for control and ischemic tissues, respectively, were compared by using Student's t-test for unpaired samples. After normothermic global ischemia, the Bmax of [3H]isradipine binding increased in the left ventricle by 81% (299% +/- 1.7% to 540% +/- 11% fmoles/mg, P < 0.01) and in the right ventricle by 33% (387% +/- 9.9% to 515% +/- 38% fmoles/mg, P < 0.01) compared with control. 5'-nucleotidase activity increased by 48% in the left ventricle and by 96% in the right ventricle (p < 0.05). Fifteen minutes of normothermic ischemia in the pig is associated with marked sarcolemmal abnormalities, including increases in specific dihydropyridine binding and 5'-nucleotidase activity, which reflect global changes in membrane function, which might contribute to the increase in myoplasmic calcium during ischemia.

Volatile anaesthetic effects on calcium conductance of planar lipid bilayers formed with synthetic lipids or extracted lipids from sarcoplasmic reticulum.

Volatile anaesthetics are known to increase leakage of calcium from the light fraction of skeletal sarcoplasmic reticulum (L-SR) which has no calcium release channels. To explore the role of the lipid environment, we have examined the effect of volatile anaesthetics on calcium conductance (gCa) of lipid membranes. Planar lipid bilayers were formed with a mixture of synthetic phospholipids and cholesterol, resembling the composition of SR membranes, or with lipids extracted from skeletal L-SR, gCa was estimated by calculating the calcium transference number (tCa) using diffusion potential measurements. Membranes formed with L-SR-extracted lipids had a higher gCa than membranes formed with synthetic lipids. Volatile anaesthetics increased total conductance and gCa in a dose-dependent manner, but did not affect tCa or membrane specific capacitance. In membranes formed with L-SR-extracted lipids, isoflurane induced the largest increase in gCa (1260 (SEM 304) % increase, n = 4, 0.94 mmol litre-1), followed by enflurane (264 (75)%, n = 5, 1.88 mmol litre-1) and halothane (53 (33)%, n = 5; 1.54 mmol litre-1). In membranes formed with synthetic lipids, volatile anaesthetic-induced increases in gCa followed the same trend but were larger. Volatile anaesthetics increased gCa without changing the ionic selectivity of membranes. However, the magnitude of the increase in gCa in the presence of volatile anaesthetics cannot account for the previously observed calcium leakage from L-SR vesicles. Therefore, the volatile anaesthetic-induced increase in calcium leakage in L-SR vesicles must be mediated via other pathways involving membrane proteins.

Halothane and isoflurane alter the Ca2+ binding properties of calmodulin.

BACKGROUND: Ca2+ plays an important role in signal transduction and anesthetic mechanisms. To date, no one has observed a direct effect of volatile anesthetics on a Ca(2+)-binding protein. We therefore examined the effects of halothane and isoflurane on the Ca(2+)-binding properties of bovine brain calmodulin. METHODS: The fluorescence emission of calmodulin was obtained over a range of Ca2+ concentrations (10(-7)-10(-4)M) in the presence and absence of halothane and isoflurane. The intrinsic tyrosine fluorescence of calmodulin was measured at an excitation wavelength of 280 nm and an emission wavelength of 320 nm. Fluorescence measurements were carried out in 50 mM hydroxyethylpiperazineethane sulfonic acid, 100 mM KC1, and 2 mM ethyleneglycol-bis-(beta-aminoethyl ether) tetraacetic acid at pH 7.0 and 37 degrees C. Experiments were performed in polytetrafluorethylene-sealed cuvettes so that the volatile anesthetic concentrations remained constant. The titration data were analyzed in two ways. The data were fit to the Hill equation by using nonlinear regression analysis to derive the Hill coefficient and the dissociation constant. The data were also analyzed by two-way analysis of variance with multiple comparisons to determine statistically significant effects. Volatile anesthetic concentrations were measured by gas chromatography. RESULTS: The presence of volatile anesthetics altered the Ca(2+)-binding affinity of calmodulin in a dose-dependent fashion. At 0.57% (0.25 mM) halothane and 1.7% (0.66 mM) isoflurane, the affinity of calmodulin for Ca2+ relative to control was decreased. However, at higher concentrations of both anesthetics, the affinity for Ca2+ was increased. When the volatile anesthetics were allowed to evaporate from the experimental solutions, the observed rightward shift of the calmodulin-Ca2+ binding curve for Ca2+ at low concentrations of the anesthetics returned to the control position. The leftward shift seen at high concentrations of the anesthetics was irreversible after evaporation of 8.7% (3.3 mM) isoflurane and 5.7% (2.5 mM) halothane. CONCLUSIONS: These data demonstrate a complex interaction of two hydrophobic volatile anesthetics with calmodulin. A biphasic effect was observed both for halothane and for isoflurane. Calmodulin, an EF-hand Ca(2+)-binding protein, undergoes a conformational shift when binding Ca2+, exposing several hydrophobic residues. These residues may be sites at which the anesthetics act.

Calcium-dependent translocation of sorcin to membranes: functional relevance in contractile tissue.

