Hydrogen Ion Regulation in Rat Cerebellar Granule Cells Studied by Single-Cell Fluorescence Microscopy.

The internal pH (pHi) regulatory mechanisms of individual rat cerebellar granule cells maintained in tissue culture have been investigated using the fluorescent indicator BCECF (2,7' bis carboxyethyl 5,6 carboxy-fluorescein) and quantitative fluorescence microscopy. The steady-state pHi was estimated as 7.27 +/- 0.25 in bicarbonate-buffered media and 7.49 +/- 0.35 in HEPES-buffered media. Buffering power was estimated at about 8 mM/pH unit from the peak alkalinization and acidification transients seen on addition and removal of NH4Cl. Bicarbonate did not appear to contribute to the buffering power estimated in this way. Following an acid load imposed by the ammonium prepulse technique, pHi recovered to steady-state values with first-order kinetics. Recovery was absolutely dependent upon extracellular sodium and, in about half of the cells tested, bicarbonate ions. In cells that did not require bicarbonate for pHi recovery, amiloride (1 mM) inhibited pHi recovery. Removal of extracellular chloride produced a reversible alkalinization of pHi in a third of the cells studied. This alkalinization persisted even when extracellular sodium had been reduced to zero. Removal of extracellular chloride did not inhibit bicarbonate-dependent pHi recovery following an acid load. These results are best explained by the existence of three independent pHi regulatory mechanisms: Na+/H+ exchange, Na+/HCO3- cotransport and Cl-/HCO3- exchange.

The action of volatile anaesthetics on stimulus-secretion coupling in bovine adrenal chromaffin cells.

1. The action of four volatile anaesthetics, ethrane, halothane, isoflurane and methoxyflurane on stimulus-secretion coupling has been studied in isolated bovine adrenal medullary cells. All four agents inhibited the secretion of adrenaline and noradrenaline evoked by 500 microM carbachol at concentrations within the anaesthetic range. Total catecholamine secretion induced by stimulation with 77 mM potassium was also inhibited but at higher concentrations. All four agents inhibited the 45Ca influx evoked by stimulation with 500 microM carbachol and the 45Ca influx in response to K+-depolarization. 2. When total catecholamine secretion in response to potassium or carbachol was modulated by varying extracellular calcium or by adding halothane or methoxyflurane to the incubation medium, the amount of catecholamine secretion for a given Ca2+ entry was the same. 3. The action of methoxyflurane on the relationship between intracellular free Ca and exocytosis was examined using electropermeabilised cells, which were suspended in solutions containing a range of concentrations of ionised calcium between 10(-8) and 10(-4)M. The anaesthetic had no effect on the activation of exocytosis by intracellular free calcium. 4. Halothane and methoxyflurane inhibited the carbachol-induced secretion of catecholamines in a non-competitive manner. 5. Halothane and methoxyflurane inhibited the increase in 22Na influx evoked by carbachol. For halothane and methoxyflurane this inhibition of Na influx appears to be sufficient to account for the inhibition of the evoked catecholamine secretion. 6. We conclude that the volatile anaesthetics ethrane, halothane, isoflurane and methoxyflurane inhibit the secretion of adrenaline and noradrenaline induced by carbachol at concentrations that lie within the range encountered during general anaesthesia. In addition all four also inhibit the secretion of catecholamines induced by depolarization with 77 mM K+ but at much higher concentrations. The decrease in Ca influx caused by methoxyflurane accounts fully for the decrease in secretion in response to depolarization with potassium. Similar actions at synapses within the CNS may underlie the general anaesthetic effects of these agents.

The action of pentobarbitone on stimulus-secretion coupling in adrenal chromaffin cells.

