Molecular actions of propofol on human 5-HT3A receptors: enhancement as well as inhibition by closely related phenol derivatives.

BACKGROUND: 5-Hydroxytryptamine type 3 (5-HT3) receptors are excitatory ligand-gated ion channels which are involved in postoperative nausea and vomiting. They are depressed by the anesthetic propofol, which, in contrast, enhances the activity of inhibitory ligand-gated ion channels such as gamma-aminobutyric acid type A receptors and glycine receptors. To investigate the molecular mechanisms responsible for these contrasting actions, we examined the kinetics of the action of propofol and its lesser hydrophobic derivatives 2-isopropylphenol and phenol on human 5-HT3A receptors. METHODS: Human embryonic kidney 293 cells containing stably transfected cDNA of the human 5-HT3A receptor subunit were patch clamped (excised outside-out patches). Drugs were applied with a fast solution exchange system (within 2 ms) and their concentrations were determined by high performance liquid chromatography. RESULTS: When applied in equilibrium (60 s before and during the 5-HT pulse), propofol inhibited human 5-HT3A receptors (IC50 = 18 +/- 1.0 microM). In equilibrium, the less hydrophobic 2-isopropylphenol was surprisingly a similarly potent inhibitor of human 5-HT3A receptors (IC50 = 17 +/- 3.2 microM), whereas phenol was considerably less potent (IC50 = 1.6 +/- 0.2 mM). Varying the duration of drug application before currents were elicited, and then applying 5-HT still in the presence of the drug revealed that fast and slow processes contributed to the (equilibrium) effects of propofol (tau(IN-1) = 35 ms and tau(IN-2) = 4.8 s), 2-isopropylphenol (tau(IN-1) = 64 ms and tau(IN-2) = 6.6 s), and phenol (tau(IN-1) < 10 ms, tau(IN-2) = 20.4 s). When applied transiently together with 5-HT (open channel application), propofol depressed currents and accelerated the 5-HT-induced desensitization significantly, whereas, in contrast, 2-isopropylphenol and phenol increased currents and slowed desensitization. Slowed desensitization was also observed for 5-hydroxyindole (1 mM), a 5-HT derivative, but not for benzene. The fast effects of phenol, 2-isopropylphenol, and propofol were more pronounced when the 5-HT concentration was decreased from 30 to 3 microM, whereas the slow effects were not sensitive to 5-HT. CONCLUSIONS: At least two separate inhibitory actions on 5-HT3A receptors could be identified for propofol, whereas the enhancing action seen for the two related smaller phenol derivatives could no longer be detected. 5-HT-dependent and 5-HT-independent interactions could be distinguished for all three drugs. Propofol was less potent than expected from its hydrophobic properties. Underlying mechanisms appear to involve the phenolic hydroxyl group, hydrophobic interactions, and steric restrictions.

The effects of fentanyl-like opioids and hydromorphone on human 5-HT3A receptors.

