RESUMO
The intracellular pH (pHi) of mammalian central neurons is tightly regulated and small pHi-fluctuations can fine-tune inter-/intracellular signaling, excitability, and synaptic plasticity. The research-gap about the pHi-regulation of human brain neurons is addressed here by testing possible influences of the anticonvulsant levetiracetam (LEV). BCECF-AM-loaded neocortical pyramidal cells were fluorometrically investigated in slice-preparations of tissue resected from the middle temporal gyrus of five adults with intractable temporal-lobe epilepsy. Recovery-slope from intracellular acidification following an ammonium prepulse (APP) was used to measure the pHi-regulation. Among twenty pyramidal cells exposed to 50⯵M LEV, the resting pHi (7.09⯱â¯0.14) was lowered in eight (40%) neurons, on average by 0.02⯱â¯0.011 pH-units. In three (15%) and nine (45%) neurons, a minimal alkaline shift (0.017⯱â¯0.004 pH-units) and no pHi-shift occurred, respectively. The LEV-induced pHi-shifts were positively correlated with the resting pHi (râ¯=â¯0.6, pâ¯=â¯0.006, nâ¯=â¯20). In five neurons, which all had responded on LEV with an acidification before, the recovery from APP-acidification was significantly delayed during LEV (pâ¯<â¯0.001). This inhibitory LEV-effect on pHi-regulation i) was similar to that of 200⯵M 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (nâ¯=â¯2) and ii) did not occur under nominal bicarbonate-free conditions (nâ¯=â¯2). Thus, LEV lowered the pHi of human neocortical pyramidal cells most likely by a weakening of the transmembrane HCO3(-)-mediated acid-extrusion. This might contribute to LEV's anticonvulsive potency. Neurons with more acidic resting pHi-values showed a minimal alkalization upon LEV providing a mechanism for paradoxical proconvulsive LEV-effects rarely observed in epilepsy patients. The significance of these subtle pHi-shifts for cortical excitability and plasticity is discussed.
Assuntos
Anticonvulsivantes/administração & dosagem , Bicarbonatos/metabolismo , Concentração de Íons de Hidrogênio , Levetiracetam/administração & dosagem , Células Piramidais/metabolismo , Lobo Temporal/efeitos dos fármacos , Lobo Temporal/metabolismo , Adulto , Epilepsia do Lobo Temporal/tratamento farmacológico , Feminino , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Masculino , Células Piramidais/efeitos dos fármacos , Adulto JovemRESUMO
The intracellular pH (pHi) in the cytosol of mammalian central neurons is tightly regulated and small pHi-fluctuations are deemed to modulate inter-/intracellular signaling, excitability, and synaptic plasticity. The resting pHi of young rodent hippocampal pyramidal neurons is known to decrease alongside aging for about 0.1 pH-units. There is no information about the relationship between age and pHi of human central neurons. We addressed this knowledge gap using 26 neocortical slices from 12 patients (1-56-years-old) who had undergone epilepsy surgery. For fluorometric recordings, the slice-neurons were loaded with the pHi-sensitive dye BCECF-AM. We found that the pyramidal cells' resting pHi (n = 26) descended linearly alongside aging (r = - 0.71, p < 0.001). This negative relationship persisted, when the sample was confined to specific brain regions (i.e., middle temporal gyrus, 23 neurons, r = - 0.68, p < 0.001) or pathologies (i.e., hippocampus sclerosis, 8 neurons, r = - 0.78, p = 0.02). Specifically, neurons (n = 9, pHi 7.25 ± 0.12) from young children (1.5 ± 0.46-years-old) were significantly more alkaline than neurons from adults (n = 17, 38.53 ± 12.38 years old, pHi 7.08 ± 0.07, p < 0.001). Although the samples were from patients with different pathologies the results were in line with those from the rodent hippocampal pyramidal neurons. Like a hormetin, the age-related mild pHi-decrease might contribute to neuroprotection, e.g., via limiting excitotoxicity. On the other hand, aging cortical neurons could become more vulnerable to metabolic overstress by a successive pHi-decrease. Certainly, its impact for the dynamics in short and long-term synaptic plasticity and, ultimately, learning and memory provides a challenge for further research.