The action of pentobarbitone on stimulus-secretion coupling was studied in bovine isolated adrenal medullary cells. Pentobarbitone inhibited catecholamine release evoked by 500 microM carbachol with half maximal inhibition (IC50) around 50 microM. It also inhibited catecholamine release induced by depolarization with 77 mM potassium (IC50 100 microM). These effects of pentobarbitone were observed with concentrations that lie within the range encountered during general anaesthesia. Evoked secretion required the presence of calcium in the extracellular medium and was associated with an influx of Ca2+ through voltage-sensitive channels. Pentobarbitone inhibited 45Ca influx in response to both carbachol (IC50 50 microM) and K+-depolarization (IC50 150 microM). The action of pentobarbitone on the relationship between intracellular free Ca and exocytosis was examined using electropermeabilised cells which were suspended in solutions containing a range of concentrations of ionised calcium between 10(-8) and 10(-4)M. Catecholamine secretion was measured in the presence of 0, 50, 200 or 500 microM pentobarbitone. The anaesthetic had no effect on the activation of exocytosis by intracellular free calcium. When catecholamine secretion in response to potassium or carbachol was modulated by varying extracellular calcium or by adding pentobarbitone to the incubation medium, the amount of catecholamine secretion for a given Ca2+ entry was the same. Pentobarbitone inhibited the secretion and 45Ca uptake induced by carbachol in a non-competitive manner. The secretion evoked by nicotinic agonists was associated with an increase in 22Na influx. Pentobarbitone inhibited this influx with an IC50 of 100 microM. We concluded that: (a) Pentobarbitone inhibits the catecholamine secretion from bovine adrenal chromaffin cells induced by nicotinic agonists by non-competitive inhibition of the nicotinic receptor. (b) The decrease in Ca influx caused by pentobarbitone accounts fully for the decrease in secretion in response to depolarization with potassium.

The mechanism by which procaine inhibits catecholamine secretion from bovine chromaffin cells.

1. We have investigated the action of procaine on stimulus-secretion coupling in bovine adrenal chromaffin cells. 2. Procaine inhibited the catecholamine secretion evoked by 500 microM carbachol (CCh) with an IC50 of 35 microM and the associated calcium influx (IC50 60 microM). It inhibited the catecholamine secretion evoked by depolarization with high potassium by less than 20% even at the highest concentrations tested (3.2 mM). 3. The secretion evoked by CCh was associated with an increase in sodium influx. This evoked influx was also inhibited by procaine (IC50 80 microM). 4. This selective action of procaine on the CCh-evoked catecholamine secretion was investigated further by patch-clamp techniques. 5. In agreement with the ion flux studies, procaine inhibited the inward current evoked by CCh. Procaine also altered the spectral characteristics of the noise associated with the agonist-induced current by adding an additional high frequency component. The amplitude of this component showed an e-fold increase for a 55 mV membrane hyperpolarization. 6. Data from cell-attached patches showed that increasing concentrations of procaine produced a progressive fall in the mean channel open time and an increase in mean blocked time. This combination led to a decrease in mean burst length. In addition, Popen was reduced by 50 microM procaine. These changes in channel conducting time were sufficient to account for the reduction in inward current. A limited study of the action of procaine on nicotinic channels in outside-out patches gave similar results. 7. The data were considered in relation to various schemes of anaesthetic-channel interactions. The data did not fit the sequential blocking model or the extended channel block model but could be fitted to a modified sequential blocking model in which the rate constant for channel reopening after block was itself subject to modulation by the anaesthetic and the blocked channel could close without passing through the open state.

Effects of pentobarbitone on the properties of nicotinic channels of chromaffin cells.

We have used the whole cell patch clamp technique to investigate the action of pentobarbitone on the nicotinic channels of bovine chromaffin cells. Application of agonists induced an inward current associated with a large increase in current noise. The noise could be fitted by Lorentzian functions with time constants of 17 +/- 2 ms for 10 microM acetylcholine and 10 +/- 1 ms for 10 microM carbachol. The single channel conductance estimated from the current variance was about 25 pS in each case. Pentobarbitone decreased the time constants in a concentration-dependent fashion, but the unit conductances were unaffected. Single channel events were recorded in chromaffin cells held under voltage clamp. Pentobarbitone did not reduce the amplitude of channel openings or the probability of channel opening but reduced the mean channel open time. This reduction was sufficient to account for the decrease in inward current produced by pentobarbitone.

