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P Grafe

Publications and source records attributed to P Grafe.

At least 37 records · Page 2Linked to original sources
PubMed1994

The effects of hyperglycaemic hypoxia on rectification in rat dorsal root axons.

1. Electrotonic responses to 150 ms current pulses were recorded from isolated rat dorsal roots incubated for at least 3 h with either normal (5 mM) or high (25 mM) D-glucose solutions, and with either normal (25 mM) or low (5 mM) bicarbonate concentrations. 2. On replacement of O2 by N2 for 50 min, all the roots depolarized, but the changes in electrotonus differed systematically. With normal glucose, the depolarization was accompanied by an increase in input conductance. In contrast, for the hyperglycaemic roots the depolarization was slower and accompanied by a fall in input conductance which was exacerbated in low bicarbonate concentrations. 3. The changes induced by hyperglycaemic hypoxia in low bicarbonate could be mimicked by exposure of the roots either to 100% CO2 or to a combination of 3 mM tetraethylammonium chloride and 3 mM 4-aminopyridine, to block both fast and slow potassium channels. 4. These results indicate that the primary mechanism of hypoxic depolarization of these sensory axons is altered by hyperglycaemia. In normoglycaemia, the changes in electrotonus are consistent with an increase in axonal potassium conductance. The block of potassium channels seen in hyperglycaemic hypoxia is attributed to intra-axonal acidification by anaerobic glycolysis and may contribute to the pathogenesis of diabetic neuropathy.

4-Aminopyridine↗
PubMed1994

Alkalinization during re-oxygenation prevents functional damage by hyperglycaemic hypoxia.

Hyperglycaemia impairs recovery from transient cerebral ischaemia: the importance of tissue acidification for this phenomenon has not been clarified in detail. We investigated this issue in a less complex in vitro preparation of isolated rat dorsal spinal roots exposed for 30 min to hyperglycaemic hypoxia. Peak height of compound action potentials recovered minimally in 5 mM bicarbonate. However, recovery was greatly improved by addition of the weak base trimethylamine during re-oxygenation. Addition of the weak acid propionate had no such effect. Cytoplasmic alkalinization improved recovery in a brief time window only: application of trimethylamine after 15 min of re-oxygenation was without beneficial effect. These data emphasize the importance of cytoplasmic acidification for neurophysiological recovery from hyperglycaemic hypoxia during the initial period of re-oxygenation.

Action Potentials↗
PubMed1994

Extracorporeal shock waves stimulate frog sciatic nerves indirectly via a cavitation-mediated mechanism.

Shock waves (SWs) are single pressure pulses with amplitudes up to over 100 MPa, a rise time of only a few nanoseconds, and a short duration of approximately 2 microseconds. Their clinical application for stone destruction causes pain, indicating nerve stimulation by SWs. To examine this phenomenon, sciatic nerves of frogs were exposed to SWs in an organ bath. The SWs were generated with an experimental Dornier lithotripter model XL1 at an operating voltage of 15 kV. The nerves were mounted in a chamber which allowed electrical nerve stimulation and the registration of electrically and SW-induced compound action potentials (SWCAPs). The chamber was filled with frog Ringer's solution. In a standardized protocol. The first experiment established that 95.0 +/- 4.7% of administered SWs induced action potentials which were lower in amplitude (1.45 +/- 1.14 versus 1.95 +/- 0.95 mV, p = 0.004) but similar in shape to electrically induced compound action potentials. In a second experiment, it was shown that the site of origin of the SWCAPs could be correctly determined by simultaneous recording of action potentials at both ends of the nerve. The mechanism of shock wave stimulation was examined by experiments 3 and 4. In experiment 3, in contrast to the previous experiments, SW exposure of the nerves was performed 6 cm outside the shock wave focus. This resulted in a mean probability of inducing a SWCAP of only 4%. After gas bubble administration, this probability increased to 86% for the first SW released immediately after bubble application and declined to 56% for the second, 21% for the third, to 0 for the 10th SW after fluid injection. This indicates that cavitation, the interaction between shock waves and gas bubbles in fluid or tissues, was involved in SWCAP generation. In experiment 4, nerves were again exposed in the focus, however, the Ringer's solution surrounding the nerve was replaced by polyvinyl alcohol (PVA). PVA is a solution with low cavitation activity.In PVA, the excitability was markedly diminished to 11.0 +/- 5.1% compared with 96.0 +/- 4.4% in control nerves exposed in Ringer's solution. In conclusion, bioeffects of SWs on nervous tissue appear to result from cavitation. It is suggested that cavitation is also the underlying mechanism of SW-related pain during extracorporeal SW lithotripsy in clinical medicine.

