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J M Ritchie

Publications and source records attributed to J M Ritchie.

At least 37 records · Page 2Linked to original sources
PubMed1993

Two types of 4-aminopyridine-sensitive potassium current in rabbit Schwann cells.

1. Delayed rectifier K+ currents were studied in Schwann cells cultured from neonatal rabbit sciatic nerves with the whole-cell patch-clamp technique. 2. Depolarizing voltage steps (40 ms duration) activated two types of K+ current: type I, whose apparent activation threshold was about -60 mV (half-maximal conductance at -40 +/- 1 mV, n = 10); and type II, whose apparent activation threshold was about -25 mV (half-maximal conductance at + 11 +/- 1 mV, n = 9). 3. Type I current was blocked by alpha-dendrotoxin (alpha-DTX) with an apparent equilibrium dissociation constant (KD) of 1.3 nM, whereas the type II current was unaffected by exposure to 500 nM toxin. The action of alpha-DTX on the type I current was reversible. 4. Most cells exhibited both types of current, but occasionally some cells displayed just type I or just type II. 5. Type I current activated rapidly and then showed a much slower fade, which became more noticeable with larger depolarizations. Activation of type II current was slower than that of type I and depended less steeply on voltage. The time constants of activation for type I and type II currents were derived with a Hodgkin-Huxley formalism (based on second-power activation and deactivation kinetics). The longest activation time constant for type II gating was more than twice the corresponding time constant for type I; however, the time constants determined from tail current decays at potentials more negative than -60 mV were shorter for the type II currents than for the type I currents. 6. Both type I and type II currents were sensitive to micromolar concentrations of 4-aminopyridine (4-AP). The KD for 4-AP blockade of type II current was 630 microM (pH 7.2, membrane potential (Em) = -10 mV), which is about 6 times higher than the corresponding value for 4-AP blockade of type I current at negative membrane potentials. The differential sensitivity of the type I and type II currents to 4-AP may account for the apparent voltage dependence of 4-AP block of delayed rectifier K+ currents. 7. In addition to types I and II, a third type of outward K+ current (type III) was generated in most cells at positive membrane potentials. This latter current was insensitive to millimolar concentrations of 4-AP. 8. Similarities between Schwann cell and neuronal potassium channels are discussed.

4-Aminopyridine↗
PubMed1992

On the block of outward potassium current in rabbit Schwann cells by internal sodium ions.

Currents through delayed rectifier-type K+ channels in Schwann cells cultured from rabbit sciatic nerve were studied with patch-clamp techniques. When the internal and external solutions contained physiological concentrations of sodium, the amplitude of these outward currents declined as the cell was depolarized to potentials above about +40 mV, despite the increased driving force. This reduction in the amplitude of outward K+ currents was observed in many cells before the subtraction of leakage currents; it was also observed for ensemble currents recorded in outside-out patches. It was therefore not the result of a leak-subtraction artefact nor of inadequate voltage-clamp control. Several lines of evidence also suggested that it was not the result of the extracellular accumulation of K+. By contrast, when the Na+ ion concentration of the internal solution was nominally zero, the reduction in the amplitude of outward K+ currents at positive membrane potentials was not observed. The apparent amplitude of single-channel currents through two types of K+ channel was reduced by 30 mM internal Na+, apparently as the result of a rapid 'flickery' block. The results suggest that channel block by internal Na+ is largely responsible for the negative slope conductance seen in current-voltage plots of whole-cell K+ currents at positive membrane potentials. In addition, our analysis of single-channel currents suggests that the current-voltage curve for a delayed rectifier channel in rabbit Schwann cells (in the absence of internal Na+) is roughly linear with internal and external K+ concentrations of 140 mM and 5.6 mM, respectively.

Animals↗
PubMed1992

Restoration of conduction and growth of axons through injured spinal cord of neonatal opossum in culture.