Sorcin, a 22 kDa calcium binding protein present in abundance in cardiac tissue and in multi-drug resistant cells and previously described as a soluble protein, is now shown to undergo a calcium-dependent translocation process from the cytosol to cellular membranes in both systems. The translocation process takes place also in E. coli BL21 cells that express recombinant sorcin, r-sorcin, and can be exploited in the purification of the protein. Calcium binding to purified r-sorcin occurs at micromolar concentrations of the metal and is accompanied by a conformational change that renders the protein soluble in the non-ionic detergent Triton X-114. This finding suggests that lipids are the target of sorcin on cellular membranes. The possible significance of the calcium-dependent translocation of sorcin in the specialized functions of sorcin-expressing cells is discussed.

The effects of halothane on voltage-dependent calcium channels in isolated Langendorff-perfused rat heart.

BACKGROUND: Halothane has been previously shown in vitro to decrease both the inward calcium current in isolated cells and the density of calcium antagonist binding sites in cardiac sarcolemmal membranes prepared from several species, including humans, presumably contributing to the negative inotropic effects seen with volatile anesthetics. In this study we examined whether halothane produced similar changes in calcium channel antagonist binding characteristics ex vivo in an intact perfused heart by using isradipine, a dihydropyridine calcium channel blocker that binds specifically to the alpha 1 subunit of the L-type voltage-dependent calcium channel. METHODS: The rat hearts were perfused by the Langendorff method in the presence of halothane and unlabeled isradipine. After the hearts were homogenized and prepared into membranes, a radioligand binding assay was performed and binding curves obtained. Data were analyzed by nonlinear regression analysis of a one-site binding equation and were evaluated by a paired t test. RESULTS: Halothane protected or inhibited the binding of unlabeled isradipine to calcium channels in a dose-dependent manner such that as the halothane is removed during the membrane preparation process, previously obscured sites were then available for specific binding of the radioligand. The sites that were protected by halothane had a lower affinity for [3H]-isradipine than controls. CONCLUSIONS: In both isolated membranes and the intact heart, halothane changes the availability of calcium channel antagonist binding sites, indicating a change in conformation of the voltage-dependent calcium channel in the presence of anesthetic. This change may result from a direct effect on the protein or from an indirect effect mediated through the membrane lipid bilayer. It also is demonstrated that halothane "protected" channels are probably a modified class of channels compared to those in control tissues as exemplified by the much lower affinity that the protected channels have for [3H]-isradipine. We conclude that a major mechanism by which halothane depresses contractility is mediated through the voltage-dependent calcium channel, and this process results from a conformational change in the channel.

Increased activation of L-type voltage-dependent calcium channels is associated with glycine enhancement of N-methyl-D-aspartate-stimulated dopamine release in global cerebral ischemia/reperfusion.

We investigated the relationships among N-methyl-D-aspartate, glycine, L-type voltage-dependent calcium channels, and [3H]dopamine release in a canine model of global cerebral ischemia/reperfusion. The binding of [3H]PN200-110 ([3H]isradipine) to L-type voltage-dependent calcium channels, that open as a consequence of N-methyl-D-aspartate-induced changes in membrane potential, was approximately doubled in striatal membranes prepared from ischemic animals relative to controls, and remained significantly elevated at 30 min and 2 h of reperfusion. These changes coincided temporally with changes in the ability of the voltage-sensitive calcium channel blocker nitrendipine to inhibit glycine enhancement of N-methyl-D-aspartate-stimulated [3H]dopamine release in striatal slices prepared from the same animals. Compared with nonischemic controls, N-methyl-D-aspartate-stimulated [3H]dopamine release was increased in ischemic animals and remained increased throughout reperfusion up to at least 24 h. Glycine enhanced N-methyl-D-aspartate-stimulated release in all treatment groups. The enhancement of N-methyl-D-aspartate-stimulated dopamine release by glycine was reduced by the inclusion of nitrendipine in striatal slices from ischemic and 30-min reperfused animals. These data suggest that glycine may facilitate opening of the voltage-dependent calcium channels activated by N-methyl-D-aspartate and that this facilitation is blocked by the antagonist nitrendipine.

Lonidamine-mediated respiratory changes in rat heart myocytes: a re-examination of the functional response of mitochondrial cytochrome c oxidase.

Respiratory activity of intact cardiac myocytes isolated from rats treated with lonidamine (LND) has been examined under conditions where cytochrome oxidase turns over at its maximal rate. Compared to myocytes isolated from control rat hearts, those treated with LND displayed a 60% increase in the cytochrome oxidase-dependent rate of respiration; electron microscopy revealed, in agreement with the literature, that the membrane structure of the mitochondrion had become disorganized. The increase in the rate of oxygen consumption was correlated with the (partial) impairment of the membrane ability to maintain the proton electrochemical potential gradient which normally inhibits oxidase activity. Results are discussed with reference to previous reports showing no effect of LND on cytochrome c oxidase activity. The evidence reported better clarifies the contribution of cytochrome oxidase to the demonstrated energetic failure displayed by cells treated with LND.