BACKGROUND: 5-HT(3) receptors are involved in various physiologic functions, including the modulation of emesis. 5-HT(3) antagonists are clinically widely used as potent antiemetics. Emesis is also a side effect of opioid analgesics. Intriguingly, the natural opioid morphine shows specific interactions with human 5-HT(3) receptors at clinically relevant concentrations. In the present study, we investigated whether this is a general effect of opioids, even when they are structurally diverse. Therefore, another morphine (phenanthrene-type) derivative, hydromorphone, and fentanyl including its (4-anilinopiperidine-type) derivatives were tested. METHODS: Whole-cell patches from human embryonic kidney-293 cells, stably transfected with the human 5-HT(3A) receptor cDNA, were used to determine the opioid effects on the 5-HT (3 microM)-induced currents using the patch clamp technique (voltage-clamp). RESULTS: None of the fentanyl derivatives affected currents through the 5-HT(3A) receptor (3 microM 5-HT) significantly in the clinically relevant nanomolar concentration range (IC(50) values >30 microM). In contrast, hydromorphone was considerably more potent (IC(50) = 5.3 microM), slowing the current activation- and desensitization-kinetics significantly (at 3 microM by a factor of 1.9 and 2.4, respectively), similar to morphine. At concentrations much higher than clinically relevant, but within the range predicted from Meyer-Overton correlations for nonspecific interactions, the fentanyl derivatives all showed at least a tendency to suppress current amplitudes, but they had diverse effects on the activation- and desensitization-kinetics of 5-HT(3A) receptors. CONCLUSIONS: Only morphine and hydromorphone, but not the fentanyl derivatives, reduced 5-HT-induced current amplitudes and slowed current kinetics near clinically relevant concentrations. The high potencies of morphine and hydromorphone, when compared to their lipophilicities, suggest a specific interaction with 5-HT(3A) receptors. In contrast, the effects of fentanyl-type opioids appear to be of unspecific nature. Because the rank order of opioid potencies for human 5-HT(3A) receptors is opposite of that for opioid receptors, the site involved is structurally different from opioid receptor binding sites. In agreement with recent data on different phenols, a phenolic OH-group (which morphine and hydromorphone possess) may contribute to specific interactions of morphine and hydromorphone with the 5-HT(3A) receptor. Future clinical studies could test whether corresponding differences in emetogenicity between different classes of opioids will be found.

Interactions of anesthetics with their targets: non-specific, specific or both?

What makes a general anesthetic a general anesthetic? We shall review first what general anesthesia is all about and which drugs are being used as anesthetics. There is neither a unique definition of general anesthesia nor any consensus on how to measure it. Diverse drugs and combinations of drugs generate general anesthetic states of sometimes very different clinical quality. Yet the principal drugs are still considered to belong to the same class of 'general anesthetics'. Effective concentrations of inhalation anesthetics are in the high micromolar range and above, and even for intravenous anesthetics they do not go below the micromolar range. At these concentrations, many molecular and higher level targets are affected by inhalation anesthetics, fewer probably by intravenous anesthetics. The only physicochemical characteristic shared by anesthetics is the correlation of their anesthetic potencies with hydrophobicity. These correlations depend on the group of general anesthetics considered. In this review, anesthetic potencies for many different targets are plotted against octanol/water partition coefficients as measure of hydrophobicity. Qualitatively, similar correlations result, suggesting several but weak interactions with proteins as being characteristic of anesthetic actions. The polar interactions involved are weak, being roughly equal in magnitude to hydrophobic interactions. Generally, intravenous anesthetics are noticeably more potent than inhalation anesthetics. They differ considerably more between each other in their interactions with various targets than inhalation anesthetics do, making it difficult to come to a decision which of these should be used in future studies as representative 'prototypical general anesthetics'.

Differential lidocaine sensitivity of human voltage-gated potassium channels relevant to the auditory system.

HYPOTHESIS: Lidocaine may lead to an alteration in the processing of hearing as observed during tinnitus by inhibiting voltage-gated potassium channels at clinically relevant concentrations. BACKGROUND: Recent molecular evidence suggests that the voltage-gated potassium channels Kv 3.1 and Kv 1.1 play an important functional role in the auditory system. Lidocaine is known to influence the auditory system and may thus exert pharmacological effects on these human potassium channels. METHODS: Patch-clamp recordings were performed on the pharmacologic action of lidocaine on Kv 3.1 channels natively expressed in SH-SY5Y cells and Kv 1.1 channels expressed in HEK 293 cells. RESULTS: Lidocaine reversibly inhibited Kv 3.1 and Kv 1.1 channels in a concentration-dependent manner. The half-maximal inhibitory concentration for conductance block was 607 micromol/L for Kv 3.1 (n=47) and 4,550 micromol/L for Kv 1.1 channels (n=56), respectively. The Hill coefficients were 0.9 and 0.8. Conductance block was voltage dependent for Kv 3.1 but not for Kv 1.1 channels. The midpoint of current activation of both channels was shifted to hyperpolarized potentials. At free plasma concentrations determined during suppression (0.5-1 mg/L; 1.75-3.5 micromol/L) or induction (>1-2 mg/L; >3.5-7 micromol/L) of tinnitus Kv 3.1 and K v1.1 channels would be suppressed by at most 1.5 to 2%. CONCLUSION: Human Kv 3.1 and Kv 1.1 channels exhibited different sensitivities to the inhibitory action of lidocaine. The small effect at clinically relevant concentrations suggests that the physiologic roles of Kv 3.1 and Kv 1.1 channels in auditory neurons seem not to be impaired during the therapeutic or diagnostic application of lidocaine in the auditory system.