The ability of neurons in the central nervous system to grow through a lesion and restore conduction has been analyzed in a developing spinal cord. The preparation consists of the entire central nervous system of the newly born opossum (Monodelphis domestica), isolated and maintained in culture. Cell division, cell migration, and reflexes are maintained in such preparations for up to 8 days in culture. In the present experiments, massive lesions were produced by crushing the spinal cord, which abolished all conduction for a day. By 2-3 days after injury, electrical conduction across the crush could be observed. After 4-5 days, clear recovery had occurred: the amplitude of the conducted volley was comparable to that in acute preparations. In such preparations, the spinal cord had largely regained its normal appearance at the crush site. Axons stained by carbocyanine dyes or horseradish peroxidase had, by 4 days, grown in profusion through the lesion and several millimeters beyond it. These experiments demonstrate that neurons in the central nervous system of newly born mammals, unlike those in adults, can respond to injury by rapid and extensive outgrowth in the absence of peripheral nerve bridges or antibodies that neutralize inhibitory factors of myelin. With rapid and reliable regeneration occurring in vitro, it becomes practicable to assay the effects of molecules that promote or inhibit the restoration of functional connections.

Action Potentials↗
PubMed1992

Voltage-gated ion channels in Schwann cells and glia.

In the past decade, particularly its last half, it has become clear that the satellite cells of the nervous system (oligodendrocytes and astrocytes in the CNS, Schwann cells in the PNS) are liberally endowed with a vast array of voltage-gated ion channels. These original observations of the various channel types have been followed by the elucidation in considerable detail of the electrophysiological characteristics of the channels. Virtually all of the types of voltage-gated channel found in neurons have now also been found in the various populations of satellite cells. In spite of this, the intriguing questions concerning the physiological function of these channels remain to be fully elucidated.

Animals↗
PubMed1992

Multiple kinetic components of sodium channel inactivation in rabbit Schwann cells.

1. We have studied the kinetics of inactivation of sodium currents evoked in Schwann cells from neonatal and adult rabbits with patch-clamp recording techniques. The decay both of whole-cell currents and of ensemble currents obtained from outside-out patches was reasonably well-described by single exponential fits which gave values for the time constant, tau h, similar to those found for such currents in nerve (about 0.5 ms at 0 mV). Although inclusion of an additional exponential component usually improved the fits to the decay of these currents, the relative amplitude of the slower component (time constant 3-6 ms) was always small. 2. The time course of recovery from steady-state inactivation clearly consisted of two exponential components. At -120 mV, recovery from steady-state inactivation at -50 mV consisted of a fast component with a time constant of 2.2 +/- 0.2 ms and a much slower component with a time constant of 1.2 +/- 0.2 s (n = 9). The relative amplitude of the slow component (expressed as a fraction of the sodium current when inactivation was removed completely) was 0.56 +/- 0.03. The corresponding amplitudes of the slow component when similar experiments were done from holding potentials of 0 and -70 mV were 0.80 +/- 0.04 and 0.36 +/- 0.03, respectively (n = 5 and 8). 3. The onset of steady-state inactivation also followed a bi-exponential time course. The time constant of the slower component was similar at each potential examined (0, -50 and -70 mV), being 6-7 s. The relative amplitude of the slow component of onset depended on membrane potential, and it was similar (at each potential examined) to the corresponding amplitude of the slow component of recovery. 4. The inclusion of 40 mM-iodate ions in the pipette solution slowed the decay of whole-cell sodium currents, as did the extracellular application of venom (10 micrograms ml-1) from the scorpion Leiurus quinquestriatus. Prolonged exposure of the cells to Leiurus venom appeared to increase the steady-state amount of slow inactivation. 5. Records of single-channel sodium currents tended to cluster into records which either did, or did not, contain openings. This apparently non-random behaviour depended on membrane potential, and on the frequency at which the test steps were repeated, in the way expected if it resulted from slow inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

On the active form of 4-aminopyridine: block of K+ currents in rabbit Schwann cells.

