Esperanza Recio-Pinto

Cytotoxicity of Local Anesthetics in Human Neuronal Cells

BACKGROUND: In addition to inhibiting the excitation conduction process in peripheral nerves, local anesthetics (LAs) cause toxic effects on the central nervous system, cardiovascular system, neuromuscular junction, and cell metabolism. Different postoperative neurological complications are ascribed to the cytotoxicity of LAs, but the underlying mechanisms remain unclear. Because the clinical concentrations of LAs far exceed their EC50 for inhibiting ion channel activity, ion channel block alone might not be sufficient to explain LA-induced cell death. However, it may contribute to cell death in combination with other actions. In this study, we compared the cytotoxicity of six frequently used LAs and will discuss the possible mechanism(s) underlying their toxicity. METHODS: In human SH-SY5Y neuroblastoma cells, viability upon exposure to six LAs (bupivacaine, ropivacaine, mepivacaine, lidocaine, procaine, and chloroprocaine) was quantitatively determined by the MTT-(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-odium bromide) colorimetry assay and qualitatively confirmed by fluorescence imaging, using the LIVE/DEAD® assay reagents (calcein/AM and ethidium homodimer-1). In addition, apoptotic activity was assessed by measuring the activation of caspase-3/-7 by imaging using a fluorescent caspase inhibitor (FLICA™). Furthermore, LA effects on depolarization- and carbachol-stimulated intracellular Ca2+-responses were also evaluated. RESULTS: 1) After a 10-min treatment, all six LAs decreased cell viability in a concentration-dependent fashion. Their killing potency was procaine ≤ mepivacaine < lidocaine < chloroprocaine < ropivacaine < bupivacaine (based on LD50, the concentration at which 50% of cells were dead). Among these six LAs, only bupivacaine and lidocaine killed all cells with increasing concentration. 2) Both bupivacaine and lidocaine activated caspase-3/-7. Caspase activation required higher levels of lidocaine than bupivacaine. Moreover, the caspase activation by bupivacaine was slower than by lidocaine. Lidocaine at high concentrations caused an immediate caspase activation, but did not cause significant caspase activation at concentrations lower than 10 mM. 3) Procaine and chloroprocaine concentration-dependently inhibited the cytosolic Ca2+-response evoked by depolarization or receptor-activation in a similar manner as a previous observation made with bupivacaine, ropivacaine, mepivacaine, and lidocaine. None of the LAs caused a significant increase in the basal and Ca2+-evoked cytosolic Ca2+-level. CONCLUSION: LAs can cause rapid cell death, which is primarily due to necrosis. Lidocaine and bupivacaine can trigger apoptosis with either increased time of exposure or increased concentration. These effects might be related to postoperative neurologic injury. Lidocaine, linked to the highest incidence of transient neurological symptoms, was not the most toxic LA, whereas bupivacaine, a drug causing a very low incidence of transient neurological symptoms, was the most toxic LA in our cell model. This suggests that cytotoxicity-induced nerve injury might have different mechanisms for different LAs and different target(s) other than neurons.

Glycosylation Affects the Protein Stability and Cell Surface Expression of Kv1.4 but Not Kv1.1 Potassium Channels A PORE REGION DETERMINANT DICTATES THE EFFECT OF GLYCOSYLATION ON TRAFFICKING

Kv1.1 and Kv1.4 potassium channels are plasma membrane glycoproteins involved in action potential repolarization. We have shown previously that glycosylation affects the gating function of Kv1.1 (Watanabe, I., Wang, H. G., Sutachan, J. J., Zhu, J., Recio-Pinto, E. & Thornhill, W. B. (2003) J. Physiol. (Lond.) 550, 51–66) and that a pore region determinant of Kv1.1 and Kv1.4 affects their cell surface trafficking negatively or positively, respectively (Zhu, J., Watanabe, I., Gomez, B. & Thornhill, W. B. (2001) J. Biol. Chem. 276, 39419–39427). Here we investigated the role of N-glycosylation of Kv1.1 and Kv1.4 on their protein stability, cellular localization pattern, and trafficking to the cell surface. We found that preventing N-glycosylation of Kv1.4 decreased its protein stability, induced its high partial intracellular retention, and decreased its cell surface protein levels, whereas it had little or no effect on these parameters for Kv1.1. Exchanging a trafficking pore region determinant between Kv1.1 and Kv1.4 reversed these effects of glycosylation on these chimeric channels. Thus it appeared that the Kv1.4 pore region determinant and the sugar tree attached to the S1–S2 linker showed some type of dependence in promoting proper trafficking of the protein to the cell surface, and this dependence can be transferred to chimeric Kv1.1 proteins that contain the Kv1.4 pore. Understanding the different trafficking programs of Kv1 channels, and whether they are altered by glycosylation, will highlight the different posttranslational mechanisms available to cells to modify their cell surface ion channel levels and possibly their signaling characteristics.