Assuntos
Envelhecimento/metabolismo , Neocórtex/citologia , Neurônios/metabolismo , Adulto , Células Cultivadas , Pré-Escolar , Epilepsia Resistente a Medicamentos/cirurgia , Feminino , Fluoresceínas/análise , Fluorometria , Humanos , Concentração de Íons de Hidrogênio , Lactente , Líquido Intracelular/química , Masculino , Pessoa de Meia-Idade , Neocórtex/metabolismo , Adulto JovemRESUMO
AIMS: In cortical mammalian neurons, small fluctuations of intracellular pH (pHi) play a crucial role for inter- and intracellular signaling as well as for cellular and synaptic plasticity. Yet, there have been no respective data about humans. Thus, we investigated the interrelation of pHi and excitability of human cortical neurons. MATERIALS AND METHODS: Intracellular electrophysiological and pH-recordings were made in neurons in slices taken from brain tissue resected from the middle temporal gyrus of two male children (26â¯months and 35â¯months old) who suffered from pharmacotherapy-resistant temporal lobe epilepsy. To excite the tissue (nâ¯=â¯13), we used the 0-Mg2+/high-K+-in vitro epilepsy model producing robust epileptiform discharges (ED). To evoke an intracellular acidification (nâ¯=â¯12), we used the well-established propionate-model and applied 10â¯mM propionate to the bath solutions. In addition, we recorded the effects of other strongly related short-chain monocarboxylates (l-lactate (10â¯mM) and the ketone body DL-ß-hydroxybutyrate (10â¯mM)) on ED and pHi. KEY FINDINGS: The ED-frequency was reversibly reduced by propionate (nâ¯=â¯5), l-lactate (nâ¯=â¯5), or DL-ß-hydroxybutyrate (nâ¯=â¯3), while the durations of EDs and their after-depolarizations increased. In parallel experiments, all three short-chain monocarboxylates (each nâ¯=â¯4) lowered the pHi of the neurons (nâ¯=â¯12) by 0.05-0.07 pH units which was temporally related to the reported changes in bioelectric activity. SIGNIFICANCE: A mild drop of the intraneuronal pH was associated with the control of even over-excited human neocortical tissue. This is identical with prior observations in non-human mammalian cortical neurons. Possible implications for neuroplasticity and the treatment of neuropsychiatric disorders are discussed.
Assuntos
Ácidos Carboxílicos/metabolismo , Neocórtex/citologia , Neocórtex/metabolismo , Neurônios/metabolismo , Ácido 3-Hidroxibutírico/metabolismo , Pré-Escolar , Fenômenos Eletrofisiológicos , Epilepsia/induzido quimicamente , Epilepsia/fisiopatologia , Epilepsia do Lobo Temporal/fisiopatologia , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Lactente , Ácido Láctico/metabolismo , Masculino , Potenciais da Membrana/fisiologia , Microbiota , Neocórtex/efeitos dos fármacos , Neocórtex/fisiopatologia , Neurônios/efeitos dos fármacos , PropionatosRESUMO
BACKGROUND AND PURPOSE: The intracellular pH (pHi) of neurones is tightly regulated by, for example, membrane-bound acid-exchangers and loaders. Nevertheless, excessive bioelectric activity lowers steady-state pHi. In turn, even a moderate acidification can inhibit neuronal activity, a process believed to be part of a negative feedback loop controlling neuronal excitation. As moclobemide, an antidepressant, and also some antiepileptic drugs can reduce neuronal pHi in hippocampus slices in vitro, we screened a panel of currently used neuropsychopharmaca for comparable effects. EXPERIMENTAL APPROACH: BCECF-AM loaded hippocampal slices were superfused with 16 different neuroleptics, antidepressants and antiepileptics under bicarbonate-buffered conditions. Changes in steady-state pHi of CA3 neurones were measured fluorometrically. KEY RESULTS: The antipsychotics haloperidol, clozapine, ziprasidone, and the antidepressants amitriptyline, doxepin, trimipramine, citalopram, mirtazapine, as well as the anticonvulsive drug tiagabine reversibly reduced the steady-state pHi by up to 0.35 pH-units in concentrations of 5-50 microM. In contrast, venlafaxine, the anticonvulsants carbamazepine, clonazepam, gabapentin, lamotrigine, zonisamide, and the mood stabilizer lithium had no effect on neuronal pHi. CONCLUSION AND IMPLICATIONS: These data substantiate the view that clinically relevant concentrations of neuroleptics and antidepressants can mediate changes in neuronal pHi, which may contribute to their pharmacological mode of action. Effects on pHi should be taken into account when therapeutic or even harmful effects of these drugs are evaluated.
