The NHE3 inhibitor AVE1599 stimulates phrenic nerve activity in the rat.

The effect of AVE1599, an inhibitor of the sodium/proton exchanger type 3 (NHE3), on phrenic nerve (PN) activity was investigated using the working heart brainstem preparation (WHBP). Hypercapnia (Delta pH: -0.1) applied for 10 min reversibly increased PN frequency (f) by 66.0 +/- 19.5% and decreased burst duration by 23.3 +/- 3% (mean +/- SE, n = 21). Similarly, AVE1599 (0.3 microM) increased f after 10 and 30 min by 75.1 +/- 13.2 and 176 +/- 36.2% (n = 10), respectively, and reduced duration of PN bursts by 24.9 +/- 10.8%. Hypercapnia-induced increases of f were attenuated by AVE1599. An elevated concentration of AVE1599 (0.9 microM) had no significant effect on PN. As AVE1599 accumulates in brain tissue and might interfere with the less affine NHE1, we furthermore tested the NHE1-inhibitor HOE642. In fact, HOE642 (0.9 microM) diminished f by 88.5 +/- 9.2 and 58.6 +/- 10.0% after 10 and 30 min (n = 6), respectively, but did not abolish hypercapnic responses. We conclude that AVE1599 augments central respiratory drive in the WHBP via NHE3 but not NHE1 inhibition.

Expression of anion exchanger 3 influences respiratory rate in awake and isoflurane anesthetized mice.

The anion exchanger 3 (AE3) is involved in neuronal pH regulation of which may include chemosensitive neurons. Here we examined the effect of AE3 expression on respiratory rate (RR) in vivo. AE3 knockout (KO, n=5) and wild type (WT, n=6) mice were subjected to body plethysmography, both while awake and during isoflurane anesthesia. RR was significantly lower in awake AE3 KO (162+/-7SE min(-1)) than in WT mice (212+/-20 min(-1), P=0.036). The same was found during isoflurane anesthesia at 0.5 MAC (KO: 123+/-9 min(-1), WT: 168+/-15 min(-1), P=0.026) and 1.0 MAC (KO: 51+/-6 min(-1), WT: 94+/-6 min(-1), P=0.001). Hypercapnia (5% CO2) increased RR in awake and decreased RR in nesthetized (1.0 MAC) mice, whereby relative changes were larger in AE3 KO mice. Recovery from isoflurane anesthesia in respect to RR regaining baseline values was more pronounced in AE3 KO. Results show that AE3 expression profoundly influences control of breathing in mice.

[Optimizing histological image data for 3-D reconstruction using an image equalizer]. / Optimierung histologischer Bilddaten für die 3D-Rekonstruktion mit Hilfe eines Image-Equalizers.

Bone cells form a wired network within the extracellular bone matrix. To analyse this complex 3D structure, we employed a confocal fluorescence imaging procedure to visualize live bone cells within their native surrounding. By means of newly developed image processing software, the "Image-Equalizer", we aimed to enhanced the contrast and eliminize artefacts in such a way that cell bodies as well as fine interconnecting processes were visible.

A reporter-cell assay for the detection of BMP-2 immobilized on porous and nonporous materials.

Human recombinant bone morphogenetic protein-2 (rhBMP-2) immobilized on the surface of metal implants can facilitate osseointegration. Here, we describe a cell reporter assay useful for quantifying small amounts of immobilized rhBMP-2 on various materials. The peptide was dotted and heat-fixed on titanium, 316L stainless steel, nitrocellulose, or glass, and its distribution was monitored by in situ biotinylation followed by detection with the avidin-biotin method. Bioactivity of rhBMP-2 was demonstrated by means of a confluent layer of osteoblastic MC3T3-E1 cells that evenly covered rhBMP-2-free and rhBMP-2-loaded surface areas, as shown with epifluorescence microscopy of calcein acetoxymethyl (AM)-loaded cells. Expression of osteocalcin, fibronectin, actin, and vimentin increased where cells were located on rhBMP-2 dotted areas, but the signal:noise ratio was too low to bioassay the peptide. However, local pronounced expression of alkaline phosphatase was used to quantify BMP-2 in the range of 5-80 ng/dot by means of a cytochemical color reaction for alkaline phosphatase and image analysis of resulting dots. The lower detection limit was in the order nitrocellulose > glass > titanium > 316L steel. We conclude that the cell reporter assay is useful to assess biological activity of rhBMP-2 even after immobilization on three-dimensional implant materials.