Animals↗
PubMed1993

A Ca(2+)- and pH-dependent K+ channel of rat C6 glioma cells and its possible role in acidosis-induced cell swelling.

The aim of the present study was to explore whether a change in membrane K+ conductance contributes to acidosis-induced swelling of cultured rat C6 glioma cells. Electrophysiological studies were performed using whole-cell and single-channel recordings in combination with cell volume measurements in cell suspension by flow cytometry. Whole-cell recordings revealed a voltage-dependent K+ conductance. The predominant K+ channel in single-channel recordings with symmetrical high K+ concentrations was inwardly rectifying and had conductances of 35 and 15 pS, respectively. A raised internal free Ca2+ concentration and membrane depolarization increased the open probability of this channel. Internal acidosis (pH 6.4-5.4), on the other hand, reduced open probability and single-channel conductance. Both whole-cell and single-channel K+ currents were blocked by quinidine (0.1-1 mM), which was therefore used to analyze the functional consequences of an inhibition of this conductance for cell volume. Thereby, quinidine (1 mM) produced a small (5%) and transient cell swelling of C6 glioma cells. In contrast, acidosis (pH 5.6) caused a much larger (about 20%) and maintained swelling. Since quinidine produced only a minor swelling of C6 cells, it is unlikely that inhibition of the K+ conductance caused acidosis-induced cell swelling. Other mechanisms, such as activation of ion transporters, must therefore be responsible.

Acidosis↗
PubMed1993

Sodium channel inactivation kinetics of rat sensory and motor nerve fibres and their modulation by glutathione.

Na+ channel currents of rat motor and sensory nerve fibres were studied with the patch-clamp technique on enzymatically demyelinated axons. Differences between motor and sensory fibres in multi-channel inactivation kinetics and the gating of late single-channel currents were investigated. In the axon-attached mode, inactivation of multi-channel Na+ currents in sensory axons was best fitted with a single time constant while for motor axons two time constants were needed. Late single-channel currents in sensory axons were characterized by short openings whereas motor axons exhibited additional long single-channel openings. In contrast, in excised, inside-out membrane patches, no differences between motor and sensory fibres were found; in both types of fibre inactivation of multi-channel Na+ currents proceeded with two time constants and late single-channel currents showed short and long openings. After application of the reducing agent glutathione to the cytoplasmic side of excised inside-out patches, inactivation of Na+ currents in both motor and sensory fibres proceeded with a single, fast exponential time constant and late currents appeared with short openings only. These data indicate that the axonal metabolism may contribute to the different inactivation kinetics of Na+ current in motor and sensory nerve fibres.

Animals↗
PubMed1993

Lactate-proton co-transport and its contribution to interstitial acidification during hypoxia in isolated rat spinal roots.