Interactions of metoclopramide and ergotamine with human 5-HT(3A) receptors and human 5-HT reuptake carriers.

The actions of metoclopramide and ergotamine, drugs which are used as a combined migraine medication, on human (h)5-HT3A receptors and 5-HT reuptake carriers, stably expressed in HEK-293 cells, were studied with patch-clamp- and ([3H]5-HT)-uptake techniques. At clinical concentrations, metoclopramide inhibited peak and integrated currents through h5-HT3A receptors concentration-dependently (IC50 = 0.064 and 0.076 microM, respectively) when it was applied in equilibrium (60 s before and during 5-HT (30 microM) exposure). The onset and offset time constants of metoclopramide action were 1.3 and 2.1 s, respectively. The potency of metoclopramide when exclusively applied during the agonist pulse decreased more than 200-fold (IC50 = 19.0 microM, peak current suppression). Metoclopramide (0.10 microM) did not alter the EC50 of 5-HT-induced peak currents. In contrast to the lack of competitive interaction between metoclopramide and 5-HT in this functional assay, metoclopramide inhibited specific [3H]GR65630 binding to human h5-HT3A receptors in a surmountable manner. This seeming discrepancy between functional studies and radioligand binding experiments may be accounted for by (1) the slow kinetics of inhibition of peak currents by metoclopramide compared with the fast onset and offset kinetics of 5-HT-induced currents and (2) the low efficacy of metoclopramide in inhibiting radioligand binding (e.g. only 20% binding inhibition compared to 79% peak current suppression by 200 nM metoclopramide). At low concentrations (1-10 nM), ergotamine had no effect on 5-HT (30 microM)-induced peak currents. Above clinical concentrations, ergotamine (>3 microM) inhibited them. When both drugs were applied together (0.10 microM metoclopramide +0.001 to 0.01 microM ergotamine), an inhibition of both, peak and integrated current responses was observed. Neither metoclopramide (< or =30 microM) nor ergotamine (< or =30 microM) had an effect on the 5-HT reuptake carrier as they did not alter the citalopram-sensitive [3H]5-HT uptake.

Single sodium channels from human skeletal muscle in planar lipid bilayers: characterization and response to pentobarbital.

PURPOSE: To investigate the response to general anesthetics of different sodium-channel subtypes, we examined the effects of pentobarbital, a close thiopental analogue, on single sodium channels from human skeletal muscle and compared them to existing data from human brain and human ventricular muscle channels. METHODS: Sodium channels from a preparation of human skeletal muscle were incorporated into planar lipid bilayers, and the steady-state behavior of single sodium channels and their response to pentobarbital was examined in the presence of batrachotoxin, a sodium-channel activator. Single-channel currents were recorded before and after the addition of pentobarbital (0.34-1.34 mM). RESULTS: In symmetrical 500 mM NaCl, human skeletal muscle sodium channels had an averaged single-channel conductance of 21.0 +/- 0.6 pS, and the channel fractional open time was 0.96 +/- 0.04. The activation midpoint potential was -96.2 +/- 1.6 mV. Extracellular tetrodotoxin blocked the channel with a half-maximal concentration (k1/2) of 60 nM at 0 mV. Pentobarbital reduced the time-averaged conductance of single skeletal muscle sodium channels in a concentration-dependent manner (inhibitory concentration 50% [IC50] = 0.66 mM). The steady-state activation was shifted to more hyperpolarized potentials (-16.7 mV at 0.67 mM pentobarbital). CONCLUSION: In the planar lipid bilayer system, skeletal muscle sodium channels have some electrophysiological properties that are significantly different compared with those of sodium channels from cardiac or from central nervous tissue. In contrast to the control data, these different human sodium channel subtypes showed the same qualitative and quantitative response to the general anesthetic pentobarbital. The implication of these effects for overall anesthesia will depend on the role the individual channels play within their neuronal networks, but suppression of both central nervous system and peripheral sodium channels may add to general anesthetic effects.