Glycosylation affects rat Kv1.1 potassium channel gating by a combined surface potential and cooperative subunit interaction mechanism

The effect of glycosylation on Kv1.1 potassium channel function was investigated in mammalian cells stably transfected with Kv1.1 or Kv1.1N207Q. Macroscopic current analysis showed that both channels were expressed but Kv1.1N207Q, which was not glycosylated, displayed functional differences compared with wild‐type, including slowed activation kinetics, a positively shifted V1/2, a shallower slope for the conductance versus voltage relationship, slowed C‐type inactivation kinetics, and a reduced extent of and recovery from C‐type inactivation. Kv1.1N207Q activation properties were also less sensitive to divalent cations compared with those of Kv1.1. These effects were largely due to the lack of trans‐Golgi added sugars, such as galactose and sialic acid, to the N207 carbohydrate tree. No apparent change in ionic current deactivation kinetics was detected in Kv1.1N207Q compared with wild‐type. Our data, coupled with modelling, suggested that removal of the N207 carbohydrate tree had two major effects. The first effect slowed the concerted channel transition from the last closed state to the open state without changing the voltage dependence of its kinetics. This effect contributed to the G‐V curve depolarization shift and together with the lower sensitivity to divalent cations suggested that the carbohydrate tree and its negatively charged sialic acids affected the negative surface charge density on the channels extracellular face that was sensed by the activation gating machinery. The second effect reduced a cooperativity factor that slowed the transition from the open state to the closed state without changing its voltage dependence. This effect accounted for the shallower G‐V slope, and contributed to the depolarized G‐V shift, and together with the inactivation changes it suggested that the carbohydrate tree also affected channel conformations. Thus N‐glycosylation, and particularly terminal sialylation, affected Kv1.1 gating properties both by altering the surface potential sensed by the channels activation gating machinery and by modifying conformational changes regulating cooperative subunit interactions during activation and inactivation. Differences in glycosylation pattern among closely related channels may contribute to their functional differences and affect their physiological roles.

The glycosylation state of Kv1.2 potassium channels affects trafficking, gating, and simulated action potentials.

We presented evidence previously that decreasing the glycosylation state of the Kv1.1 potassium channel modified its gating by a combined surface potential and a cooperative subunit interaction mechanism and these effects modified simulated action potentials. Here we continued to test the hypothesis that glycosylation affects channel function in a predictable fashion by increasing and decreasing the glycosylation state of Kv1.2 channels. Compared with Kv1.2, increasing the glycosylation state shifted the V(1/2) negatively with a steeper G-V slope, increased activation kinetics with little change in deactivation kinetics or in their voltage-dependence, and decreased the apparent level of C-type inactivation. Decreasing the glycosylation state had essentially the opposite effects and shifted the V(1/2) positively with a shallower G-V slope, decreased activation kinetics (and voltage-dependence), decreased deactivation kinetics, and increased the apparent level of C-type inactivation. Single channel conductance was not affected by the different glycosylation states of Kv1.2 tested here. Hyperpolarized or depolarized shifts in V(1/2) from wild type were apparently due to an increased or decreased level of channel sialylation, respectively. Data and modeling suggested that the changes in activation properties were mostly predictable within and between channels and were consistent with a surface potential mechanism, but those on deactivation properties were not predictable and were more consistent with a conformational mechanism. Moreover the effect on the deactivation process appeared to be channel-type dependent as well as glycosylation-site dependent. The glycosylation state of Kv1.2 also affected action potentials in simulations. In addition, preventing N-glycosylation decreased cell surface Kv1.2 expression levels by approximately 40% primarily by increasing partial endoplasmic reticulum retention and this effect was completely rescued by Kv1.4 subunits, which are glycosylated, but not by cytoplasmic Kvbeta2.1 subunits. The nonglycosylated Kv1.2 protein had a similar protein half-life as the glycosylated protein and appeared to be folded properly. Thus altering the native Kv1.2 glycosylation state affected its trafficking, gating, and simulated action potentials. Differential glycosylation of ion channels could be used by excitable cells to modify cell signaling.

Allowed N-glycosylation sites on the Kv1.2 potassium channel S1-S2 linker: implications for linker secondary structure and the glycosylation effect on channel function.