Exposure of nervous tissue to hypoxia results in interstitial acidification. There is evidence for concomitant decrease in extracellular pH to the increase in tissue lactate. In the present study, we used double-barrelled pH-sensitive microelectrodes to investigate the link between lactate transport and acid-base homeostasis in isolated rat spinal roots. Addition of different organic anions to the bathing solution at constant bath pH caused transient alkaline shifts in extracellular pH; withdrawal of these compounds resulted in transient acid shifts in extracellular pH. With high anion concentrations (30 mM), the largest changes in extracellular pH were observed with propionate > L-lactate approximately pyruvate > 2-hydroxy-2-methylpropionate. Changes in extracellular pH induced by 10 mM L- and D-lactate were of similar size. Lactate transport inhibitors alpha-cyano-4-hydroxycinnamic acid and 4,4'-dibenzamidostilbene-2,2'-disulphonic acid significantly reduced L-lactate-induced extracellular pH shifts without affecting propionate-induced changes in extracellular pH. Hypoxia produced an extracellular acidification that was strongly reduced in the presence of alpha-cyano-4-hydroxycinnamic acid and 4,4'-dibenzamidostilbene-2,2'-disulphonic acid. In contrast, amiloride and 4,4'-di-isothiocyanostilbene-2,2'-disulphonate were without effect on hypoxia-induced acid shifts. The results indicate the presence of a lactate-proton co-transporter in rat peripheral nerves. This transport system and not Na+/H+ or Cl-/HCO3- exchange seems to be the dominant mechanism responsible for interstitial acidification during nerve hypoxia.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

Hyperglycaemic hypoxia alters after-potential and fast K+ conductance of rat axons by cytoplasmic acidification.

1. The effects of hyperglycaemic hypoxia (a condition possibly involved in the pathogenesis of diabetic neuropathy) on the depolarizing after-potential and the potassium conductance of myelinated rat spinal root axons were investigated using electrophysiological recordings from intact spinal roots and from excised, inside-out axonal membrane patches. 2. Isolated spinal roots were exposed to hypoxia in solutions containing normal or high glucose concentrations. The depolarizing after-potential of compound action potentials was only enhanced in spinal roots exposed to hyperglycaemic (25 mM D-glucose) hypoxia. A maximal effect was seen in bathing solutions with low buffering power. 3. The depolarizing after-potential was also enhanced by cytoplasmic acidification after replacement of 10-30 mM chloride in the bathing solution by propionate. 4. Multi-channel current recordings from excised, inside-out axonal membrane patches were used to study the effects of cytoplasmic acidification on voltage-dependent K+ conductances with fast (F channels) and intermediate (I channels) kinetics of deactivation. 5. F channels were blocked by small changes in cytoplasmic pH (50% inhibition at pH 6.9). I channels were much less sensitive to intra-axonal acidification. 6. In conclusion, our data show that hyperglycaemic hypoxia enhances the depolarizing after-potential in peripheral rat axons. The underlying mechanism seems to be an inhibition of a fast, voltage-dependent axonal K+ conductance by cytoplasmic acidification. This alteration in membrane conductance may contribute to positive symptoms in diabetic neuropathy.

4-Aminopyridine↗
PubMed1993

The paradox between resistance to hypoxia and liability to hypoxic damage in hyperglycemic peripheral nerves. Evidence for glycolysis involvement.

Isolated ventral and dorsal rat spinal roots incubated in normal (2.5 mM) or high glucose (25 mM) concentrations or in high concentrations of other hexoses were exposed transiently to hypoxia (30 min) in a solution of low buffering power. Compound nerve action potentials, extracellular direct current potentials, and interstitial pH were continuously recorded before, during, and after hypoxia. Ventral roots incubated in 25 mM D-glucose showed resistance to hypoxia. Dorsal roots, on the other hand, revealed electrophysiological damage by hyperglycemic hypoxia as indicated by a lack of posthypoxic recovery. In both types of spinal roots, interstitial acidification was most pronounced during hyperglycemic hypoxia. The changes in the sensitivity to hypoxia induced by high concentrations of D-glucose were imitated by high concentrations of D-mannose. In contrast, D-galactose, L-glucose, D-fructose, and L-fucose did not have such effects. Resistance to hypoxia, hypoxia-generated interstitial acidification, and hypoxia-induced electrophysiological damage were absent after pharmacological inhibition of nerve glycolysis with iodoacetate. These observations indicate 1) that enhanced anaerobic glycolysis produces resistance to hypoxia in hyperglycemic peripheral nerves and 2) that acidification may impair the function of peripheral axons when anaerobic glycolysis proceeds in a tissue with reduced buffering power.