Monitoring of immobility to noxious stimulation during sevoflurane anesthesia using the spinal H-reflex.

BACKGROUND: The spinal H-reflex has been shown to correlate with surgical immobility, i.e., the absence of motor responses to noxious stimulation, during isoflurane anesthesia. Here, the authors established individual concentration-response functions for H-reflex amplitude and tested the predictive power of the H-reflex for movement responses during sevoflurane anesthesia in comparison to electroencephalographic parameters. In addition, they investigated the effect of noxious stimulation on the H-reflex itself. METHODS: The authors studied 12 female patients during sevoflurane anesthesia before surgery. The sevoflurane concentration was increased, a laryngeal mask was inserted, and then the sevoflurane concentration was decreased until H-reflex amplitude (recorded over the soleus muscle) recovered. Thereafter, the end-tidal sevoflurane concentration was kept at a constant value close to the minimum alveolar concentration for suppression of movement responses after tetanic stimulation (MACtetanus), determined by the Dixon up-down method. Pharmacodynamic modeling of H-reflex amplitude and of the Bispectral Index was performed, and predictive values for motor responses to noxious electrical stimulation (50 Hz, 60 mA tetanus, volar forearm) were compared using the prediction probability. RESULTS: Concentration-dependent depression of H-reflex amplitude by sevoflurane was well modeled (median r2 = 0.97) by a sigmoid function with a median EC50 of 1.5 vol% and a median slope parameter of 3.7, much steeper than the slope for the Bispectral Index. MACtetanus calculated by logistic regression was 1.6 vol%. H-reflex amplitude predicted motor responses to noxious stimulation with a prediction probability of 0.76, whereas the prediction probability for Bispectral Index and spectral edge frequency (SEF95) were not different from chance alone. Noxious stimulation was followed by a substantial increase of H-reflex amplitude for several minutes, whereas the Bispectral Index and SEF95 exhibited no significant changes. CONCLUSIONS: Suppression of movement to noxious stimulation and suppression of H-reflex amplitude by sevoflurane follow similar concentration-response functions. Although this does not imply a causal relation, it explains the high predictive value of H-reflex amplitude for motor responses to noxious stimuli, even in a narrow concentration range around the MACtetanus.

Anticonvulsant pharmacology of voltage-gated Na+ channels in hippocampal neurons of control and chronically epileptic rats.