Assuntos
Anticonvulsivantes/farmacologia , Antidepressivos/farmacologia , Antipsicóticos/farmacologia , Hipocampo/efeitos dos fármacos , Líquido Intracelular/química , Neurônios/efeitos dos fármacos , Animais , Cobaias , Hipocampo/química , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Neurônios/químicaRESUMO
Veratridine is believed to cause epileptiform discharges via its effects on sodium channels. We addressed the question whether calcium currents, known to contribute to the generation of paroxysmal depolarization shifts (PDS) in most models of epilepsies, also contribute to veratridine-induced epileptiform activity. Therefore, we recorded from CA3 neurons (n=50) of veratridine-treated hippocampal slices and analyzed the effects of two calcium antagonists. Veratridine (0.5-1.0 microM) elicited spontaneous epileptiform bursts, paroxysmal depolarization shifts (PDS) lasting 100-300 ms, and depolarizations (LD) lasting up to several minutes. Most often PDS directly preceded LD which resulted in typical composite depolarizations termed veratridine-induced complexes (VC). VC persisted even in the presence of CNQX and APV (25 micromol/l, both), or in nominally calcium-free saline, revealing the non-synaptic nature of these potentials. Cobalt (1-2mM) abolished VC within minutes, but allowed LD type-like potentials to be elicited by depolarizing current pulses. Verapamil (50 microM) also diminished or abolished amplitudes of VC. All inhibitory effects of cobalt and verapamil were at least partly reversible. Due to the effects of both calcium antagonists we conclude that veratridine-induced epileptiform activity depends not only on sodium, but also on calcium currents.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Convulsivantes/antagonistas & inibidores , Convulsivantes/farmacologia , Epilepsia/induzido quimicamente , Epilepsia/prevenção & controle , Hipocampo/fisiopatologia , Neurônios/fisiologia , Veratridina/antagonistas & inibidores , Veratridina/farmacologia , Animais , Cálcio/fisiologia , Cobalto/farmacologia , Epilepsia/fisiopatologia , Cobaias , Hipocampo/efeitos dos fármacos , Técnicas In Vitro , Neurônios/efeitos dos fármacos , Verapamil/farmacologiaRESUMO
Previous studies have demonstrated an inverse correlation between the degree of respiratory drive and NHE3 mRNA expression in the brainstem of awake rabbits. Here we show that the levels of NHE3 mRNA extractable from kryo-conserved tissue are highly variable also in the human brainstem. As an insufficient drive to breath may be a final event causing sudden infant death, we compared the expression of NHE3 mRNA in a collective of children who died from non-natural causes to an equal number of SIDS victims. Evaluation of signals from NHE3 RT-PCR showed higher values for the SIDS collective than for the control group. We suggest that the level of NHE3 expression in brainstem tissue may contribute to the vulnerability of infants for SIDS.
Assuntos
Tronco Encefálico/fisiopatologia , Trocadores de Sódio-Hidrogênio/genética , Morte Súbita do Lactente/genética , Idoso , Autopsia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Lactente , Masculino , RNA Mensageiro/genética , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trocador 3 de Sódio-HidrogênioRESUMO
Bone cells contacting nickel (Ni)-containing implant materials may be affected by Ni species via disturbed signaling pathways involved in bone cell development. Here we analyze effects of the Ni-containing steel 316L and major metal constituents thereof on bone morphogenetic protein-2 (BMP-2)-induced alkaline phosphatase (ALP) of MC3T3-E1 cells. While cells grew normally on 316L, cellular Ni content increased 10-fold vs. control within 4 days. With respect to the major components of 316L, Ni2+ (3-50 microM) was most inhibitory to BMP-2-induced ALP, whereas even 50 microM Fe3+, Cr3+, Mo5+, or Mn2+ had no such effect. In line with this, BMP-2-induced ALP was significantly reduced in cells on 316L. This effect was not prevented by the metal ion chelator diethylenetriaminepentaacetic acid (DTPA). Instead, DTPA abolished the stimulatory effect of BMP-2 on ALP, pointing to chelatable metal ions involved. Zn2+, as one possible candidate, antagonized the Ni2+ inhibition of BMP-2-induced ALP in both MC3T3-E1 and human bone marrow stromal cells. Results show that cells contacting 316L steel are exposed to increased concentrations of Ni which suffice to impair BMP-2-induced ALP activity. Zn2+, as a competitor of this inhibition, may help to restore normal osteoblastic function and bone development under these conditions.