Action Potentials↗
PubMed1992

Differences in sensitivity to hyperglycemic hypoxia of isolated rat sensory and motor nerve fibers.

We explore whether the prevalence of sensory deficits in diabetic neuropathy can be explained by diffuse endoneurial hypoxia. Isolated ventral and dorsal rat spinal roots incubated in 2.5 or 25 mM extracellular glucose were transiently exposed to hypoxia (30 min) in a solution of low buffering power. Compound nerve action potentials and extracellular direct current potentials were continuously recorded before, during, and after hypoxia. In both ventral and dorsal roots incubated in 2.5 mM glucose, sensitivity to hypoxia and posthypoxic recovery were similar. In contrast, hypoxia in 25 mM glucose preferentially induced electrophysiological damage in dorsal roots as indicated by a lack of posthypoxic recovery. This observation was not made in the presence of 25 mM bicarbonate, which suggests involvement of nerve acidosis. In conclusion, the different sensitivity of sensory and motor fibers to hyperglycemic hypoxia supports the hypothesis that hypoxia has an important role in the pathogenesis of diabetic neuropathy.

Action Potentials↗
PubMed1992

High conductance anion channel in Schwann cell vesicles from rat spinal roots.

Potassium uptake, possibly together with chloride, is one of the presumed functions of Schwann cells in the peripheral nervous system. However, the presence of chloride channels has not been demonstrated in adult Schwann cells. We present here a new method which allows single channel recordings to be made from Schwann cells in situ without enzymatic treatment. Isolated rat spinal roots were split mechanically into several bundles. Within about 30 min after this procedure small bleb-like vesicles (approximately 20-30 microns in diameter) with a clean surface appeared at the edges of the fibre bundles. Immunofluorescence microscopy with a surface marker for Schwann cell membranes (monoclonal antibody O4) revealed that the vesicles originate from Schwann cells. In standard patch clamp recordings with symmetrical bath and pipette solutions (excised inside-out configuration) an anion channel with the following characteristics was mainly observed: 1) single channel slope conductance of 337 +/- 5 pS in 125 mM KCl and 209 +/- 6 pS in 125 mM K+ methylsulphate; 2) ion permeability ratio: PCl/PK/Pgluconate = 1/0.12/0.06; 3) linear current-voltage relationship (range +/- 60 mV); and 4) voltage- and time-dependent inactivation (the channel was most active at potentials +/- 20 mV). Pharmacologically, the channel was completely blocked with zinc (1 mM) and barium (10 mM). A similar anion channel, showing characteristics 1-4), has been described in cultured Schwann cells of newborn rats (Gray et al., 1984). We now demonstrate that this channel is also present in adult Schwann cells in situ.

Action Potentials↗
PubMed1992

Glutathione accelerates sodium channel inactivation in excised rat axonal membrane patches.

The effects of glutathione were studied on the gating behaviour of sodium channels in membrane patches of rat axons. Depolarizing pulses from -120 to -40 mV elicited sodium currents of up to 500 pA, indicating the simultaneous activation of up to 250 sodium channels. Inactivation of these channels in the excised, inside-out configuration was fitted by two time constants (tau h1 = 0.81 ms; tau h2 = 5.03 ms) and open time histograms at 0 mV revealed a biexponential distribution of channel openings (tau short = 0.28 ms; tau long = 3.68 ms). Both, the slow time constant of inactivation and the long lasting single channel openings disappeared after addition of the reducing agent glutathione (2-5 mM) to the bathing solution. Sodium channels of excised patches with glutathione present on the cytoplasmatic face of the membrane had inactivation kinetics similar to channels recorded in the cell-attached configuration. These observations indicate that redox processes may contribute to the gating of axonal sodium channels.

