Roger C. Thomas

The role of bicarbonate, chloride and sodium ions in the regulation of intracellular pH in snail neurones

1. Intracellular pH (pHi), Cl− and Na+ levels were recorded in snail neurones using ion‐sensitive micro‐electrodes, and the mechanism of the pHi recovery from internal acidification investigated.

The effect of calcium injection on the intracellular sodium and pH of snail neurones

1. Ion‐sensitive glass micro‐electrodes were used to measure the intracellular pH (pHi) and the intracellular sodium ion concentration, [Na+]i, in identified Helix aspersa neurones. 2. The injection of small volumes of 0‐1 McaCl2, which increased the membrane potential by 10‐15 mV for 1‐2 min, had little or no effect on [Na+]i. Increases of up to 1 mM in [Na+]i could be reversibly induced by larger injections. 3. Calcium injection caused an immediate decrease in pHi, which appeared to be directly proportional to the amount of calcium injected. Injections causing hyperpolarizations of 10‐20 mV which recovered in 2‐5 min caused pHi decreases of 0‐04‐0‐15 units. After each of these injections both pHi and the membrane potential recovered exponentially but with different time constants. 4. The injection of calcium at a low rate could decrease pHi without affecting the membrane potential. 5. Neither membrane potential nor pHi were affected by the injection of small volumes of 0‐1 M‐MgCl2, Injection of CoCl2 produced a large transient decrease in pHi but no significant change in membrane potential. 6. Exposure of the cell to saline equilibrated with 2‐5% CO2 greatly reduced the pHi decrease caused by calcium injection but had only small effects on the membrane potential response. 7. It is concluded that most of the injected calcium is exchanged for protons inside the cell.

AN ELECTROGENIC SODIUM PUMP IN SNAIL NERVE CELLS.

Abstract 1. 1. If sodium ions are injected at the rate of 4·4 mM/min into a snail neurone, there is an increase in the membrane potential by about 30 mV in 10 min. 2. 2. This marked hyperpolarization is not brought about if potassium ions are injected instead. 3. 3. The hyperpolarization is inhibited by ouabain, parachloromercuribenzoate or reduction in the potassium concentration. 4. 4. It is concluded that the hyperpolarization is due to the stimulation of an electrogenic sodium pump in the nerve cell.

Calcium–hydrogen exchange by the plasma membrane Ca-ATPase of voltage-clamped snail neurons

The submicromolar levels of free Ca2+ ions in animal cells are believed to be maintained in the long term by two different plasma membrane transport mechanisms. These are Na–Ca exchange, driven by the sodium gradient, and a Na-independent Ca pump, driven by ATP. There is good evidence from red blood cells, and indirect evidence from other non-neuronal preparations, that the Ca-ATPase exchanges internal Ca2+ for external H +. Although Ca extrusion from nerve cells is inhibited by high external pH , there as yet is no evidence for the counter-transport of H+. We have used both pH- and calcium-sensitive microelectrodes on the cell surface, and the Ca indicator fura-2 intracellularily, to investigate how snail neurons regulate cytoplasmic free Ca2+. We now report that in snail neurons the recovery of intracellular Ca2+ after an increase coincides with both the expected increase in surface Ca2+ and a decrease in surface H+. Recovery of intracellular Ca and the changes in surface pH and Ca are all blocked by intracellular vanadate. We conclude that snail neurons regulate intracellular Ca mainly by a Ca–H ATPase, and suggest that this Ca–H exchange may account for many of the reported extracellular pH changes seen with neuronal excitation.

The effect of anion injection and changes in the external potassium and chloride concentration on the reversal potentials of the IPSP and acetylcholine

Abstract 1. 1. Certain neurones in the abdominal ganglion of the snail Helix aspersa show spontaneous inhibitory post-synaptic potentials (IPSP). 2. 2. Addition of acetylcholine normally causes a hyperpolarization of such neurones. 3. 3. Fifteen different anions, of increasing hydrated volume, were injected into the neurone. Those with a hydrated diameter greater than 1·24 times the hydrated potassium ion had no effect on the reversal potential of the IPSP and ACh. These anions were fluoride, bisulphite, bicarbonate, acetate, chromate, sulphate, phosphate and propionate. 4. 4. Injection of small anions, such as bromide, chloride, nitrate, thiocyante, chlorate, formate, altered the reversal potentials of the IPSP and ACh. 5. 5. Alteration of the external chloride concentration or the external potassium concentration affected the reversal potentials of the IPSP and ACh. 6. 6. Experiments in which the external concentrations of potassium and chloride were altered so that they either opposed or augmented each other indicate that the effect of the chloride ion was more marked and more rapid than that of the potassium ion on the IPSP and ACh reversal potentials. 7. 7. It is suggested that during the IPSP or when ACh is applied to the cell, pores open in the membranes so that approximately 90 per cent of the current is carried by the chloride ion and 10 per cent by the potassium ion. 8. 8. It is probable that acethylcholine is the transmitter at the snail inhibitory synapse.