1. The blocking action of 4-aminopyridine (4-AP) on the outward potassium currents evoked by depolarization of rabbit Schwann cells in short-term primary culture was studied with the whole-cell configuration of the patch-clamp method. 2. We have determined the apparent equilibrium dissociation constant, K, for the action of 4-AP to block potassium currents at a series of different extracellular and intracellular pH values. 4-Aminopyridine is an organic base and exists in both charged and uncharged forms in aqueous solution. Changes in the pH of the extracellular and intracellular solutions therefore also change the extracellular and intracellular proportions of these two forms, and the values of K that were obtained were found to depend in a consistent way on both the extracellular and the intracellular pH. 3. At alkaline extracellular pH, K was decreased. At acidic extracellular pH, K was increased. In contrast, increasing the intracellular pH from 7.2 to 8.1 reduced the apparent potency of extracellularly applied 4-AP (i.e. increased K), and decreasing the intracellular pH (to 6.4) increased this apparent potency (i.e. decreased K). 4. The 4-AP analogues, 2-aminopyridine, 3-aminopyridine and 3,4-diaminopyridine, were also tested. At half-block of the potassium current, the intracellular concentration of the cationic form of the various aminopyridines (applied extracellularly at pH 7.2) varied by a factor of less than five, whereas that of the uncharged form varied by a factor of over 700. 5. The results are inconsistent with the hypothesis that the cationic form of the aminopyridines, acting from the extracellular solution, contributes in any substantial way to potassium channel block. It also seems unlikely that the uncharged form, acting either extracellularly or intracellularly, is solely responsible for the block. However, the results as a whole are consistent with the idea that it is the cation acting from the intracellular side that blocks the 4-AP-sensitive potassium channel and that the affinity with which 4-AP blocks the channel depends on the intracellular pH. The results would be explained if the cation competes with protons for a binding site that has an apparent pKa of about 7.0. 6. The results, nevertheless, are not inconsistent with the possibility that both the uncharged form and the intracellular charged form of 4-AP are active in blocking the 4-AP-sensitive potassium channel.(ABSTRACT TRUNCATED AT 400 WORDS)

4-Aminopyridine↗
PubMed1990

Sodium channels in the cytoplasm of Schwann cells.

Immunoblotting, ultrastructural immunocytochemistry, and tritiated saxitoxin ([3H]STX) binding experiments were used to study sodium channel localization in Schwann cells. Polyclonal antibody 7493, which is directed against purified sodium channels from rat brain, specifically recognizes a 260-kDa protein corresponding to the alpha subunit of the sodium channel in immunoblots of crude glycoproteins from rat sciatic nerve. Electron microscopic localization of sodium channel immunoreactivity within adult rat sciatic nerves reveals heavy staining of the axon membrane at the node of Ranvier, in contrast to the internodal axon membrane, which does not stain. Schwann cells including perinodal processes also exhibit antibody 7493 immunoreactivity, localized within both the cytoplasm and the plasmalemma of the Schwann cell. To examine further the possibility that sodium channels are localized within Schwann cell cytoplasm, [3H]STX binding was studied in cultured rabbit Schwann cells, both intact and after homogenization. Saturable binding of STX was significantly higher in homogenized Schwann cells (410 +/- 37 fmol/mg of protein) than in intact Schwann cells (214 +/- 21 fmol/mg of protein). Moreover, the equilibrium dissociation constant was higher for homogenized preparations (1.77 +/- 0.37 nM) than for intact Schwann cells (1.06 +/- 0.29 nM). These data suggest the presence of an intracellular pool of sodium channels or channel precursors in Schwann cells.

Animals↗
PubMed1990

Sodium currents in Schwann cells from myelinated and non-myelinated nerves of neonatal and adult rabbits.

