To our knowledge, the study presented here is the first to identify glycine receptors as novel targets for inhibition by TXA and EACA, but not aprotinin. Because TXA is a competitive antagonist, inhibition mediated by this drug was expected to be highly dependent on the concentration of glycine applied, and this expectation was corroborated by the results. The potency of TXA was 10-fold higher for current evoked by a low concentration of glycine (IC50 = 93.1 μM) in spinal cord neurons than for postsynaptic glycinergic currents (IC50 = 1 mM) or currents activated by the EC50 of glycine in cortical neurons (IC50 = 1.1 mM) and spinal cord neurons (IC50 = 1.4 mM). Interestingly, the potency of TXA inhibition was similar for glycine receptors and GABAA receptors activated by the EC50 of agonist in cortical neurons and spinal cord neurons (Table ).
We also show that TXA inhibition of glycine receptors activated by a low concentration of glycine (EC6
) was reversed by isoflurane at clinically relevant concentrations. Propofol also reversed TXA-mediated glycine blockade, but only at concentrations 3-fold higher than those that occur during anesthesia (63
). Consistent with these results, TXA-induced enhancement of excitatory synaptic drive in cortical slices was more effectively reversed by isoflurane than by propofol. Finally, we showed that the peak concentration of TXA in the CSF of patients undergoing major cardiovascular surgery (220.8 μM) occurred after CPB and termination of drug infusion. A similar concentration of TXA increased the frequency of SLEs and enhanced evoked field responses in cortical slices, which suggests that TXA has proconvulsive properties.
Inhibitors of glycine receptors are known to induce convulsions and proconvulsive behavior (78
). For example, strychnine causes myoclonic spasms (79
), twitching of muscles, convulsions, and hyperreactivity (78
). Genetic disorders characterized by reduced expression of glycine receptors cause hyperekplexia, convulsions, and startle disorders in infants (80
). In animals with naturally occurring mutations of the glycine receptor, the magnitude of the reduction in glycine receptor function directly correlates with the severity of the convulsive symptoms (81
). Thus, it is plausible that the reduction in function of these receptors caused by TXA leads to disinhibition and proconvulsive effects.
The potency of TXA was much greater for currents evoked by a low concentration of glycine than for synaptic glycinergic mIPSCs. The demonstration that TXA attenuates tonic current is of great interest, since a reduction in tonic current enhances neuronal excitability (36
). Tonic current reduces neuronal excitability via two mechanisms. First, this type of current allows for the transfer of large quantities of negative charge, a process that hyperpolarizes the cell membrane (36
). Second, tonic conductance can attenuate the excitatory drive to generate action potentials by decreasing membrane resistance, thereby reducing the amplitude of excitatory postsynaptic potentials (83
As anticipated for a competitive antagonist, the potency of TXA inhibition of glycine receptors was highly dependent on the concentration of agonist used in each experiment. However, this difference in potency might also have been due, at least in part, to the subunit composition of glycine receptors. Glycine receptors are composed of multiple α1–4
and β subunits, which form homomeric (5α) or heteromeric (2α3β) ion channels (32
). Synaptic glycinergic currents are generated by heteromeric receptors that are clustered at the postsynaptic sites (35
). Tonic glycinergic currents are mediated by homomeric receptors, which are located predominantly extrasynaptically (35
). Glycine receptor antagonists, such as picrotoxin, show higher potency for homomeric than heteromeric glycine receptors (37
). Post-transitional factors such as glycine receptor phosphorylation and the clustering of receptors in the postsynaptic domain can also alter the pharmacological properties of glycine receptors (52
). TXA potency in relation to various combinations of glycine receptor subunits as well as the effects of cytosolic regulatory factors on TXA potency are topics worthy of future study.
Our results and those of others (38
) showed that TXA is a competitive antagonist of GABAA
receptors in both cortical and spinal cord neurons (IC50
= 1.5 ± 0.1 mM for both). Interestingly, the potency of TXA for current evoked by a low concentration of GABA (EC4
) in cortical (IC50
= 1.0 mM) and spinal cord (IC50
= 0.9 mM) neurons was similar to its potency for current evoked by higher concentrations of GABA (EC50
). This result was unexpected and may reflect differences in the subunit composition of receptors activated by the various concentrations of GABA (56
). In addition, our results showed that TXA inhibition of GABAA
receptors increase evoked field responses in neocortical slices in the presence of strychnine. Thus, TXA inhibition of GABAA
receptors may increase network excitability.