Spontaneously active and InsP3-activated ion channels in cell nuclei from rat cerebellar Purkinje and granule neurones

Increases in Ca2+ concentration in the nucleus of neurones modulate gene transcription and may be involved in activity‐dependent long‐term plasticity, apoptosis, and neurotoxicity. Little is currently known about the regulation of Ca2+ in the nuclei of neurones. Investigation of neuronal nuclei is hampered by the cellular heterogeneity of the brain where neurones comprise no more than 10% of the cells. The situation is further complicated by large differences in properties of different neurones. Here we report a method for isolating nuclei from identified central neurones. We employed this technique to study nuclei from rat cerebellar Purkinje and granule neurones. Patch‐clamp recording from the nuclear membrane of Purkinje neurones revealed numerous large‐conductance channels selective for monovalent cations. The nuclear membrane of Purkinje neurones also contained multiple InsP3‐ activated ion channels localized exclusively in the inner nuclear membrane with their receptor loci facing the nucleoplasm. In contrast, the nuclear membrane of granule neurones contained only a small number of mainly anion channels. Nuclear InsP3 receptors (InsP3Rs) were activated by InsP3 with EC50= 0.67 μm and a Hill coefficient of 2.5. Ca2+ exhibited a biphasic effect on the receptors elevating its activity at low concentrations and inhibiting it at micromolar concentrations. InsP3 in saturating concentrations did not prevent the inhibitory effect of Ca2+, but strongly increased InsP3R activity at resting Ca2+ concentrations. These data are the first evidence for the presence of intranuclear sources of Ca2+ in neurones. Ca2+ release from the nuclear envelope may amplify Ca2+ transients penetrating the nucleus from the cytoplasm or generate Ca2+ transients in the nucleus independently of the cytoplasm.

High-molecular-weight organic matter in the particles of comet 67P/Churyumov–Gerasimenko

The presence of solid carbonaceous matter in cometary dust was established by the detection of elements such as carbon, hydrogen, oxygen and nitrogen in particles from comet 1P/Halley. Such matter is generally thought to have originated in the interstellar medium, but it might have formed in the solar nebula—the cloud of gas and dust that was left over after the Sun formed. This solid carbonaceous material cannot be observed from Earth, so it has eluded unambiguous characterization. Many gaseous organic molecules, however, have been observed; they come mostly from the sublimation of ices at the surface or in the subsurface of cometary nuclei. These ices could have been formed from material inherited from the interstellar medium that suffered little processing in the solar nebula. Here we report the in situ detection of solid organic matter in the dust particles emitted by comet 67P/Churyumov–Gerasimenko; the carbon in this organic material is bound in very large macromolecular compounds, analogous to the insoluble organic matter found in the carbonaceous chondrite meteorites. The organic matter in meteorites might have formed in the interstellar medium and/or the solar nebula, but was almost certainly modified in the meteorites’ parent bodies. We conclude that the observed cometary carbonaceous solid matter could have the same origin as the meteoritic insoluble organic matter, but suffered less modification before and/or after being incorporated into the comet.

The plasma membrane calcium ATPase (PMCA) of neurones is electroneutral and exchanges 2 H+ for each Ca2+ or Ba2+ ion extruded

