No 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 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.

The structure of gap junctions in osteoblast-like cells (OBs) and the connexins (cx) that build up these structures were characterized by ultrastructural, immunocytochemical, and molecular techniques. Ultrastructural studies revealed numerous gap junctions which were mostly located on processes of neighboring cells. Immunofluorescence labeling using two different antibodies (specific to mouse live cx26 and cx32 and to a peptide-specific rat heart gap junction protein cx43) gave evidence that in OBs, gap junctions consist mainly of cx43. The presence of cx43 in cultured OB was also confirmed by Western blot analysis. Dye-coupling with Lucifer yellow led to a staining of up to 30 neighboring cells. Parallel intracellular recordings showed that membrane potential amplitude changes (4-5 mV) are typically related to those in the coupled cells. Thus, there is morphological and functional evidence for intercellular communication between OB in culture. OBs in culture express the same connexins as observed in vivo and may serve as a model to investigate electrophysiological events in response to different stimulation signals.

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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 GABA A receptors) have been identified, there is no report on how harmala alkaloids interact with voltagegated membrane channels. 3 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 patchclamp 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 I Ca(V) , I Na(V) and I K(V) concentration-dependent (10-500 mM) over the voltage range tested. I Ca(V) was reduced with an IC 50 of 100.6 mM for harmaline and by a significantly lower concentration of 75.8 mM (Po0.001, t-test) for harmane. The Hill coefficient was close to 1. Threshold concentration was around 10 mM for both substances. 5 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. 6 It was mainly due to a reduction in the sustained calcium channel current (I Ca (L þ N) ), while the transient voltage-gated calcium channel current (I Ca(T) ) was only partially affected. 7 We conclude that harmaline and harmane are modulators of I Ca(V) in vitro. This might be related to their neuroprotective effects.

For a detailed analysis of the oxygen supply of hippocampal slices, tissue PO2 (Pt,O2) was recorded polarographically in the neural layers of thick and thin slice preparations from the guinea pig. The experiments showed that the Pt,O2-gradients were extremely steep in the outer zones of vital slices. In an air equilibrated salt solution the surface PO2 was reduced to less than 50% within ca. 25 micron. Minimum values were measured at a depth of ca. 150 micron. A rise of temperature lowered the oxygen supply in the deeper layers of the excised tissue. An elevation of the surface PO2 hardly improved Pt,O2 in the deep structures, because the O2-consumption of the hippocampal slices increased with rising PO2.

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 (pH i ) that might develop along with sulthiame's anticonvulsant properties.Methods: The effects of sulthiame (a) on pH i of 2Ј,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymetyl ester (BCECF-AM) loaded CA3 neurones as well as (b) on epileptiform activity (induced by 50 M 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 ‫ס‬ 8) as well as the membrane permeant acetazolamide (0.5-1.0 mM; n ‫ס‬ 6) reversibly decreased pH i 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 pH i (n ‫ס‬ 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 ‫ס‬ 9, n ‫ס‬ 7), whereas benzolamide (1.0-2.0 mM) had no effect (n ‫ס‬ 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.

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.