Assuntos
Fosfatase Alcalina/biossíntese , Proteínas Morfogenéticas Ósseas/farmacologia , Comunicação Celular/efeitos dos fármacos , Níquel/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/enzimologia , Aço Inoxidável , Fator de Crescimento Transformador beta/farmacologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Proteína Morfogenética Óssea 2 , Proliferação de Células/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Humanos , Camundongos , Osteoblastos/citologia , Espectrofotometria Atômica , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Zinco/metabolismoRESUMO
Bone cells seeded directly on depots of bone morphogenetic protein-2 (BMP-2) increase alkaline phosphatase (ALP) expression. Heating of such BMP-2 depots to 100 degrees C augmented the intensity of this local ALP induction. To understand this unexpected finding, we investigated the effect of heat treatment on BMP-2 depots more closely. Using a novel bioassay based on ALP-induction of remote cells, we found that the amount of released bioactive BMP-2 from heat-treated depots decays within days and could be described by an exponential function. From this function, we expected that pre-incubation of BMP-2 depots in culture medium for 4 weeks renders them insufficient to induce ALP. However, preincubated, heat-treated depots still induced maximal ALP, unless treated with the selective BMP-2 inhibitor noggin. Furthermore, heat treatment of BMP-2 depots generated a layer of immunoreactive BMP-2 at the surface of the carrier. In contrast, BMP-2 was washed off completely if heat treatment of adsorbed protein was omitted. Results show that heat treatment generates both a soluble pool of BMP-2 and a material-bound layer of BMP-2 in which the protein is protected against degradation. Therefore, heat treatment appears useful to locally immobilize BMP-2 on various implant surfaces.
Assuntos
Proteínas de Bactérias/biossíntese , Proteínas Morfogenéticas Ósseas , Osso e Ossos/enzimologia , Materiais Revestidos Biocompatíveis , Endopeptidases/biossíntese , Próteses e Implantes , Fator de Crescimento Transformador beta , Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas/química , Osso e Ossos/citologia , Técnicas de Cultura de Células/métodos , Linhagem Celular , Preparações de Ação Retardada/química , Temperatura Alta , Humanos , Fatores de Tempo , Fator de Crescimento Transformador beta/químicaRESUMO
RATIONALE: In vivo inhibition of the sodium/proton exchanger 3 (NHE3) in chemosensitive neurons of the ventrolateral brainstem augments central respiratory drive in anesthetized rabbits. OBJECTIVES: To further explore the possible role of this exchanger for the control of breathing, we examined the individual relationship between brainstem NHE3 abundance and ventilation in rabbits during wakefulness. METHODS: In 32 adult male rabbits on standard nutritional alkali load, alveolar ventilation, metabolic CO2 production, and blood gases were determined, together with arterial and urinary acid-base status and renal base control functions. Expression of NHE3 in brainstem tissue from the obex region was determined by quantitative real-time reverse-transcription polymerase chain reaction analysis. MEASUREMENTS AND MAIN RESULTS: Regarding the distribution above and below the median, we classified high and low brainstem NHE3 animals, expressing a mean (+/- SEM) NHE3 mRNA of 2.08 +/- 0.28 and 0.72 +/- 0.06 fg cDNA/mg RNA, respectively. Alveolar ventilation of high brainstem NHE3 animals was lower than that of low brainstem NHE3 animals (715 +/- 36 vs. 919 +/- 41 ml . minute(-1); p < 0.01), a finding also reflected by a marked difference in Pa(CO2) (5.24 +/- 0.16 vs. 4.44 +/- 0.15 kPa; p < 0.01). Among possible secondary factors, CO2 production, systemic base excess, and fractional renal base reabsorption were not found to be different. CONCLUSIONS: We conclude that the level of brainstem NHE3 expression-most likely via intracellular pH modulation-contributes to the individual control of breathing and Pa(CO2) in conscious rabbits by adjusting the set point and the loop gain of the system.