The coupling between Ca2+ extrusion and H+ uptake by the ubiquitous plasma membrane calcium ATPase (PMCA) has not been measured in any neurone. I have investigated this with Ca2+‐ and pH‐sensitive microelectrodes in large voltage‐clamped snail neurones, which have no Na+–Ca2+ exchangers. The recovery of [Ca2+]i and surface pH after a brief depolarization or Ca2+ injection was not slowed by hyperpolarization to −90 mV from a holding potential of −50 mV, consistent with a 1 Ca2+ : 2 H+ coupling ratio. Since Ca2+ injections proved difficult to quantify, and Ca2+ currents through channels were obscured by K+ currents, Ba2+ was used as a substitute. When the cell was bathed in Ca2+‐free Ba2+ Ringer solution, the K+ currents were blocked and large inward currents were revealed on depolarization. The Ca2+‐sensitive microelectrodes were sensitive to intracellular Ba2+ as well as Ca2+. With equal depolarizations Ba2+ entry appeared larger than Ca2+ entry and generated similar but slower pH changes. Ba2+ extrusion was insensitive to hyperpolarization, blocked by eosin or high pH, and about 5 times slower than Ca2+ extrusion. The ratio of the pH change caused by the extrusion of unit charge of Ba2+ influx to that caused by unit charge of H+ injection was 0.85 ± 0.08 (s.e.m., n= 8), corresponding to a Ba2+ : H+ ratio of 1 : 1.7. Both this ratio and the electroneutrality of the PMCA suggest that the Ca2+ : H+ ratio is 1 : 2, ensuring that after a Ca2+ influx [Ca2+]i recovery is not influenced by the membrane potential and maximizes the conversion of Ca2+ influxes into possible pH signals.

The effects of intracellular pH changes on resting cytosolic calcium in voltage-clamped snail neurones

1 We have investigated the effects of changing intracellular pH on intracellular free calcium concentration ([Ca2+]i) in voltage‐clamped neurones of the snail Helix aspersa. Intracellular pH (pHi) was measured using the fluorescent dye 8‐hydroxypyrene‐1,3,6‐trisulphonic acid (HPTS) and changed using weak acids and weak bases. Changes in [Ca2+]i were recorded using either fura‐2 or calcium‐sensitive microelectrodes. 2 Acidification of the neurones with 5 mM or 20 mM propionate (≈0.2 or 0.3 pH units acidification, respectively) caused a small reduction in resting [Ca2+]i of 5 ± 2 nM (n = 4) and 7 ± 16 nM (n = 4), respectively. The removal of the 20 mM propionate after ≈40 min superfusion resulted in an alkalinization of ≈0.35 pH units and an accompanying rise in resting [Ca2+]i of 31 ± 9 nM (n = 4, P < 0.05). The removal of 5 mM propionate did not significantly affect [Ca2+]i. 3 Alkalinizations of ≈0.2‐0.4 pH units of Helix neurones induced by superfusion with 3 mM concentrations of the weak bases trimethylamine (TMA), ammonium chloride (NH4Cl) and procaine were accompanied by significant (P < 0.05) increases in resting [Ca2+]i of 42 ± 4 nM (n = 26), 30 ± 7 nM (n = 5) and 36 ± 4 nM (n = 3), respectively. The effect of TMA (0.5‐6 mM) on [Ca2+]i was dose dependent with an increase in [Ca2+]i during pHi increases of less than 0.1 pH units (0.5 mM TMA). 4 Superfusion of neurones with zero calcium (1 mM EGTA) Ringer solution inhibited depolarization‐induced calcium increases but not the calcium increase produced by the first exposure to TMA (3 mM). In the prolonged absence of extracellular calcium (≈50 min) TMA‐induced calcium rises were decreased by 64 ± 10% compared to those seen in the presence of external calcium (P < 0.05). 5 The calcium rise induced by TMA (3 mM) was reduced by 60 ± 5% following a 10 min period of superfusion with caffeine (10 mM) to deplete the endoplasmic reticulum (ER) stores of calcium (P < 0.05). 6 Cyclopiazonic acid (10‐30 μM CPA), an inhibitor of the ER calcium pump, inhibited the calcium rise produced by TMA (3 mM) and NH4Cl (3 mM) by 61 ± 4% compared to controls (P < 0.05). 7 These data are consistent with physiological intracellular alkaline shifts stimulating release of calcium, or inhibiting re‐uptake of calcium by an intracellular store. The calcium increase was much reduced following application of caffeine, treatment with CPA or prolonged removal of external calcium. Hence the ER was likely to be the source of mobilized calcium.

ACETYLCHOLINE AND THE SPONTANEOUS INHIBITORY POST SYNAPTIC POTENTIALS IN THE SNAIL NEURONE.

Abstract 1. 1. Acetylcholine can hyperpolarize certain snail neurones and inhibit the action potentials. 2. 2. The spontaneous inhibitory post-synaptic potentials (ipsp) have a different reversal potential to that for acetylcholine. 3. 3. The ipsp is less sensitive than the acetylcholine response to chloride changes. 4. 4. The possible relationship between the ipsp and acetylcholine is discussed.