N-glycosylation is a post-translational modification that plays a role in the trafficking and/or function of some membrane proteins. We have shown previously that N-glycosylation affected the function of some Kv1 voltage-gated potassium (K+) channels [Watanabe, Wang, Sutachan, Zhu, Recio-Pinto and Thornhill (2003) J. Physiol. (Cambridge, U.K.) 550, 51-66]. Kv1 channel S1-S2 linkers vary in length but their N-glycosylation sites are at similar relative positions from the S1 or S2 membrane domains. In the present study, by a scanning mutagenesis approach, we determined the allowed N-glycosylation sites on the Kv1.2 S1-S2 linker, which has 39 amino acids, by engineering N-glycosylation sites and assaying for glycosylation, using their sensitivity to glycosidases. The middle section of the linker (54% of linker) was glycosylated at every position, whereas both end sections (46% of linker) near the S1 or S2 membrane domains were not. These findings suggested that the middle section of the S1-S2 linker was accessible to the endoplasmic reticulum glycotransferase at every position and was in the extracellular aqueous phase, and presumably in a flexible conformation. We speculate that the S1-S2 linker is mostly a coiled-loop structure and that the strict relative position of native glycosylation sites on these linkers may be involved in the mechanism underlying the functional effects of glycosylation on some Kv1 K+ channels. The S3-S4 linker, with 16 amino acids and no N-glycosylation site, was not glycosylated when an N-glycosylation site was added. However, an extended linker, with an added N-linked site, was glycosylated, which suggested that the native linker was not glycosylated due to its short length. Thus other ion channels or membrane proteins may also have a high glycosylation potential on a linker but yet have similarly positioned native N-glycosylation sites among isoforms. This may imply that the native position of the N-glycosylation site may be important if the carbohydrate tree plays a role in the folding, stability, trafficking and/or function of the protein.

Changes in sodium channel function during postnatal brain development reflect increases in the level of channel sialidation

Developmental changes of forebrain sodium channels were studied at three postnatal ages: P0, P15 and adult (P30/P180). Electrophysiological analysis determined that the midpoint potential of activation was -64, -75 and -81 mV for P0, P15 and adult channels, respectively. At negative potentials, gating state changes were observed in all channels; at positive potentials they were observed in most P0 (72%) and to a lower extent in older channels (25%). A long non-conductive state was displayed with a higher frequency in P0 than in older channels. Immunoblot analysis determined that the apparent molecular weight was approximately 227, approximately 241 and approximately 246 kDa for P0, P15 and adult channels, respectively. Upon neuraminidase treatment, which cleaves sialic acids, these differences in molecular weight were abolished. The data suggest that these developmental changes in the function of forebrain sodium channels correlate with changes in the channels sialidation level.

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

We investigated halothanes 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[degree sign]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. (Anesth Analg 1998;86:885-95)

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 Mg²⁺ 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.

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.

Local anesthetics modulate neuronal calcium signaling through multiple sites of action

Background Local anesthetics (LAs) are known to inhibit voltage-dependent Na+ channels, as well as K+ and Ca2+ channels, but with lower potency. Since cellular excitability and responsiveness are largely determined by intracellular Ca2+ availability, sites along the Ca2+ signaling pathways may be targets of LAs. This study was aimed to investigate the LA effects on depolarization and receptor-mediated intracellular Ca2+ changes and to examine the role of Na+ and K+ channels in such functional responses. Methods Effects of bupivacaine, ropivacaine, mepivacaine, and lidocaine (0.1–2.3 mm) on evoked [Ca2+]i transients were investigated in neuronal SH-SY5Y cell suspensions using Fura-2 as the intracellular Ca2+ indicator. Potassium chloride (KCl, 100 mm) and carbachol (1 mm) were individually or sequentially applied to evoke increases in intracellular Ca2+. Coapplication of LA and Na+/K+ channel blockers was used to evaluate the role of Na+ and K+ channels in the LA effect on the evoked [Ca2+]i transients. Results All four LAs concentration-dependently inhibited both KCl- and carbachol-evoked [Ca2+]i transients with the potency order bupivacaine > ropivacaine > lidocaine ≥ mepivacaine. The carbachol-evoked [Ca2+]i transients were more sensitive to LAs without than with a KCl prestimulation, whereas the LA-effect on the KCl-evoked [Ca2+]i transients was not uniformly affected by a carbachol prestimulation. Na+ channel blockade did not alter the evoked [Ca2+]i transients with or without a LA. In the absence of LA, K+ channel blockade increased the KCl-, but decreased the carbachol-evoked [Ca2+]i transients. A coapplication of LA and K+ channel blocker resulted in larger inhibition of both KCl- and carbachol-evoked [Ca2+]i transients than by LA alone. Conclusions Different and overlapping sites of action of LAs are involved in inhibiting the KCl- and carbachol-evoked [Ca2+]i transients, including voltage-dependent Ca2+ channels, a site associated with the caffeine-sensitive Ca2+ store and a possible site associated with the IP3-sensitive Ca2+ store, and a site in the muscarinic pathway. K+ channels, but not Na+ channels, seem to modulate the evoked [Ca2+]i transients, as well as the LA-effects on such responses.