Non-cholinergic effects of acetylcholinesterase in the substantia nigra: a possible role for an ATP-sensitive potassium channel

Summary Within the substantia nigra acetylcholinesterase has non-cholinergic actions that can be demonstrated at both behavioural and cellular levels: the aim of this study was, thus, to explore, in the in vitro guinea pig substantia nigra, the ionic mechanisms which mediate these non-classical phenomena. Acetylcholinesterase had a reversible hyperpolarizing action, via an opening of potassium channels, on a selective population of nigral neurons. These neurons could be identified by an ability to generate bursts of action potentials and by a sensitivity to either amphetamine or to a reduction of glucose in the perfusing medium. The acetylcholinesterase-induced hyperpolarization could not be attributed to a contaminant in the exogenous solution, since a highly purified preparation was even more potent. Furthermore, enzymatic action of any kind could be eliminated as boiled acetylcholinesterase was equally efficacious. The effect of acetylcholinesterase was not subject to tachyphylaxis and was resistant to blockade of potassium channels with tetraethylammonium: since both these phenomena are features of the D₂ autoreceptor for dopamine within the substantia nigra, it seems unlikely that acetylcholinesterase is operating on the same target as dendritically released local dopamine. On the other hand, the actions of acetylcholinesterase were enhanced by low glucose and blocked by the sulfonylurea, tolbutamide. These results strongly suggest that acetylcholinesterase can exert a nonenzymatic action and that this action, in the substantia nigra, is mediated by an ATP-sensitive potassium channel.     

Effects of intra- and extracellular H+ and Na+ concentrations on Na+-H+ antiport activity in the lacrimal gland acinar cells

Kinetic properties of the Na⁺-H⁺ antiport in the acinar cells of the isolated, superfused mouse lacrimal gland were studied by measuring intracellular pH (pH_i) and Na⁺ activity (aNa_i) with the aid of double-barreled H⁺- and Na⁺-selective microelectrodes, respectively. Bicarbonate-free solutions were used throughout. Under untreated control conditions, pH_i was 7.12±0.01 and aNa_i was 6.7±0.6 mmol/l. The cells were acid-loaded by exposure to an NH ₄ ⁺ solution followed by an Na⁺-free N-methyl-d-glucamine (NMDG⁺) solution. Intracellular Na⁺ and H⁺ concentrations were manipulated by changing the duration of exposure to the above solutions. Subsequent addition of the standard Na⁺ solution rapidly increased pH_i. This Na⁺-induced increase in pH_i was almost completely inhibited by 0.5 mmol/l amiloride and was associated with a rapid, amiloride-sensitive increase in aNa_i. The rate of pH_i recovery induced by the standard Na⁺ solution increased in a saturable manner as pH_i decreased, and was negligible at pH_i 7.2–7.3, indicating an inactivation of the Na⁺-H⁺ antiport. The apparent K _m for intracellular H⁺ concentration was 105 nmol/l (pH 6.98). The rate of acid extrusion from the acid-loaded cells increased proportionally to the increase in extracellular pH. Depletion of aNa_i to less than 1 mmol/l by prolonged exposure to NMDG⁺ solution significantly increased the rate of Na⁺-dependent acid extrusion. The rate of acid extrusion increased as the extracellular Na⁺ concentration increased following Michaelis-Menten kinetics (V _max was 0.55 pH/min and the apparent K _m was 75 mmol/l at pH_i 6.88). The results clearly showed that the Na⁺-H⁺ antiport activity is dependent on the chemical potential gradient of both Na⁺ and H⁺ ions across the basolateral membrane, and that the antiporter is asymmetric with respect to the substrate affinity of the transport site. The data agree with the current model of activation and inactivation of the antiporter by an intracellular site through changes in the intracellular Na⁺ and H⁺ concentrations.     

Electrical and molecular coupling between sodium and proton fluxes in basolateral membrane vesicles of rat liver

Mechanisms of Na⁺–H⁺ exchange in the hepatocyte were studied utilizing isolated basolateral membrane vesicles prepared by two different methods: Evidence was obtained for the existence of molecular coupling of Na⁺ and H⁺ fluxes (Na⁺/H⁺-antiport) which exhibits saturation kinetics (Km 7 mmol/l Na⁺) and is inhibited by amiloride (1.0 mmol/l). Although the two membrane preparations showed differences with respect to ionic permeabilities, our data suggest that a relatively high H⁺ conductance exists in the basolateral plasma membrane. Hence, electrical coupling of conductive H⁺ and Na⁺ fluxes in the opposite direction could contribute to net Na⁺–H⁺ exchange across the basolateral hepatocyte plasma membrane.     