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.

Kava pyrones exert effects on neuronal transmission and transmembraneous cation currents similar to established mood stabilizers--a review.

1. Antiepileptic drugs that are successful as mood stabilizers, e.g. carbamazepine, valproate and lamotrigine, exhibit a characteristic pattern of action on ion fluxes. As a common target, they all affect Na+- and Ca2+ inward and K+ outward currents. 2. Furthermore, they have a variety of interactions with the metabolism and receptor occupation of biogenic amines and excitatory and inhibitory amino acids, and, by this, also influence long- term potentiation (LTP) to different degrees. 3. The kava pyrones (+/-)-kavain and dihydromethysticin are constituents of Piper methysticum. Anticonvulsant, analgesic and anxiolytic properties have been described in small open trials. 4. In the studies summarized in this article the effects mainly of (+/-)-kavain were tested on neurotransmission and especially on voltage gated ion channels. It is assumed that effects on ion channels may significantly contribute to clinical efficacy. 5. Experimental paradigms included current and voltage clamp recordings from rat hippocampal CA 1 pyramidal cells and dorsal root ganglia as well as field potential recordings in guinea pig hippocampal slices. 6. The findings suggest that (i) kava pyrones have a weak Na+ antagonistic effect that may contribute to their antiepileptic properties (ii) that they have pronounced L- type Ca2+ channel antagonistic properties and act as an positive modulator of the early K+ outward current. These two actions may be of importance for mood stabilization. (iii) Furthermore, kava pyrones have additive effects with the serotonin-1A agonist ipsapirone probably contributing to their anxiolytic and sleep- inducing effects. (iv) Finally, they show a distinct pattern of action on glutamatergic and GABAergic transmission without affecting LTP. The latter, however, seems not to be true for the spissum extract of Kava where suppression of LTP was observed. 7. In summary, kava pyrones exhibit a profile of cellular actions that shows a large overlap with several mood stabilizers, especially lamotrigine.

A novel inhibitor of the Na+/H+ exchanger type 3 activates the central respiratory CO2 response and lowers the apneic threshold.

Cultured CO2-sensitive neurons from the ventrolateral medulla of newborn rats enhanced their bioelectric activity upon intracellular acidification induced by inhibition of the Na+/H+ exchanger type 3 (NHE3). Now we detected NHE3 also in the medulla oblongata of adult rabbits. Therefore, this animal model was employed to determine whether NHE3 inhibition also affects central respiratory chemosensitivity in vivo. Seven anesthetized (pentobarbital), vagotomized, paralyzed rabbits were artificially ventilated with O2-enriched air. From the phrenic nerve compound discharge, integrated burst amplitude (IPNA), respiratory rate (fR), and phrenic minute activity (IPNA. fR) were taken as measures of central respiratory rhythm and drive. Effects of potent NHE3 inhibition with the novel brain permeant substance S8218 were studied by comparing respiratory characteristics before and after up to 9.2 +/- 1.1 mg/kg cumulative drug application, yielding average plasma concentrations of 0.9 +/- 0.2 microg/ml. In response to S8218, the baseline level of IPNA. fR was significantly enhanced by an average of 51.0 +/- 6.4% (n = 27, p < 0.0001). The influence of NHE3 inhibition on the respiratory CO2 response was studied at plasma concentrations of S8218 maintained in the range of 0.3 microg/ml (10(-6) M). Although the metabolic acid-base status thereby remained widely unchanged, the group mean apneic threshold PaCO2 was significantly lowered by 0.45 +/- 0.11 kPa (n = 7, p < 0.01), whereby in four of seven animals even strong hyperventilation failed to suppress phrenic nerve rhythmicity completely. Likewise, S8218 significantly augmented IPNA. fR, in the range of PaCO2 between 1 and 6 kPa above threshold, by an average of 38.0 +/- 8.5% (n = 35, p < 0.0001). These in vivo results are compatible with the effects of NHE3 inhibition on chemosensitive brainstem neurons in vitro. Moreover, rhythmogenesis is supported through NHE3 inhibition by lowering the threshold PCO2 for central apnea.