Assuntos
Bulbo/metabolismo , Mecânica Respiratória , Trocadores de Sódio-Hidrogênio/metabolismo , Absorção , Animais , Artérias , Bicarbonatos/metabolismo , Tronco Encefálico/metabolismo , Dióxido de Carbono/sangue , Humanos , Rim/metabolismo , Masculino , Pressão Parcial , RNA Mensageiro/metabolismo , Coelhos , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/genéticaRESUMO
Harmala alkaloids are endogenous substances, which are involved in neurodegenerative disorders such as M. Parkinson, but some of them also have neuroprotective effects in the nervous system. While several sites of action at the cellular level (e.g. benzodiazepine receptors, 5-HT and GABA(A) receptors) have been identified, there is no report on how harmala alkaloids interact with voltage-gated membrane channels. The aim of this study was to investigate the effects of harmaline and harmane on voltage-activated calcium- (I(Ca(V))), sodium- (I(Na(V))) and potassium (I(K(V)))-channel currents, using the whole-cell patch-clamp method with cultured dorsal root ganglion neurones of 3-week-old rats. Currents were elicited by voltage steps from the holding potential to different command potentials. Harmaline and harmane reduced I(Ca(V)), I(Na(V)) and I(K(V)) concentration-dependent (10-500 microM) over the voltage range tested. I(Ca(V)) was reduced with an IC(50) of 100.6 microM for harmaline and by a significantly lower concentration of 75.8 microM (P<0.001, t-test) for harmane. The Hill coefficient was close to 1. Threshold concentration was around 10 microM for both substances. The steady state of inhibition of I(Ca(V)) by harmaline or harmane was reached within several minutes. The action was not use-dependent and at least partly reversible. It was mainly due to a reduction in the sustained calcium channel current (I(Ca(L+N))), while the transient voltage-gated calcium channel current (I(Ca(T))) was only partially affected. We conclude that harmaline and harmane are modulators of I(Ca(V)) in vitro. This might be related to their neuroprotective effects.
Assuntos
Canais de Cálcio/fisiologia , Harmalina/farmacologia , Harmina/análogos & derivados , Harmina/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Neurônios Aferentes/efeitos dos fármacos , Canais de Potássio/fisiologia , Canais de Sódio/fisiologia , Animais , Canais de Cálcio/genética , Estimulantes do Sistema Nervoso Central/farmacologia , Relação Dose-Resposta a Droga , Gânglios Espinais/citologia , Concentração Inibidora 50 , Ativação do Canal Iônico/fisiologia , Neurônios Aferentes/fisiologia , Técnicas de Patch-Clamp , Canais de Potássio/genética , Ratos , Ratos Wistar , Canais de Sódio/genética , Fatores de TempoAssuntos
Tronco Encefálico/metabolismo , Respiração , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Animais Recém-Nascidos , Tronco Encefálico/citologia , Tronco Encefálico/fisiologia , Dióxido de Carbono/sangue , Células Cultivadas , RNA Mensageiro/genética , Coelhos , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/genéticaRESUMO
The novel anticonvulsant levetiracetam (LEV) was tested for effects on bioelectric activity and intracellular pH (pHi) regulation of hippocampal CA3 neurons from adult guinea-pigs. In 4-aminopyridine-treated slices, LEV (10-100 microm) reduced the frequency of action potentials and epileptiform bursts of CA3 neurons by 30-55%, while the shape of these potentials remained largely unchanged. Suppressive effects were reversed by an increase of pHi with trimethylamine (TMA). Using BCECF-AM-loaded slices, we found that LEV (10-50 microm) reversibly lowered neuronal steady-state pHi by 0.19 +/- 0.07 pH units in the presence of extracellular CO2/HCO3- buffer. In the nominal absence of extracellular CO2/HCO3- or in Na+-free CO2/HCO3(-)-buffered solution, LEV had no effect on steady-state pHi. Recovery of pHi subsequent to ammonium prepulses remained unchanged in the absence of CO2/HCO3- buffer, but was significantly reduced by LEV in the presence of CO2/HCO3- buffer. These findings show that LEV inhibits HCO3(-)-dependent acid extrusion, but has no effect on Na+/H+ exchange. LEV did not affect Na+-independent Cl-/HCO3- exchange because intracellular alkalosis upon withdrawal of extracellular Cl- remained unchanged. These data show that LEV at clinically relevant concentrations inhibits Na+-dependent Cl-/HCO3- exchange, lowers neuronal pHi, and thereby may contribute to its anticonvulsive activity.