Ionic and metabolic requirements for stimulation of secretion by ouabain in bovine adrenal medullary cells.

The effect of ouabain on the secretion of catecholamines from isolated bovine adrenal medullary cells was investigated. Ouabain enhances the basal rate of secretion approximately 2-fold, with half-maximal stimulation occurring at a glycoside concentration of around 5 X 10(-7) M. Parallel measurements of the release of dopamine beta-hydroxylase (EC 1.14.17.1) (an enzyme associated with chromaffin granules) and lactate dehydrogenase (EC 1.1.1.27) (which is confined to the cytosolic compartment) suggest that this increase in secretion occurs as a result of an enhanced rate of exocytosis rather than by any other route. The stimulatory effect of ouabain is dependent on extracellular sodium but is maintained in the nominal absence of calcium and is unaffected by changes in the major external anion. Neither tetrodotoxin nor phenoxybenzamine alters the response to glycoside treatment, but the calcium channel blocker methoxyverapamil reduces the catecholamine secretion evoked by ouabain in a dose-dependent fashion. This study serves to characterize the secretory action of ouabain in isolated chromaffin cells and to provide a foundation for the ion flux studies reported in the following paper.

Ion movements in isolated bovine adrenal medullary cells treated with ouabain.

In the previous paper [Mol. Pharmacol. 23:671-680 (1983)] it was shown that ouabain enhances the exocytotic release of catecholamines from isolated bovine adrenal medullary cells. This effect is dependent upon extracellular sodium, but persists in the nominal absence of calcium. In this paper the study has been extended to include an investigation of the effects of ouabain on the fluxes of 86Rb, 42K, 24Na, and 45Ca in these cells. The basic features of the chromaffin cell sodium pump are characterized, and it is shown for the first time that both the pump itself (i.e., the kinetics and properties of transport) and its inhibition by ouabain resemble those of squid axons and erythrocytes. However, serious doubts are cast upon the often-stated possibility that there is a direct link between sodium pump inhibition and exocytotic secretion because parallel measurements of both phenomena have, for example, shown that while the secretory effect of ouabain is sodium-dependent, pump inhibition is not. Instead, an entirely different explanation is suggested by the discovery that ouabain produces a marked decrease in the rate of active calcium extrusion from chromaffin cells, under all conditions in which catecholamine secretion is enhanced. This inhibition seems not to be accompanied by any change in calcium influx, and may therefore provide a direct explanation for the rise in free calcium which is required to stimulate exocytosis in this tissue.

Excitatory and inhibitory synaptic mechanisms in anaesthesia.

To understand the cellular and molecular basis of the anaesthetic state, it is important to remember that, in the intact CNS, synapses operate within elaborate nerve networks. From the data presented above, it is evident that block of impulse conduction in presynaptic fibres does not explain the effects of most anesthetics on synaptic activity. This is not surprising since some anaesthetics, the barbiturates in particular, may both depress excitation and enhance inhibition. General anaesthetics modulate the activity of presynaptic voltage-gated calcium channels and this appears to be sufficient to account for the reduction in transmitter secretion they produce. Transmitter operated ion channels in the postsynaptic membrane are modulated by smaller concentrations of anaesthetics than are required to modulate the presynaptic voltage-gated calcium channels. For this reason, transmitter operated channels appear to represent a major target site for anaesthetics. Finally, there are subtle effects of anaesthetics on the patterns of impulse propagation in nerve axons and on action potential generation in the cell body which result from modulation of membrane excitability. The overall effect of an anaesthetic agent depends on summation of events occurring at the many individual synapses and neurones that make up the network. The effects of anaesthetics on different neuronal pathways may therefore depend on the nature of the receptors and ion channels of the cells that comprise the network. The anaesthetic state may be the result of all these actions, but the characteristics of the state may differ somewhat from agent to agent.

Effects of lead ions on events associated with exocytosis in isolated bovine adrenal medullary cells.