Enhancement of connexin 43 expression increases proliferation and differentiation of an osteoblast-like cell line.

Bone cells form a functional syncytium as they are coupled by gap junctions composed mainly of connexin 43 (Cx43). To further understand the role of Cx43 in bone cell growth and differentiation, we stably transfected Cx45-expressing UMR 106-01 cells with Cx43 using an expression vector containing rat Cx43 cDNA. Three stably transfected clones were analyzed, all of which showed altered expression of Cx43 and/or Cx45 as was obvious from immunocytochemistry and Northern blotting. Double whole-cell patch clamping revealed single-channel conductances of 20 (Cx45) and 60 pS (Cx43). The overexpression of Cx43 led to an increase in dye coupling concomitant with elevated gap-junctional conductance. The phenotype of the transfected clones was characterized by an increased proliferation (4- to 7-fold) compared to controls. Moreover, a transfectant clone with 10- to 12-fold enhanced Cx43 expression showed a significantly increased calcium content of the extracellular matrix and enlarged mineralization nodules, while alkaline phosphatase was moderately increased. We conclude that enhanced gap-junctional coupling via Cx43 significantly promotes proliferation and differentiation of UMR cells.

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.

Different effects of GABAergic anticonvulsants on 4-aminopyridine-induced spontaneous GABAergic hyperpolarizations of hippocampal pyramidal cells--implication for their potency in migraine therapy.

Clinical studies indicate anti-migrane efficacy of the probably GABAergic anticonvulsants valproate and gabapentin. For the GABAergic anticonvulsants vigabatrin and tiagabine, studies about antimigrane efficacy are missing. The aim of this study was to test the GABAergic potency of these drugs in vitro before further clinical studies. Intracellular recordings were obtained from hippocampal pyramidal cells. Spontaneous GABAergic hyperpolarizations (SGH) elicited by 75 microM 4-aminopyridine were used to test the effect of these drugs on GABA-dependent potentials. Tiagabine (0.1 mM) prolonged the duration of SGH. Furthermore, monophasic SGH turned over into triphasic typical GABAergic membrane potential fluctuations within 20 min. In contrast, valproate, gabapentin, and vigabatrin failed to affect SGH up to 60 min of application. The reason for the fast action of tiagabine on SGH may be caused by a faster increase of synaptic GABA levels compared with other drugs. As migraine therapy benefits from an augmentation of GABA activity, we recommend clinical studies of tiagabine as a fast-acting agent in migraine attacks.

Cutting of living hippocampal slices by a highly pressurised water jet (macromingotome).

Living brain slices are usually cut with razor blades, which compress a ca. 50-microm-thick layer of tissue. This results in cell debris and lesioned cells which, e.g. form diffusion barriers between the bath and living neurons underneath, thereby prolonging response times of neurons to drugs in the bath saline and impeding the experimental access to intact neurons. To avoid such drawbacks, a macromingotome was developed which cuts nervous tissue with water jets. Physiological saline under pressures of 100-1800 bar was ejected through nozzles of 35-100 microm to cut 300-500-microm-thick hippocampal slices. Systematic variations of pressure and nozzle diameter revealed best results at 400-600 bar and with nozzle diameters of 60-80 microm. Under these conditions, intact CA1- and CA3-neurons as well as granule cells were detected with infrared microscopy at less than 10 microm underneath the surface of the slice. Superficial neurons with intact fine structures were also seen when the slices were studied by light-microscopy. Intra- and extracellular recordings from superficial neurons showed normal membrane- and full action potentials and the development of stable epileptiform discharges in 0 Mg(2+)-saline. These results indicate that the macromingotome offers an alternative way of cutting slices which may facilitate electrophysiological/neuropharmacological or fluorometric studies on superficial neurons.

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.