Animals↗
PubMed1991

A chloride channel in rat and human axons.

Current recordings from single chloride channels were obtained from excised and cell-attached patches of rat and human axons. In rat axons the channels showed an outwardly rectifying current-voltage relationship with a slope conductance of 33 pS at negative membrane potentials and 65 pS at positive potentials (symmetrical 150 mM CsCl). They were measurably permeable for cations (PNa/PCs/PCl = 0.1/0.2/1). Channel currents were independent of cytoplasmatic calcium concentration. Inactivation was not observed and gating was weakly voltage dependent. Cl- channels in human axons showed similar gating behavior but had a lower conductance.

Animals↗
PubMed1991

Changes in excitability and accommodation of human motor axons following brief periods of ischaemia.

1. The mechanism of post-ischaemic ectopic impulse generation in nerve is not known, and previous measurements of excitability changes in human motor axons have appeared to conflict. We have used automatic threshold tracking and different stimulus-response combinations to follow the effects on excitability of brief (5-10 min) periods of ischaemia, too short to induce motor fasciculations. Excitability changes have been compared at different sites in axons innervating hand, arm and foot muscles. 2. Threshold was determined as the percutaneous stimulus current required to excite a single motor unit, or to evoke a constant multiunit response, after rectifying and integrating the electromyogram (EMG). Three different waveforms of stimulus current were compared: short (less than or equal to 2 ms) pulses, long (100-200 ms) pulses to measure rheobase, and 100 ms current ramps. We also measured accommodation by recording the effects of subthreshold depolarizing currents on excitability. 3. Ischaemic and post-ischaemic excitability changes were greatest in the proximal parts of the longest motor axons, and greater if the sphygmomanometer cuff was inflated over, rather than proximal to, the stimulating site. 4. Using integrated EMG responses from abductor digiti minimi, the ulnar nerve stimulated above the elbow became rapidly much less excitable after ischaemia when tested with short pulses, but more excitable when tested with current ramps. The rheobase rose briefly, but then fell, often below resting level, always staying below the pulse and ramp thresholds. 5. The latency of the response to a rheobasic stimulus altered in parallel with the threshold to short current pulses, and increased dramatically after ischaemia. This latency increase was associated with a prolonged phase of 'negative accommodation', i.e. the continued increase in excitability to a maintained subthreshold depolarizing current. 6. Changes in excitability and accommodation similar to those occurring after ischaemia were recorded following high frequency trains of stimuli. They were attributed primarily to hyperpolarization by the electrogenic sodium pump, since comparable changes could be induced by passing a steady hyperpolarizing current through the stimulating electrode. 7. Threshold and latency recordings from single motor units during and after ischaemia resembled in most respects the multiunit responses, but single unit rheobase did not show a post-ischaemic fall below the resting level. Repetitive firing contributed to the low multiunit thresholds recorded with long current pulses during the post-ischaemic period. 8. We conclude that human motor nerves become simultaneously both more and less excitable than normal after 10 min of ischaemia, depending on the choice of stimulus and response.(ABSTRACT TRUNCATED AT 400 WORDS)

Axons↗
PubMed1991

Glucose availability and sensitivity to anoxia of isolated rat peroneal nerve.