Lead buffers (citrate and Tiron) were used to investigate the effects of low concentrations (0.1-6 microM) of Pb2+ on stimulus-secretion coupling in isolated bovine chromaffin cells. Nicotinic agonists and high K elicit secretion by enhancing Ca2+ influx into chromaffin cells. Pb2+ inhibited the catecholamine secretion in response to 500 microM carbachol and 77 mM K+ depolarization but was without significant effect on basal secretion. Pb2+ also inhibited the influx of 45Ca occurring in response to these agents. The K0.5 values for inhibition suggest that the carbachol-evoked flux is more sensitive to Pb2+ than influx in response to a direct depolarization. When extracellular calcium was lowered in the absence of Pb2+, both secretion and 45Ca entry were reduced. The effects of Pb2+ were comparable to those of lowered Ca2+. 22Na influx through nicotinic receptor-mediated channels, measured in the presence of tetrodotoxin (2 microM) and ouabain (50 microM), was inhibited by Pb2+. The results suggest that Pb2+ inhibits exocytotic catecholamine secretion by inhibiting Ca2+ influx. The differential sensitivity to Pb2+ of K- and carbachol-evoked 45Ca flux, coupled with the 22Na measurements, indicates that Pb2+ inhibits the movement of ions through acetylcholine-induced channels as well as through voltage-sensitive calcium channels.

Lead enters bovine adrenal medullary cells through calcium channels.

Agents that stimulate secretion also accelerate the rate of Pb uptake into adrenal medullary cells. For example, when cells are suspended in a medium containing 5 microM Pb2+, depolarization by 77 mM K increases the rate of Pb uptake from 12 +/- 1 to 47 +/- 5 mumol/(L cells X min). K-induced Pb uptake has an apparent Km for Pb2+ of 2.6 microM, and is antagonized by Ca2+ with a K0.5 of 1.4 mM. The Ca channel blocker D-600 inhibits Pb entry with a K0.5 of 0.4 microM. Pb uptake is also stimulated by the Ca channel agonist BAY K 8644. These observations suggest that Pb passes through Ca channels. The permeability of the channels to Pb appears to be at least 10 times the permeability to Ca.

The action of anaesthetics on stimulus-secretion coupling and synaptic activity.

1. Anaesthetics are known to depress excitatory synaptic transmission and the mechanism of this inhibition has been investigated using bovine adrenal chromaffin cells as an experimental model. 2. These cells are homologous with post-ganglionic sympathetic neurons and have well characterized receptor and secretory mechanisms. They are amenable both to the direct measurement of evoked secretion with its associated ion fluxes, and to electrophysiological investigation using the patch clamp technique. 3. These approaches have been used to study the influence of anaesthetics on pre- and post-synaptic mechanisms involved in stimulus-secretion coupling. 4. A variety of agents inhibited secretion evoked by direct depolarization, and this was shown to be due to a reduction in calcium influx. 5. Direct inhibition of voltage-gated calcium currents was confirmed by whole-cell patch clamp measurements. 6. In addition, anaesthetics powerfully modulated nicotinic receptor mediated events: carbachol-evoked secretion was more sensitive to anaesthetics than that stimulated by high potassium. 7. The mechanism of anaesthetic action on the nAChR was examined in more detail with patch-clamp experiments. 8. These showed that anaesthetics reduced the probability of channels being in the open state, largely as a result of reduction in mean channel open time. 9. The data are discussed in relation to excitatory synaptic transmission.

A clinical study of forces generated by the intrinsic muscles of the index finger and the extrinsic flexor and extensor muscles of the hand.

A study of the forces generated by the intrinsic muscles of the index finger and coordinating muscles of the hand found that the intrinsic muscles of the index finger contributed combined forces equivalent to approximately 80% of those generated by the flexor profundus and superficialis, and to 73% of the moment for the motion of metacarpopalangeal flexion with simultaneous interphalangeal joint extension. No current tendon transfer operation can correct this deficit, though several supply sufficient force at the metacarpophalangeal joint to counterbalance the extrinsic extensors.

On the role of bicarbonate as a hydrogen ion buffer in rat CNS neurones.