Evidence of Delayed Mesangial Transport of Human IgA in Glomeruli of ddY Mice Pretreated with Sheep Anti-type IV Collagen Serum

In order to investigate whether mesangial transport by glomeruli is delayed in ddY mice pretreated with sheep anti type IV collagen serum, the mice were administered an overload of human IgA myeloma serum. Non pretreated ddY mice used as controls and both experimental and control BALB/c mice were also processed in a similar manner. The intensities of mesangial deposition of human IgA were examined periodically and were found to correlate well with deposition of mouse IgA. Both mouse and human IgAs showed a gradual increase for up to 8 experimental weeks. In the control young ddY mice, however, the overloaded mesangial human IgA quickly disappeared, presenting no appreciable mesangial deposition of autologous IgA. In sharp contrast, both the experimental and control BALB/c mice showed an initially prolonged and rather heavy mesangial deposition of human IgA, followed by a gradual decrease and somewhat light mesangial deposition of autologous mouse IgA. These results obtained using experimental ddY mice appear to confirm the possibility that non immunological local trapping, due to retardation of mesangial transport function, causes mesangial deposition of autologous mouse IgA in this particular strain. Acta Pathol Jpn 39: 289 295, 1989.     

A simple device to aid impalement of cells using conventional microelectrode drives

Consistent penetration of cell membranes by micropipettes is facilitated by using electrode accelerators or high velocity step drives. Notwithstanding, much intracellular work is still done with conventional mechanical or hydraulic drives; cell membrane penetration is achieved by means of gentle taps on any convenient part of the set up. A remote control device is described which performs this function and is compact enough to be fixed on either the microelectrode holder or the preparation mounting. It consists of a small magnetized rod freely suspended in a pot-core coil. A current pulse through the coil jolts the rod; the inertial reaction of the coil frame provides the sudden movement required by the micropipette tip to overcome the elastic resistance of the cell membrane.     

Ischemic disruption of glutamate homeostasis in brain: quantitative immunocytochemical analyses

More than 10 years ago, it was shown by microdialysis that the excitatory transmitter glutamate accumulates in the interstitial space of brain subjected to ischemic insult. This was one of the key observations leading to the formulation of the `glutamate hypothesis' of ischemic cell death. It is now assumed that even a transient glutamate overflow may set in motion a number of events that ultimately cause cell loss in vulnerable neuronal populations. The aim of the present review is to discuss the intracellular changes that underlie the dysregulation of extracellular glutamate during and after ischemia, with emphasis on data obtained by postembedding, electron microscopic immunogold cytochemistry. While the time resolution of this approach is necessarily limited, it can reveal, quantitatively and at a high level of spatial resolution, how the intracellular pools of glutamate and metabolically related amino acids are perturbed during and after an ischemic insult. Moreover, this can be done in animals whose extracellular amino acid levels are monitored by microdialysis, allowing a direct correlation of extra- and intracellular changes. Immunogold analyses of brains subjected to ischemia have identified dendrites and neuronal somata as likely sources of glutamate efflux, probably mediated by reversal of glutamate uptake. The vesicular glutamate pool has been found to be largely unchanged after 20 min of ischemia. Ischemia causes an increased glutamate content and an increased glutamate/glutamine ratio in glial cells, as revealed by double immunogold labelling. This argues against the idea that glial cells contribute to the extracellular overflow of glutamate in the ischemic brain.     

Effect of amiloride on electrolyte transport parameters of the main duct of the rabbit mandibular salivary gland

We have studied the response of the rabbit mandibular main duct perfused in vitro to luminally administered amiloride. The half-maximal inhibitory concentrations (K_I) when the duct was bathed in Cl solutions were: for net Na⁺ transport, 3×10^–6 mol l^–1; for transepithelial potential difference, 6×10^–6 mol l^–1; and for transepithelial conductance, 3×10^–7 mol l^–1. Substitution of the impermeant SO ₄ ^2– anion for Cl^– changed the K_I for conductance to 3×10^–6 mol l^–1. Within Cl^–-containing media, the time course of the amiloride effect on potential difference showed an early rapid fall of 10 mV with a half-time 2 s, followed by a slower depolarization of 9 mV, and the conductance change followed the slower component of the potential change. In SO ₄ ^2– -containing media, the potential difference and conductance changes followed time courses similar to one another. Finally, experiments on the effect of serosal applications of ouabain revealed that, although, in general, ouabain reduced resistance, it caused an increase in resistance in those ducts where the initial resistance was low. We conclude that: i) luminal Na⁺ transport occurs via amiloride-sensitive, conductive Na⁺ channels; ii) the Cl^– conductance is the major determinant of transepithelial conductance; iii) the first phase of the potential response is due to blocking of the Na⁺ conductive channels, whilst the slow phase reflects secondary inhibition of an electrogenic Na⁺ pump; and iv) duct resistance changes are secondary to alterations in intracellular Cl^– concentration.     

