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.
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.
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.
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 5001iM carbachol at concentrations within the anaesthetic range. Total catecholamine secretion induced by stimulation with 77mM potassium was also inhibited but at higher concentrations. All four agents inhibited the 45Ca influx evoked by stimulation with 500pM carbachol and the 45Ca influx in response to K+-depolarization. 2 When total catecholaIiine 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-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.
1 The action ofpentobarbitone on stimulus-secretion coupling was studied in bovine isolated adrenal medullary cells. 2 Pentobarbitone inhibited catecholamine release evoked by 5001M carbachol with half maximal inhibition (IC50) around 50 pM. It also inhibited catecholamine release induced by depolarization with 77 mM potassium (IC50 100 gM). These effects of pentobarbitone were observed with concentrations that lie within the range encountered during general anaesthesia. 3 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 gM) and K+-depolarization (IC50 150 pM).4 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 ofionised calcium between 10-8 and 10-4 M. Catecholamine secretion was measured in the presence of 0, 50, 200 or 500 pM pentobarbitone. The anaesthetic had no effect on the activation of exocytosis by intracellular free calcium. 5 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.6 Pentobarbitone inhibited the secretion and 45Ca uptake induced by carbachol in a non-competitive manner.7 The secretion evoked by nicotinic agonists was associated with an increase in 22Na influx.Pentobarbitone inhibited this influx with an ICm of IOO1M. 8 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.
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