Reduced signal complexity of intracellular recordings: a precursor for epileptiform activity?

Recent studies have shown that time windowed extraction of nonlinear parameters like an effective correlation dimension from intracranially recorded EEG of epileptic patients often allows to detect and identify an unequivocal "pre-ictal phase" preceding an epileptic seizure. In another study, however, such an anticipation could not be made. These conflicting findings may indicate that observed changes in nonlinear parameters probably depend on the type of elementary mechanisms underlying epileptic processes and/or the spatial distribution of neurons primarily involved in generation of epileptiform discharges. To test the existence of such dependencies, the transition from normal to epileptiform activity (EA) of CA3-neurons in hippocampal slices was analyzed in four epilepsy models, using a time windowed computation of an effective correlation dimension. Indeed, in xanthine and penicillin models, signal complexity in intracellular recordings was reduced before manifestation of paroxysmal depolarization shifts (PDS), whereas a preceding loss of complexity was missing in low-magnesium and veratridine models. These findings indicate that interictal-like EA is predictable only in some epilepsy models.

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

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 micromol/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 micromol/l S1611. It is concluded that the selective inhibition of NHE3 acidifies and activates CO2/H+-sensitive neurones within the ventrolateral medulla oblongata.

Effects of (+/-)-kavain on voltage-activated inward currents of dorsal root ganglion cells from neonatal rats.

Kava pyrones extracted from pepper Piper methysticum are pharmacologically active compounds. Since kava pyrones exhibit anticonvulsive, analgesic and centrally muscle relaxing properties, the influence of a synthetic kava pyrone, (+/-)-kavain, on voltage-dependent ion channel currents was studied. Effects of (+/-)-kavain on voltage-activated inward currents were analysed in cultured dorsal root ganglion cells derived from neonatal rats. Voltage-activated Ca2+ and Na+ currents were elicited in the whole-cell configuration of the patch clamp technique. Extracellularly applied (+/-)-kavain dissolved in hydrous salt solutions reduced voltage-activated Ca2+ and Na+ channel currents within 3-5 min. As the solubility of (+/-)-kavain in hydrous solutions is low, dimethyl sulfoxide (DMSO) was added to the saline as a solvent for the drug in most experiments. When (+/-)-kavain was dissolved in DMSO, the drug induced a fast and pronounced reduction of both Ca2+ and Na+ currents, which partly recovered within 2-5 min even in the presence of the drug. The present study indicates that (+/-)-kavain reduces currents through voltage-activated Na+ and Ca2+ channels.

Effects of vitamin D3, 17beta-estradiol, vasoactive intestinal peptide, and glutamate on electric coupling between rat osteoblast-like cells in vitro.

Osteoblast-like cells express receptors for various hormones and neurotransmitters that induce widespread actions in the bone to which intercellular communication and its modulation may contribute. Therefore, we examined the effects of the osteotropic hormones vitamin D3 (vitD3) and 17beta-estradiol (17beta-E2) as well as the neurotransmitter vasoactive intestinal peptide (VIP) and the excitatory amino acid glutamate (Glu) on gap junctions between rat osteoblast-like (ROB) cells in vitro. Electric coupling was measured by simultaneous intracellular recordings from neighboring cells. The coupling factor (cf) was calculated from membrane potential changes induced by alternate current injections into both cells. In ROB cells cf was increased by 5 x 10(-8) mol/L vitD3 to 130 +/- 13% (mean +/- SD; n = 6) of the initial value within 5-20 min. This effect was not reversible after washing with control saline for 10-15 min. In six cell pairs, cf was not affected by vitD3 (94 +/- 5%). In three cell pairs superfusion of 10(-8) mol/L E2 reduced cf to 80 +/- 6% within 10 min, whereas, in two cell pairs, this hormone improved cf to 140% within 20 min. Exposure of VIP (3 x 10(-8) mol/L) did not alter cf in the majority of cells (99 +/- 3%; n = 11). In five cell pairs, cf was improved within 5-15 min to 133 +/- 12%, whereas, in one cell pair, cf was reduced to 22% by VIP. In contrast, brief application of Glu (5 x 10(-3) mol/L) decreased cf to 75 +/- 5% (n = 5), whereas, in nine other cell pairs, cf was not affected (96 +/- 5%). The findings indicate that cell-cell coupling of gap junctions between bone cells can be altered by actions of hormones and transmitters in a cell-pair-specific way, which may depend on their functional state.

