Dieter Bingmann

Intracellular pH modulates spontaneous and epileptiform bioelectric activity of hippocampal CA3-neurones

A growing body of evidence hints at intracellular free protons to be involved in the modulation of electric activity of cortical neurones. In this study we demonstrate that application of the weak acid propionate (2.5-20 mM) transiently lowers intracellular pH (pH(i)) of BCECF-AM loaded CA3-neurones in hippocampal slices. The predictability of this acidification prompted us to use propionate as a tool to investigate effects of pH(i) on spontaneous bioelectric activity (SBA) and epileptiform activity (EA, induced by bicuculline, caffeine or low magnesium) of CA3 neurones: SBA and EA were transiently suppressed by 2-20 mM propionate - coinciding with the transient neuronal acidification. As activation of Na(+)/H(+)-exchangers (NHE) is involved in the recovery from neuronal acidosis and NHE-inhibition alone is known to increase the activity of intracellular free protons of hippocampal neurones, we tested the effect of the NHE-blockers amiloride (0.5-1 mM) or HOE642 (200 microM) on SBA and EA of CA3-neurones. Long-term application of NHE-inhibitors alone continuously suppressed SBA and EA, which recovered during additional exposure to the weak base trimethylamine (5-10 mM). Simultaneous administration of propionate and NHE-blockers intensified the inhibition of neuronal activity. Together, these results indicate that intracellular acidification inhibits bioelectric activity of hippocampal CA3-neurones. This supports the hypothesis that pH(i) contributes to the control of cortical excitability.

Selective inhibition of the Na+/H+ exchanger type 3 activates CO2/H+-sensitive medullary neurones.

Abstract Hypercapnia as well as lowered intracellular pH (pHi) increase the bioelectric activity of CO2/H+-sensitive neurones (VLNcs) of the ventrolateral medulla oblongata. Here we describe that immunoreactive Na+/H+ exchanger (NHE3) is present in ventrolateral neurones from medullary organotypic cultures (obex level). To test whether VLNcs can be acidified and thereby activated by inhibition of NHE3, we used the novel high-affinity NHE3-inhibitors S1611 and S3226. Both drugs raised the firing rates of VLNcs to at least 150% of the control values, and depolarized membrane potential by up to 15 mV at concentrations (0.5–1 µmol/l) suitable for selective inhibition of NHE3. The changes in bioelectric activity strongly resembled the responses to hypercapnia (PCO2: 60–100 mmHg). In BCECF-AM-loaded cultures a subfraction of ventrolateral VLNcs was found to be intracellularly acidified by 0.05–0.1 pH units following treatment with S1611; the time course of this acidification was similar to that evoked by hypercapnia. All drug effects were sustained and readily reversible upon washing. Non-CO2/H+-responsive medullary neurones as well as hippocampal CA3 neurones were unaffected by up to 20 µmol/l S1611. It is concluded that the selective inhibition of NHE3 acidifies and activates CO2/H+-sensitive neurones within the ventrolateral medulla oblongata.

Carbonic anhydrase inhibitor sulthiame reduces intracellular pH and epileptiform activity of hippocampal CA3 neurons.

Summary:  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 sulthiames anticonvulsant properties.

Calcium antagonistic effects of carbamazepine as a mechanism of action in neuropsychiatric disorders: studies in calcium dependent model epilepsies

Carbamazepine (CBZ) is used in neurology for the treatment of epilepsies and trigeminal neuralgia and in psychiatry for the prophylactic treatment of affective and schizoaffective psychoses. Since a common mechanism of epilepsies and affective psychoses might be increased intracellular calcium ion levels, CBZ action was analyzed in penicillin, caffeine and low Mg2+ induced model epilepsies which have been shown to be suppressed specifically by organic calcium antagonists. In CA3 and CA1 areas of hippocampal slice preparations of guinea pigs CBZ reduced paroxysmal depolarizations and extracellular field potentials (EFP) in a typical time and concentration dependent manner as it is known from calcium antagonists. Furthermore, subthreshold concentrations of the organic calcium antagonist verapamil intensified the action of CBZ. NMDA induced increases of the discharge rate of EFP were, however, unaffected by CBZ.