Voltage-gated Na+ channels are a main target of many first-line anticonvulsant drugs and their mechanism of action has been extensively investigated in cell lines and native neurons. Nevertheless, it is unknown whether the efficacy of these drugs might be altered following chronic epileptogenesis. We have, therefore, analysed the effects of phenytoin (100 micro m), lamotrigine (100 micro m) and valproate (600 micro m) on Na+ currents in dissociated rat hippocampal granule neurons in the pilocarpine model of chronic epilepsy. In control animals, all three substances exhibited modest tonic blocking effects on Na+ channels in their resting state. These effects of phenytoin and lamotrigine were reduced (by 77 and 64%) in epileptic compared with control animals. Phenytoin and valproate caused a shift in the voltage dependence of fast inactivation in a hyperpolarizing direction, while all three substances shifted the voltage dependence of activation in a depolarizing direction. The anticonvulsant effects on Na+ channel voltage dependence proved to be similar in control and epileptic animals. The time course of fast recovery from inactivation was potently slowed by lamotrigine and phenytoin in control animals, while valproate had no effect. Interestingly, the effects of phenytoin on fast recovery from inactivation were significantly reduced in chronic epilepsy. Taken together, these results reveal that different anticonvulsant drugs may exert a distinct pattern of effects on native Na+ channels. Furthermore, the reduction of phenytoin and, to a less pronounced extent, lamotrigine effects in chronic epilepsy raises the possibility that reduced pharmacosensitivity of Na+ channels may contribute to the development of drug resistance.

Propofol and sevoflurane in subanesthetic concentrations act preferentially on the spinal cord: evidence from multimodal electrophysiological assessment.

BACKGROUND: Animal experiments in recent years have shown that attenuation of motor responses by general anesthetics is mediated at least partly by spinal mechanisms. Less is known about the relative potency of anesthetic drugs in suppressing cortical and spinal electrophysiological responses in vivo in humans, particularly those, but not only those, connected with motor responses. Therefore, we studied the effects of sevoflurane and propofol in humans using multimodal electrophysiological assessment. METHODS: We studied nine healthy volunteers in two sessions during steady state sedation with 0.5, 1.0, and 1.5 microg/l (targeted plasma concentration) propofol or 0.2 and 0.4 vol% (end-tidal) sevoflurane. Following a 15-min equilibration period, motor responses to transcranial magnetic stimulation and peripheral (H-reflex, F-wave) stimulation were recorded, while electroencephalography and auditory evoked responses were recorded in parallel. RESULTS: At concentrations corresponding to two thirds of C(50 awake), motor responses to transcranial magnetic stimulation were reduced by approximately 50%, H-reflex amplitude was reduced by 22%, F-wave amplitude was reduced by 40%, and F-wave persistence was reduced by 25%. No significant differences between sevoflurane and propofol were found. At this concentration, the Bispectral Index was reduced by 7%, and the middle-latency auditory evoked responses were attenuated only mildly (N(b) latency increased by 11%, amplitude P(a)N(b) did not change). In contrast, the postauricular reflex was suppressed by 77%. CONCLUSIONS: The large effect of both anesthetics on all spinal motor responses, compared with the small effect on electroencephalography and middle-latency auditory evoked responses, assuming that they represent cortical modulation, may suggest that the suppression of motor responses to transcranial magnetic stimulation is largely due to submesencephalic effects.

A novel mechanism underlying drug resistance in chronic epilepsy.

The development of resistance to pharmacological treatment is common to many human diseases. In chronic epilepsy, many patients develop resistance to anticonvulsant drug treatment during the course of their disease, with the underlying mechanisms remaining unclear. We have studied cellular mechanisms underlying drug resistance in resected hippocampal tissue from patients with temporal lobe epilepsy by comparing two groups of patients, the first displaying a clinical response to the anticonvulsant carbamazepine and a second group with therapy-resistant seizures. Using patch-clamp recordings, we show that the mechanism of action of carbamazepine, use-dependent block of voltage-dependent Na(+) channels, is completely lost in carbamazepine-resistant patients. Likewise, seizure activity elicited in human hippocampal slices is insensitive to carbamazepine. In marked contrast, carbamazepine-induced use-dependent block of Na(+) channels and blocked seizure activity in vitro in patients clinically responsive to this drug. Consistent with these results in human patients, we also show that use-dependent block of Na(+) channels by carbamazepine is absent in chronic experimental epilepsy. Taken together, these data suggest that a loss of Na(+) channel drug sensitivity may constitute a novel mechanism underlying the development of drug-resistant epilepsy.