The role of bicarbonate as a hydrogen ion buffer has been investigated using the fluorescent dye BCECF in individual rat cerebellar, hippocampal and neocortical neurones maintained in culture. The steady-state intracellular pH (pHi) was estimated to be 7.07 +/- 0.05 (n = 22) in CO2-HCO3(-)-buffered media. Buffering power (beta) estimated from the addition and removal of weak bases was ca 10 mM (pH unit)-1 and was found to be similar in both CO2-HCO3(-)- and Hepes-buffered media. The membrane-permeant carbonic anhydrase inhibitor, acetazolamide (10-20 microM), did not affect estimates of beta. The results indicate that CO2-HCO3- does not act as an open buffer system in these neurones.

Calcium channel currents in bovine adrenal chromaffin cells and their modulation by anaesthetic agents.

1. The calcium channel currents of bovine adrenal chromaffin cells were characterized using a variety of voltage pulse protocols and selective channel blockers before examination of their modulation by anaesthetic agents. 2. All the anaesthetics studied (halothane, methoxyflurane, etomidate and methohexitone) inhibited the calcium channel currents in a concentration-dependent manner and increased the rate of current decay. 3. The anaesthetics did not shift the current-voltage relation nor did they change the voltage for half-maximal channel activation derived from analysis of the voltage dependence of the tail currents. None of the anaesthetics appeared to alter the time constant of tail current decay. 4. To complement earlier studies of the inhibitory actions of anaesthetics on K(+)-evoked catecholamine secretion and the associated Ca2+ uptake, the IC50 values for etomidate and methohexitone were determined using a biochemical assay. The IC50 values for anaesthetic inhibition of calcium channel currents corresponded closely with those for inhibition of K(+)-evoked calcium uptake and catecholamine secretion. 5. The inhibitory effect of the volatile anaesthetics and etomidate is best explained by dual action: a reduction in the probability of channel opening coupled with an increase in the rate of channel inactivation. Methohexitone appeared to inhibit the currents by a use-dependent slow block.

The determination of moments for extension of the wrist generated by muscles of the forearm.

In deciding on suitable tendon transfers to replace denervated muscle-tendon units, important considerations are the strength and effectiveness of possible substitutes. A method is presented by which the strength of the wrist extensor muscles and their moment arms can be determined. The method can be applied to other muscles at other joints. It involves the use of a force transducer which measures the combined forces of the three wrist extensors in an isometric contraction. This moment for wrist extension, measured in the living intact arm, is the same as the sum of the moments of the three wrist extensor muscles. The contribution of each muscle to the total moment is calculated from ratios that have been developed from a quantitative study of moment arms and muscle masses in sixteen cadaver limbs. It is suggested that the ratio of one moment arm to another is fairly constant from subject to subject, and that muscle masses also have sufficiently similar ratios to each other to serve as the basis for practical estimations by the surgeon. Thus the surgeon needs only one or two direct measurements of moments externally and only one or two skeletal measurements on any living subject to be able to estimate the effectiveness of a number of muscles on the basis of cadaver studies such as this, and to project the behavior of a muscle after it has been transferred to a position where it will have new moment arms.

A novel method for absolute calibration of intracellular pH indicators.

In this paper we present methods to measure intracellular pH (pHi) with fluorescent indicators. These methods are based on the change in intracellular pH following the addition of weak acids and weak bases to the extracellular medium. The first method requires that the fluorescence of the indicator is proportional to the change in pHi that follows the addition of a weak acid or weak base to the extra-cellular medium. The second is a null method which uses a mixture of weak acid and weak base that does not change the fluorescent signal. This null method can be used in situations in which the fluorescent signal is a monotonic but non-linear function of pH. The first method depends upon four assumptions. (i) That only the uncharged forms of the weak acids and bases cross the surface membrane. (ii) That the pKa is the same inside and outside the cell. (iii) That the buffering power is constant. (iv) That there is no significant pH regulation on the time scale of the change in pHi. The null method only requires the first two assumptions. We have made estimates of pHi in four different cell types and compared the results obtained with these methods with those obtained from other methods of pHi calibration.