Modulation of voltage-gated channel currents by harmaline and harmane

1 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. 2 While several sites of action at the cellular level (e.g. benzodiazepine receptors, 5‐HT and GABAA receptors) have been identified, there is no report on how harmala alkaloids interact with voltage‐gated membrane channels. 3 The aim of this study was to investigate the effects of harmaline and harmane on voltage‐activated calcium‐ (ICa(V)), sodium‐ (INa(V)) and potassium (IK(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. 4 Harmaline and harmane reduced ICa(V), INa(V) and IK(V) concentration‐dependent (10–500 μM) over the voltage range tested. ICa(V) was reduced with an IC50 of 100.6 μM for harmaline and by a significantly lower concentration of 75.8 μM (P<0.001, t‐test) for harmane. The Hill coefficient was close to 1. Threshold concentration was around 10 μM for both substances. 5 The steady state of inhibition of ICa(V) by harmaline or harmane was reached within several minutes. The action was not use dependent and at least partly reversible. 6 It was mainly due to a reduction in the sustained calcium channel current (ICa(L+N)), while the transient voltage‐gated calcium channel current (ICa(T)) was only partially affected. 7 We conclude that harmaline and harmane are modulators of ICa(V) in vitro. This might be related to their neuroprotective effects.

CO2/HCO3-withdrawal from the bath medium of hippocampal slices: biphasic effect on intracellular pH and bioelectric activity of CA3-neurons

Many studies analyzing interactions of pH and bioelectric activity focus on changes of the extracellular pH, whereas data concerning central neuronal excitability and intracellular pH (pHi) are rare. Here, we report on the spontaneous bioelectric activity and epileptiform activity of CA3-neurons during a procedure which changed pHi. As monitored in BCECF-AM loaded cells, the change from a CO2/HCO3(-)-buffered to a HEPES-buffered medium (CO2/HCO3(-)-withdrawal, hereafter termed W) was associated with a transient intracellular alkalosis (delta pH = 0.2 +/- 0.04) which preceded a sustained intracellular acidosis (delta pH = 0.4 +/- 0.04). Coinciding with this W-induced biphasic shift of pHi a biphasic alteration of spontaneous bioelectric activity was recorded: as a rule, an up to 30 min lasting increase (excitatory phase) preceded a typical sustained suppression (inhibitory phase). This biphasic action was also observed using various in vitro-epilepsy-models (bicuculline, penicillin, caffeine): epileptiform discharges were completely suppressed after an initial increase in frequency. This modulation of bioelectric activity was unlikely due to alterations of the postsynaptic GABA-system as hyperpolarizing GABAA- and GABAB-responses of CA3-neurons were hardly affected. In the majority of the neurons, the initial increase of spontaneous bioelectric activity (excitatory phase) culminated in transient burst periods lasting 5-30 min. These transient burst periods were blocked by NMDA- or AMPA-antagonists: DL-2-amino-5-phosphonovalerate (APV, 50 microM) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 50 microM). The calcium-antagonist verapamil (50 microM) reduced amplitudes of depolarizations and duration of the transient burst periods. The results suggest that the biphasic alteration of pHi modulates the susceptibility of glutamate receptors and voltage-gated calcium-channels, which leads to respective changes of bioelectric activity.

Ventrolateral neurons of medullary organotypic cultures: intracellular pH regulation and bioelectric activity.

The hypothesized role of the intracellular pH (pH(i)) as a proximate stimulus for central chemosensitive neurons is reviewed on the basis of data obtained from organotypic cultures of the medulla oblongata (obex level) of new born rats (OMC). Within OMC a subset of neurons responds to hypercapnia as do neurons in the same (or similar) brain areas in vivo. Maneuvers altering intra- and/or extracellular pH (pH(o)) such as hypercapnia, bicarbonate-withdrawal, or ammonium pre-pulses, evoked well defined changes of the neuronal pH(i). During hypercapnia (pH(o) 7.0) or bicarbonate-withdrawal (pH(o) 7.4) most ventrolateral neurons adopted a pH(i) which was < or = 0.2 pH units below the steady state pH(i), while signs of pH(i)-regulation occurred only in a small fraction of neurons. During all treatments leading to intracellular acidosis, bioelectric activity of chemosensitive neurons increased and was often indistinguishable from the response to hypercapnia, regardless of whether pH(o) was unchanged, decreased or increased during the treatment. These data strongly suggest that the pH(i) acts as proximate stimulus. The mode of acid extrusion of chemosensitive neurons is, therefore, of major importance for the control of central chemosensitivity. Immunocytochemical data, pH(i) measurements and neuropharmacological studies with novel drugs pointed to the Na(+)/H(+) exchanger subtype 3 (NHE3) as a main acid extruder in ventrolateral chemosensitive neurons. Possible functions and neuropharmacological strategies arising from this very local NHE3 expression are discussed.