A previous study showed that TXA has an IC50
value of 7.1 ± 3.1 mM for recombinant GABAA
receptors (α1β2γ2), transfected into human embryonic kidney cells when receptors are activated by GABA 30 μM (EC70
). Also, binding assays for TXA with [3
H]muscimol showed that the IC50
value for TXA binding to GABAA
receptors was 2.1 ± 0.2 mM (38
). In our study, the potency of TXA for GABA currents evoked by EC50
concentrations of agonist was lower. This difference may be attributable to our use of cell cultures with native receptors rather than recombinantly expressed receptors (87
The peak TXA concentration in the CSF as measured in the current study was higher than that measured in the CSF of 4 patients undergoing CPB in a previous study (220.8 μM versus 31 μM) (88
). In the earlier study, the TXA dose was significantly lower, and samples were taken at only two time points: 15 and 90 minutes after the start of TXA infusion (88
). The discrepancy in results is probably related to two findings from the current study, specifically, that serum concentrations of TXA do not correlate well with CSF concentrations and that TXA concentrations peak in the CSF long after the start of drug infusion. Thus, the previous study may not have detected the true peak concentrations of TXA in the CSF.
The timing and magnitude of peak TXA concentrations after CPB may be attributable to increased permeability of the blood-brain barrier during and after surgery (89
). In a study that used a protein marker (S100) to measure damage to the blood-brain barrier during CPB, the highest levels of the marker occurred after termination of the bypass (90
). The blood-brain barrier appears to be particularly “leaky” at the end of CPB, which could account for the delayed rise of TXA concentrations in the CSF. Thus, the CSF may act as a slow compartment, which could explain the observed time course of TXA concentrations. A detailed pharmacokinetic analysis of TXA in the plasma and CSF would be of interest in future studies.
The results of this translational preclinical study have important clinical implications. First, we have shown that TXA is a competitive antagonist of both glycine and GABAA
receptors. Thus, higher brain concentrations of TXA likely increase the risk of seizure. Indeed a higher incidence of seizures occurs in patients with preoperative renal failure (91
) and those receiving higher doses of TXA (30
). Currently, there is no consensus regarding optimum TXA dosing, and studies are urgently needed to determine the minimal effective dose of this drug (92
Second, our results also suggest that isoflurane or propofol may prevent or treat TXA-induced seizures in the early postoperative period. Given the variable clinical presentation and incidence of TXA-associated seizures, clinical trials comparing the efficacy of various anticonvulsant treatments are unlikely to be feasible. Our results suggest that it may be possible to use isoflurane or propofol to prevent or treat TXA-induced seizures in the early postoperative period. Inhaled anesthetics, including isoflurane, are already being used for sedation of intubated patients in the intensive care unit (93
). It must be emphasized, however, that anticonvulsants with no direct effects on glycine receptors, such as midazolam, may nonetheless be effective for treating TXA-associated seizures.
Third, because glycine receptors regulate numerous functions in the CNS, their inhibition by TXA could have adverse behavioral consequences other than seizures. One potential implication is that TXA and EACA may have pro-nociceptive properties, since antagonists of glycine receptors increase nociceptive responses in both laboratory animals and patients (94
). Consistent with this prediction, patients with subarachnoid hemorrhage who were treated with TXA and EACA required higher doses of analgesics than placebo-treated patients (95
). Also, patients with menorrhagia treated with TXA experienced a higher incidence of headaches, abdominal pain, and back pain than placebo-treated patients (96
). Furthermore, inhibition of glycine receptors causes excessive or involuntary motor activity (hyperkinesia) in laboratory animals (97
) and patients (98
). Myoclonic activity observed in some patients treated with TXA may result from inhibition of glycine receptors (99
In summary, these results show that TXA and EACA inhibit glycine receptors and suggest a novel mechanism for seizures associated with cardiovascular surgery. Isoflurane may be effective for preventing or treating TXA-induced excitability in the early postoperative period. As the indications for TXA and EACA increase, we hope that these results will aid in the prevention and management of serious neurological side effects associated with these drugs.