1. Patch-clamp methods were used to study sodium channels in Schwann cells obtained from four different tissue sources. Primary cultures of Schwann cells were prepared from the sciatic nerve and from the vagus nerve of neonatal and of adult rabbits. In the adult, the sciatic is predominantly myelinated whereas the vagus is predominantly non-myelinated. Whole-cell currents, and single-channel currents in outside-out membrane patches, were analysed. 2. No substantial differences were noted in the passive electrical properties (input resistance, cell capacitance, resting membrane potential) of the four groups of cells. Similarly, no substantial differences were found in the average properties of sodium currents (maximum current, maximum conductance, time-to-peak current, current-voltage relation, h infinity relation) recorded from each type of cell in cultures less than 8 days old. At 10-17 days a fall in the size of the sodium currents recorded from cells in the vagal cultures was found. 3. Exposure of the cells to proteolytic enzymes or collagenase, under conditions similar to those used when the cells were put in culture initially, substantially reduced the size of the peak sodium currents recorded from the cells 24 h later. 4. The results of experiments on Schwann cells with retracted processes indicated that sodium channels are present in the processes extending from each pole of the cell soma and that the plasmalemmal density of these channels in the processes is about the same as it is at the soma. 5. Recordings from outside-out patches revealed no apparent differences in the properties of single-channel sodium currents in patches from cells obtained from the four different sources. The single-channel conductance was about 20 pS for each of the four groups. Ensemble currents from single-channel records were similar in time course to those of whole-cell currents. 6. Saxitoxin reduced the maximum sodium conductance in Schwann cells and bound to the cells with equally high affinity. The equilibrium dissociation constant was about 2 nM at 20-22 degrees C. 7. It is argued that the expression of sodium channels in myelinating Schwann cells does not differ substantially from that of non-myelinating Schwann cells.

Animals↗
PubMed1989

Low density of sodium channels supports action potential conduction in axons of neonatal rat optic nerve.

The density of sodium channels in premyelinated axons was estimated from measurements of the binding of [3H]saxitoxin to neonatal rat optic nerve. The maximum saturable binding capacity of the nerve was 16.2 +/- 1.2 fmol/mg of wet weight, with an equilibrium dissociation constant of 0.88 +/- 0.18 nM (mean +/- SEM). These values correspond to a high-affinity saxitoxin-binding site density of approximately 2/microns 2 within premyelinated axon membrane. Action potential propagation in neonatal rat optic nerve is completely blocked by 5 nM saxitoxin, indicating that action potential electrogenesis is mediated by channels that correspond to high-affinity saxitoxin-binding sites. These results demonstrate that action potential conduction is supported by a low density of sodium channels in this system. Since the internodal axon membrane of myelinated fibers may contain a low density of sodium channels, it is possible that restoration of conduction in some demyelinated fibers may not require additional sodium channel incorporation into the demyelinated axon membrane.

Action Potentials↗
PubMed1988

Two types of potassium current in rabbit cultured Schwann cells.

Voltage-gated outward currents were studied in rabbit cultured Schwann cells with the 'whole-cell' configuration of the patch-clamp method. Four components of such currents were identified. The first, which was abolished by replacement of the external chloride ions by the large impermeant anion gluconate, was identified as a chloride current. The second and third were identified as potassium currents. One type of potassium current was reduced substantially by either 4-aminopyridine (4-AP) or tetraethylammonium ion (TEA). Its sensitivity to blocking by 4-AP was highly voltage-dependent: the equilibrium dissociation constant (K) was threefold greater when measured at +10 mV than when measured at -40 mV (where it was about 80 microM). The second type of potassium current was relatively insensitive to 4-AP, but was blocked by TEA. The TEA sensitivity of the two types of potassium currents was similar and displayed no obvious voltage-dependence (K approximately 200 microM). The fourth component of current was not reduced by 4-AP or TEA at concentrations less than 10 mM. Whether or not this last component is a potassium current is unclear.

Animals↗
PubMed1988

Sodium-channel turnover in rabbit cultured Schwann cells.