Effects of lead, mercury, and methyl mercury on gap junctions and [Ca2+]i in bone cells.

Heavy metals such as lead (Pb), mercury (Hg), and methyl mercury (MeHg) impair cell functions. For bone it is known that Pb changes bone formation rates, which depend on intracellular free calcium concentration ([Ca2+]i). Since heavy metals compete with Ca2+ at multiple sites and increased [Ca2+]i reduces gap junctional coupling between bone cells, we analyzed the effects of extracellular (e) and intracellular (i) application of Pb, Hg, and MeHg on these channels. Using primary cultures of osteoblast-like cells, relative changes of [Ca2+]i were studied in Fura-2/AM loaded cells. Parallel intracellular recordings of neighboring cells were obtained using a conventional and a patch electrode. Pb(e) (5 mumol/liter; n = 3) and Hg(e) (5 mumol/liter; n = 3) as well as Pb(i) (25 mumol/liter; n = 7) did not change the coupling (delta MP2/delta MP1). In contrast, MeHg(e) (1-10 mumol/liter; n = 6) and Hg(i) (> or = 5 mumol/liter; n = 8) reduced the coupling to 79.5 +/- 19.3% and 62.4 +/- 15.3%, respectively, within 15-20 minutes. The reduction of coupling followed individual time courses, and in no case was a steady state of decoupling reached within 20 minutes. Extracellular application of Pb(e) (5 mumol/liter, n = 74) for 20 minutes, linearly elevated the Fura emission ratio reflecting transmembrane Pb permeation rather than [Ca2+]i increase. Hg(e) (n = 48) slightly increased [Ca2+]i from 100 to < or = 200 nmol/liter, whereas MeHg(e) (5 mumol/liter, n = 52) released Ca2+ from internal stores, thus increasing [Ca2+]i up to 2 mumol/liter. In conclusion, Pb(e), Pb(i) and Hg(e) do not affect gap junctional coupling per se. Since MeHg(e) and Hg(i) deplete calcium stores, the decrease of the electric coupling is attributable to increased [Ca2+]i, which affects gap junction channels.

A calcium release activated calcium influx in primary cultures of rat osteoblast-like cells.

Osteoblast-like (OBL) cells in primary culture were tested for their ability to generate a calcium release activated calcium flux (CRAC). Influx of Ca2+ was optically detected by fura-2. Intracellular calcium stores (ICS) were emptied in the absence of extracellular calcium ([Ca2+]e) by 5 microM thapsigargin (TG) or 2 microM A23187. Readdition of 1.8 mM [Ca2+]e increased the free intracellular Ca2+ ([Ca2+]i) after a delay of 30-60 seconds at a rate of 2.3 nM/s due to CRAC. This rate depended on [Ca2+]e and was substantially lowered if readdition of 1.8 mM [Ca2+]e was preceded by, e.g., 0.72 mM [Ca2+]e. CRAC-induced [Ca2+]i peaks were correlated (r = 0.543) with [Ca2+]i peaks during the complete depletion of ICS with A23187. Ca2+ influx due to CRAC could be blocked by flufenamic acid (100 microM) but not verapamil (20 microM). Ni2+ (1 mM), although reversibly blocking CRAC, accelerated the initial [Ca2+]i influx rate. Induction of CRAC enhanced the influx of Mn2+ 4.3-fold, as measured by quenching of fura-2 fluorescence. In summary, OBL cells exhibit a CRAC which allows for the permeation of ions other than Ca2+. This Ca2+ flux may be activated by transmembraneous gradients of Ca2+ and Ni2+.

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.

CO2-sensitive medullary neurons: activation by intracellular acidification.

Bioelectric activity of CO2-sensitive, ventrolateral medullary neurons (VLN(CS)) 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 VLN(CS). 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 extracellular acidification activates CO2-sensitive neurons.