Contraluminal transport of hexoses in the proximal convolution of the rat kidney in situ

In order to study contraluminal hexose transport, concentration and time-dependent influx of³H-2-deoxy-d-glucose from the interstitium into cortical tubular cells has been measured. The influx curves fit to a two parameter kinetics (K _m 1.3±0.2 mmol/l,J _max 0.67±0.16 pmol/s · cm) plus an additional diffusion term (withP=6·10^–8 cm²/s) and a distribution ratio extracellular to intracellular amount of 2-deoxy-d-glucose of 10.6. Since the extracellular to intracellular free water space as estimated from morphological data was 12, one must conclude that glucose has only free access to 1/3 of the cell water. The intracellularly accessible space was augmented when the tubules were preperfused for 10 s with hypotonic saline. Thereby an increase of the compartment into which diffusion occurs was revealed and a final rupture of this intracellular compartment at 1/4 isotonic solutions was observed. Total replacement of ions in the peritubular perfusate by mannitol did not change 2-deoxy-d-glucose influx, indicating that it is Na⁺-independent. By adding isotonic concentrations of the respective sugars to the capillary perfusate, three degrees of inhibition of 2-deoxy-d-glucose influx could be revealed: strong inhibition byd-glucose, methyl--d-glucoside,d-mannose, 3-O-methyl-d-glucose, 2-deoxy-d-galactose, methyl--d-galactoside and 6-deoxy-d-glucose, moderate inhibition byd-galactose,l-glucose,l-mannose andd-fructose, no or borderline inhibition by methyl -d-glucoside, 2-deoxy-methyl--d-galactoside, 1-thio--d-glucose, 1-thio--d-galactose, 5-thio--d-glucose, myo-inositol and mannitol. The contraluminal 2-deoxy-d-glucose influx was also inhibited by phloretin, chlormerodrin and preperfusion with cytochalasin B. Starvation as well as streptozotocin diabetes has no influence on contraluminal 2-deoxy-d-glucose transport. Thus, in contrast to the luminal hexose transport system the contraluminal system is Na⁺-independent, does not require on OH-group at C-atom 2, acceptsl-glucose and fructose, but not an -methyl group at C-atom 1.     

Comparison of simultaneous pH measurements made with 8-hydroxypyrene-1,3,6-trisulphonic acid (HPTS) and pH-sensitive microelectrodes in snail neurones

 We have evaluated the pyrene-based ratiometric fluorescent dye, 8-hydroxypyrene-1,3,6-trisulphonic acid (HPTS), by using it in conjunction with glass pH-sensitive microelectrodes to measure intracellular pH (pH_i) in voltage-clamped snail neurones. Intracellular acidification with propionic acid, and alkalinization following the activation of H⁺ channels allowed the calibration of the dye to be compared with that of the pH microelectrode over the pH range 6.50–7.50. HPTS calibrated in vitro and glass pH-sensitive microelectrodes produced similar absolute resting pH_i values, 7.16±0.05 (n=10) and 7.17±0.06 (n=9) respectively in nominally CO₂/HCO₃ ^–-free saline. At both extremes of the pH range there were small discrepancies. At acidic pH_i, 6.87±0.09 (n=5), the intracellular HPTS measurement differed by –0.08±0.03 pH units from the pH-sensitive microelectrode measurement. At alkaline pH_i,7.32±0.10 (n=5), HPTS measurements produced pH values that differed by +0.07±0.04 pH units from those of the pH-sensitive microelectrode. Some of the discrepancy could be accounted for by the slow response of the recessed-tip pH-sensitive microelectrode (time constant 77±15 s, n=3). Further experiments showed that HPTS, used at an intracellular concentration of 200 μM to 2 mM, did not block activity-dependent pH_i changes. The intracellular HPTS concentration was calculated by measurement of intracellular chloride during a series of HPTS-KCl injections. Comparison of HPTS with 2′,7′-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF), at the same concentration, showed that HPTS produces a larger change in ratio over the pH range 6.00–8.00. Received: 19 February 1998 / Received after revision and accepted: 22 April 1998     

Postsynaptic fibers reaching the dorsal column nuclei in the rat

Postsynaptic fibers reaching the dorsal column nuclei were investigated in rat by means of retrograde transport of wheat germ agglutinin-horseradish peroxidase conjugate. Each nucleus received only ipsilateral afferents with most of the labeled cells forming a band which covered the mediolateral extent of the dorsal horn in an area that resembled lamina IV in the cat. The labeling excluded the reticular extension of the neck of the dorsal horn. Lumbosacral afferents were restricted to the gracilis nucleus and cervicothoracic afferents to the cuneatus nucleus. Cervical and anterior lumbar levels showed additional projections coming from their most medial parts. The organization of this second-order pathway in rat is similar to that in cat and monkey.