CO2-sensitive medullary neurons : activation by intracellular acidification

BIOELECTRIC activity of CO2-sensitive, ventrolateral medullary neurons (VLNCS) in organotypic cultures from the obex level of newborn rats was tested during changes of the intracellular pH (pHi) measured in BCECF-AM loaded cultures. Hypercapnia (pCO2 80–100 mmHg) reduced pHi by 0.15 ± 0.06 units and stimulated neuronal discharges. Replacement of CO2/HCO3− in the bath by HEPES (26 mM, pH 7.4) for 10 min acidified pHi (0.07 ± 0.03 units) and also excited VLNCS. Ammonium chloride (10 mM, 1 min) initially alkalized (0.1 ± 0.04) and thereafter acidified pHi (0.06 ± 0.03), while the extracellular pH was first acidified and then alkalized. This resulted in neuronal discharge which were first suppressed and then accelerated. The findings strongly suggest that intracellular rather than extra-cellular acidification activates CO2-sensitive neurons.

Intercellular communication between bone marrow stromal cells and CD34+ haematopoietic progenitor cells is mediated by connexin 43-type gap junctions

The existence of functional gap junctions between haematopoietic progenitor cells (HPCs) and stromal cells of the haematopoietic microenvironment in the human system is a controversial issue. Primary CD34+ HPCs isolated from leukapheresis products were co‐incubated with the human fibroblastoid bone marrow stromal cell line L87/4 in short‐term liquid culture. Using the highly sensitive double whole‐cell patch‐clamp technique, we found that the majority (91%) of CD34+ HPCs are electrically coupled to L87/4 cells. Importantly, efficient coupling was observed within 1 h of the attachment of CD34+ HPCs to plastic adherent L87/4 cells. By comparison, homologous cell pairs formed by L87/4 cells exhibited a significantly higher electric coupling. Analysis of single‐channel conductances revealed an electric profile characteristic of connexin 43 (Cx43)‐type gap junctions for both homologous and heterologous cell pairs. The Cx phenotype was confirmed using Cx43‐specific monoclonal antibodies in a flow cytometric assay and reverse transcription polymerase chain reaction (RT‐PCR) for the detection of Cx43 mRNA. Finally, the electrophysiological studies were complemented by dye‐transfer experiments using the recently described ‘parachute’ technique that allows the monitoring of dye diffusion without disruption of the plasma membrane. Taken together, our data indicate that functional Cx43‐type gap junctions exist between stromal cells and immature HPCs and, thus, may provide an important regulatory pathway in haematopoiesis.

Paroxysmal depolarization shifts induced by bicuculline in CA3 neurons of hippocampal slices : suppression by the organic calcium antagonist verapamil

Organic calcium antagonists have been reported to abolish epileptic neuronal discharges elicited by pentylenetetrazol and penicillin. It was tested whether the organic calcium antagonist verapamil is able to suppress also paroxysmal depolarization shifts (PDS) induced by bicuculline. This is of special interest, since bicuculline is assumed to produce PDS by blocking GABAergic synaptic inhibition. The experiments were performed on CA3 neurons of hippocampal slices (guinea pig). Verapamil (40, 60, and 80 microM) reduced amplitude, duration and frequency of appearance of PDS until the generation of PDS failed. The results indicate that calcium currents are also involved in bicuculline PDS, and that bicuculline exerts its epileptogenic action, at least in part, on extrasynaptic sites.