Radiolabelled saxitoxin has been used as a chemical marker for the voltage-dependent sodium channels expressed in the plasmalemma of rabbit Schwann cells in culture. Proteolytic enzymes destroy this saxitoxin-binding capacity, which gradually reappears with an exponential time constant of about 3.1 days. Exposure of cultured Schwann cells to tunicamycin, an inhibitor of glycosylation, leads to a progressive exponential fall in saxitoxin-binding capacity, again with a time constant of about 3.1 days. The assumption that the steady-state density of Schwann cell sodium channels is maintained by a constant synthesis of channels in the face of a rate of loss from the membrane proportional to the amount of channel already present, leads to the conclusion that these channels have an average lifetime of about 3.1 days. The metabolic consequences of this rapid turnover of Schwann cell sodium channels is discussed.

Animals↗
PubMed1988

Depolarization of nonmyelinated fibers of the rat vagus nerve produced by activation of protein kinase C.

The effect of activation of protein kinase C by phorbol esters has been studied on the nonmyelinated (C) fibers of the rat vagus nerve. Grease-gap recording at room temperature was used to monitor changes in the resting and action potentials. Effects of phorbol esters on the rate of efflux of 86Rb and 14C-guanidinium were also measured. The active isomer beta-phorbol 12,13-dibutyrate (PDBu), applied for 10 min at concentrations of 10 nM to 3 microM, caused a slowly developing depolarization, which persisted after the drug was washed out. The action potential was concomitantly reduced. These effects did not occur with the inactive isomer alpha-phorbol 12,13-didecanoate. The PDBu-induced depolarization was reduced by about 75% if Na+ was replaced by the impermeant cation N-methyl-(+)-glucamine (NMG); the residual effect was almost abolished if the nerves were presoaked in a solution containing gluconate in place of Cl-. It was concluded that increases in conductance mainly to Na+ and Cl- were responsible for the depolarization. The response was unaffected by tetrodotoxin or calcium-channel blockers. Omission of Ca2+, surprisingly, enhanced the PDBu-induced depolarization 3-5-fold; furthermore, addition of 2 mM Ca2+ following a PDBu-induced depolarization recorded in Ca2+-free solution caused a pronounced repolarization. This effect of Ca2+ occurred also with Sr2+ and Ba2+, but not with other divalent cations or with La3+. Divalent cations known to block Ca channels inhibited the repolarizing action of Ca2+. These results suggested that Ca2+ acts intracellularly, either to block Na channels opened by PDBu or to activate protein phosphatases. The PDBu-induced response in Ca2+-free solution was increased 2-fold by a reduction in pH from 7.4 to 6.5. Under normal conditions the nerve was reversibly depolarized by this pH change; after PDBu this pH sensitivity was enhanced, and depolarization occurred at a less acidic pH. PDBu caused a 3-4-fold increase in the rate of efflux of 86Rb (a marker for K+ ions) and of 14C-guanidinium (a marker for Na+ ions) from preloaded nerves. These effects, in contrast to the depolarization, were transient.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Optical recording of action potential propagation in demyelinated frog nerve.

Conduction in focally demyelinated frog nerves has been measured optically using potential-sensitive dyes. Absorption changes were recorded with an array of photodiodes positioned in the image plane of a microscope. Both the amplitude and conduction velocity of the optical signals decreased in the demyelinated region. Conduction was improved after exposure to the potassium channel blocking agent 4-aminopyridine.

Action Potentials↗
PubMed1987

Voltage-gated cation and anion channels in mammalian Schwann cells and astrocytes.