Assuntos
Anticonvulsivantes/farmacologia , Antiportadores de Cloreto-Bicarbonato/antagonistas & inibidores , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Piracetam/análogos & derivados , Piracetam/farmacologia , Sódio/metabolismo , Potenciais de Ação , Animais , Soluções Tampão , Cátions Monovalentes , Cobaias , Hipocampo/citologia , Hipocampo/fisiologia , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Levetiracetam , Neurônios/fisiologia , Fatores de TempoAssuntos
Células Quimiorreceptoras/fisiologia , Mecânica Respiratória , Trocadores de Sódio-Hidrogênio/fisiologia , Animais , Tronco Encefálico/fisiopatologia , Dióxido de Carbono/fisiologia , Hipercapnia/fisiopatologia , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio/genéticaRESUMO
Contingent negative variations (CNV) after acoustic stimuli (S1) followed by optical ones (S2) were recorded using electroencephalography in 22 healthy students both under control conditions and during ischemic pain to study the effects of sustained pain on CNV. Mean negative CNV-amplitudes and integrated areas below CNV were significantly larger during periods of ischemic pain than under control conditions (16.53 versus 13.11 microV, respectively (P=0.0028) and 8.318 versus 6.357 microV*s, respectively (P=0.00071)). We conclude that deep somatic pain augments CNV. Reduced CNV amplitudes occurring during migraine attacks, however, reflect other mechanisms which may mask the effects of migraine headache on CNV.
Assuntos
Variação Contingente Negativa/fisiologia , Isquemia/complicações , Dor/fisiopatologia , Adulto , Mapeamento Encefálico , Eletroencefalografia/métodos , Eletroculografia/métodos , Feminino , Lobo Frontal/fisiopatologia , Humanos , Masculino , Dor/etiologia , Medição da Dor/métodos , Estimulação Física , Fatores de TempoRESUMO
Various hypotheses try to explain the anticonvulsive and mood stabilizing effects of valproate. Among them, amplification of GABAergic inhibition and reduction of membrane excitability is favored. Here we show that superfusion with 0.1-1 mM valproate induced a moderate intracellular acidification of BCECF-AM-loaded CA3-neurons (hippocampal slices, guinea pig) which was measured as the difference between intracellular pH before (baseline pH(i)) and during valproate treatment (deltapH(i)). In two groups of neurons treated with 1 mM and 0.1-0.5 mM, deltapH(i) values amounted to 0.20 +/- 0.10 and 0.10 +/- 0.04 (deltapH(i) +/- S.D.), respectively, suggesting a dependence on the used valproate-concentration. DeltapH(i) did not correlate with the baseline pH(i). Furthermore, the acidification seems to be independent from an activation of postsynaptic GABA-A receptors, as it was not influenced by 0.1 mM picrotoxin. Since our previous studies clearly demonstrated a reduction of membrane excitability during moderate intracellular acidification, we suggest that the valproate-mediated intracellular acidification may substantially contribute to its anticonvulsive and mood stabilizing properties.
Assuntos
Anticonvulsivantes/farmacologia , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Ácido Valproico/farmacologia , Animais , Relação Dose-Resposta a Droga , Cobaias , Hipocampo/citologia , Hipocampo/fisiologia , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Modelos Biológicos , Neurônios/fisiologia , Receptores de GABA-A/efeitos dos fármacos , Receptores de GABA-A/metabolismoRESUMO
PURPOSE: Sulthiame is a carbonic anhydrase (CA) inhibitor with an anticonvulsant effect in the treatment of benign and symptomatic focal epilepsy in children. The aim of the study was to elucidate the mode of action of sulthiame with respect to possible changes of intracellular pH (pHi) that might develop along with sulthiame's anticonvulsant properties. METHODS: The effects of sulthiame (a) on pHi of 2',7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymetyl ester (BCECF-AM) loaded CA3 neurones as well as (b) on epileptiform activity (induced by 50 microM 4-aminopyridine) were compared with those of the CA inhibitors acetazolamide and benzolamide. RESULTS: In the majority of neurons, sulthiame (1.0-1.5 mM; n = 8) as well as the membrane permeant acetazolamide (0.5-1.0 mM; n = 6) reversibly decreased pHi by 0.18 +/- 0.05 (SD) and 0.17 +/- 0.10 (SD) pH units, respectively, within 10 min. The poor membrane permeant benzolamide (1.0-2.0 mM) had no influence on pHi (n = 8). Sulthiame (1.0-2.5 mM) and acetazolamide (1.0-2.0 mM) reversibly reduced the frequency of action potentials and epileptiform bursts after 10-15 min (n = 9, n = 7), whereas benzolamide (1.0-2.0 mM) had no effect (n = 6). CONCLUSIONS: The results suggest that sulthiame acts as a membrane-permeant CA inhibitor whose beneficial effect on epileptiform activity results at least in part from a modest intracellular acidosis of central neurons.