The contrast between resistance to ischemia and ischemic lesions in peripheral nerves of diabetic patients was explored by in vitro experiments. Isolated and desheathed rat peroneal nerves were incubated in the following solutions with different glucose availability: 1) 25 mM glucose, 2) 2.5 mM glucose, and 3) 2.5 mM glucose plus 10 mM 2-deoxy-D-glucose. Additionally, the buffering power of all of these solutions was modified. Compound nerve action potential (CNAP), extracellular pH, and extracellular potassium activity (aKe) were measured simultaneously before, during, and after a period of 30 min of anoxia. An increase in glucose availability led to a slower decline in CNAP and to a smaller rise in aKe during anoxia. This resistance to anoxia was accompanied by an enhanced extracellular acidosis. Postanoxic recovery of CNAP was always complete in 25 mM HCO3(-)-buffered solutions. In 5 mM HCO3- and in HCO3(-)-free solutions, however, nerves incubated in 25 mM glucose did not recover functionally after anoxia, whereas nerves bathed in solutions 2 or 3 showed a complete restitution of CNAP. We conclude that high glucose availability and low PO2 in the combination with decreased buffering power and/or inhibition of HCO3(-)-dependent pH regulation mechanisms may damage peripheral mammalian nerves due to a pronounced intracellular acidosis.

Action Potentials↗
PubMed1990

Cytosolic calcium during glucose deprivation in hippocampal pyramidal cells of rats.

Glucose deprivation (GD) results in a hyperpolarization by turning on a potassium conductance (gK,GD) in hippocampal CA3 pyramidal cells. We used combined intracellular and microfluorometric recording techniques to evaluate whether gK,GD is activated by a rise in the concentration of intracellular calcium ([Ca2+]i). We found that the activation of gK,GD is only followed, but not preceded by a rise in [Ca2+]i. Furthermore, gK,GD is not blocked by the sulfonylurea glibenclamide, a blocker of ATP-regulated potassium conductance. We conclude that activation of gK,GD does not simply reflect breakdown of the calcium of ATP homeostasis, but on the contrary might represent an active restoring mechanism which delays the pathological consequences of sustained glucose deficiency.

Adenosine Triphosphate↗
PubMed1990

K+ channel openers suppress myotonic activity of human skeletal muscle in vitro.

Isolated fibre bundles from myotonic human skeletal muscle showed after-contractions and spontaneous mechanical activity. The K+ channel openers cromakalim (10-100 mumols/l) and EMD 52962 (1-10 mumols/l) completely suppressed these abnormalities in mechanical activity. Voltage-clamp experiments revealed that cromakalim (100 mumols/l) increased the membrane K+ conductance of isolated, non-myotonic human skeletal muscle fibres 4-fold; Cl- conductance was not altered. The data show that myotonia is suppressed by an increase in in membrane K+ conductance.

Benzopyrans↗
PubMed1990

Electrophysiological measurements of volume changes in leech neuropile glial cells.

Double-barrelled microelectrodes, sensitive to quaternary ammonium ions, were used for simultaneous measurements of the intracellular free concentrations of choline ([Ch]i) or tetramethylammonium ([TMA]i) as well as membrane potential (Em) in neuropile glial cells of the leech, Hirudo medicinalis. Bath application of Ch or TMA (5 mM, 1 min) resulted in a transient membrane depolarization accompanied by a long-lasting (0.5-1 h) intracellular accumulation of these compounds to levels of between 5 and 15 mM. Changes in [Ch]i or [TMA]i were used for the calculation of changes in relative cell volume. Elevation of the extracellular K+ concentration [( K+]e) from 4 to 9, 15, 21, 27.5, or 40 mM elicited a membrane depolarization and a reversible cell swelling by about 7.5, 14, 18.5, 27 and 50%, whereas reduction of [K+]e to 1.5 mM as well as bath application of serotonin (5-HT) produced a membrane hyperpolarization and a concomitant shrinkage by about 6 and 14.3%, respectively. The measured alterations in cell volume were compared with calculated data based on the assumption of an osmotic equilibrium disturbed by potential-dependent changes of the intracellular Cl- concentration. The results indicate, that K(+)- and serotonin-induced changes in the cell volume of the neuropile glial cells are due to passive KCl and water fluxes.

Animals↗