This article reviews some recent studies on the voltage-gated ion channels in the plasmalemma of the satellite cells of the peripheral and central nervous systems. Following the finding that rabbit Schwann cells exhibit a large binding capacity for saxitoxin (Ritchie and Rang, 1983) electrophysiological studies have shown that these cells not only express plasmalemmal voltage-gated sodium channels but also voltage-gated potassium channels (Chiu, Shrager and Ritchie, 1984; Shrager, Chiu and Ritchie, 1985). Whole-cell recording with the patch-clamp method reveals that the properties of these two kinds of channel are quite similar to those of the corresponding channels in the nodal axolemma, except that the peak current-voltage relation of the sodium channels is shifted about 30 mV in the depolarizing direction. Similar voltage-gated sodium and potassium channels exist in rat cultured astrocytes (Bevan et al., 1985). Furthermore, the outward current on depolarization in astrocytes has a component other than that carried in the potassium channels. About 75% of the total outward current is blocked by external TEA or internal caesium; and it is presumed to be a potassium current. The remainder, however, is insensitive to these potassium channel blocking agents; but it is abolished by exposure to the two stilbene sulphonates, DIDS and SITS (Gray and Ritchie, 1986). This remaining current persists in the presence of the large organic cation N-methyl-(+)-glucamine in the patch pipette. It is, however, reduced when the chloride of the external medium is replaced by methyl-sulphate, sulphate, or isethionate; and it is abolished when the external anion is gluconate (M.W., 190). The TEA-insensitive component of outward current in astrocytes thus seem to involve an influx of chloride ions through a voltage-gated channel whose diameter is such that anions larger than gluconate cannot pass. The current in the channels is neglible at potentials more negative than about -40 mV.

Animals↗
PubMed1986

A voltage-gated chloride conductance in rat cultured astrocytes.

Large voltage-dependent outward currents are recorded with the whole-cell patch-clamp technique from rat cultured astrocytes under conditions where an outward movement of potassium ions is excluded (either by blockage of the potassium channels pharmacologically or by replacement of the internal potassium by the impermeant large organic cation N-methyl-(+)-glucamine). The current, which is activated at potentials more positive than -40 to -50 mV, is normally carried by an inward movement of chloride ions. Its reversal potential is the same as the chloride equilibrium potential. With depolarization to +60 mV (for 225 ms) little or no inactivation of the current occurs: with depolarizations to +90 to +110 mV a time-dependent decay is seen. The current, which is often not marked immediately after formation of the whole-cell clamp, generally increases over a period of a few minutes to a maximum (after which it usually declines), as if some as yet unknown intracellular factor keeping the channels closed were being washed away from the membrane. The time course of this phenomenon is not affected by changing of the internal free calcium concentration (from 10(-8)M to 10(-6)M) or by an intracellular mixture of cyclic AMP (1 mM), ATP (4 mM) and Mg+ (2 mM). The conductance is slightly increased when the chloride of the bathing medium is replaced by bromide; is much reduced on replacement by methylsulphate, sulphate, isethionate, or acetate; and is virtually abolished on replacement by the large anion gluconate. The outward current is inhibited by the disulphonate stilbenes DIDS and SITS; this blocking action was initially partly reversible, although never completely so. It is suggested that the chloride conductance plays a role in the spatial buffering of potassium by astrocytes.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1986

Ion channels in rabbit cultured fibroblasts.

Large outward currents are recorded with the whole-cell patch-clamp technique on depolarization of rabbit cultured fibroblasts. Our findings suggest that these outward currents consist of two voltage-dependent components, one of which also depends on cytoplasmic calcium concentration. Total replacement of external Cl- by the large anion ascorbate does not affect the amplitude of the currents, indicating that both components must be carried by K+. Consistent with these findings with whole-cell currents, in single channel recordings from fibroblasts we found that most patches contain high-conductance potassium-selective channels whose activation depends on both membrane potential and the calcium concentration at the cytoplasmic surface of the membrane. In a smaller number of patches, a second population of high-conductance calcium-independent potassium channels is observed having different voltage-dependence. The calcium- and voltage-dependence suggest that these two channels correspond with the two components of outward current seen in the whole-cell recordings. The single channel conductance of both channels in symmetrical KCl (150 mM) is 260-270 pS. Both channels are highly selective for K+ over both Na+ and Cl-. The conductance of the channels when outward current is carried by Rb+ is considerably smaller than when it is carried by K+. Some evidence is adduced to support the hypothesis that these potassium channel populations may be involved in the control of cell proliferation.

4-Aminopyridine↗