On the sodium and potassium currents of a human neuroblastoma cell line.

1. The patch-clamp method was applied to the study of ionic currents activated by depolarization of undifferentiated IMR-32 human neuroblastoma cells. Whole-cell sodium and potassium currents and single potassium ion channel currents from cell-attached patches were investigated. 2. Cells had a mean resting potential of -38 mV and mean input resistance of 1.6 G omega. Single action potentials were evoked under current clamp during the injection of depolarizing currents. 3. A voltage-dependent inward sodium current was observed which reversed at +44 mV. A Boltzmann fit to the activation curve gave a half-maximal activation voltage of -41.6 mV and a 'slope' of 3.9 mV. The steady-state inactivation curve had a half-maximal inactivation voltage of -81 mV and a 'slope' of 9.7 mV. 4. The time-dependent activation and inactivation of the current displayed classical Hodgkin-Huxley kinetics. Values for the time constants tau m and tau h of 0.16 and 0.63 ms were calculated for a voltage jump from -80 to -10 mV; tau m and tau h decreased as the step potential was changed from -30 to +20 mV. 5. Outward currents were activated in bathing solutions substantially free of anions and could thus be attributed to potassium ions. The tail current reversed in direction on repolarization to -60 mV when the potassium concentration in the bathing solution was increased from 6 to 30 mM. When the bathing solution contained 145 mM-potassium, and the patch pipette, 95 mM, a depolarization to -10 mV from a holding potential of -60 mV evoked an inward current. 6. Outward currents were examined by using voltage pulses which depolarized the cell to -20 mV, or more positive values, from a holding potential of -80 mV and by pulses which depolarized the cell to 0 mV, or to positive values, from a holding potential of -30 mV. A Boltzmann fit of typical activation data gave a half-maximal activation voltage of 17 mV and a 'slope' of 14 mV. 7. The time course of the rising phase of the current was described by a function of the form A(1-exp[-(t-delta t)/tau]), where delta t varied between 1 and 4 ms and tau varied between 4 and 27 ms, decreasing with increasing depolarization. There was no evidence for a fast transient component. 8. The amplitude of outward currents was reduced by extracellular calcium ions, cobalt ions, tetraethylammonium and 4-aminopyridine.(ABSTRACT TRUNCATED AT 400 WORDS)

Cyclic AMP as a possible mediator of dopamine stimulation of cockroach gland cells.

Isolated salivary glands of the cockroach Nauphoeta cinerea Olivier secrete fluid in response to nerve stimulation or application of dopamine, the acinar cells undergoing a hyperpolarization during secretion. The aim of the present work was to examine whether cyclic AMP acts as a second messenger in the acinar cells to cause the secretory and electrical responses to the transmitter dopamine. Cyclic AMP (10-500 microM) in the bathing solution of isolated glands caused a dose-dependent secretory response but no change in the membrane potential of acinar cells. The time courses and magnitudes of the secretory responses to cyclic AMP resembled those features of responses to dopamine. Forskolin, an adenylate cyclase activator, caused fluid secretion but the responses were small and irregular. The phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine (IBMX)(1-1000 microM) produced fluid secretion in a dose-dependent manner, the maximal response being equal to that of dopamine. Maintained responses to cyclic AMP or IBMX required the presence of extracellular calcium ions. An inhibitor (MDL 12,330A) of adenylate cyclase suppressed the secretory responses to dopamine, cyclic AMP, IBMX, the ionophore A23817 or the readmission of calcium ions to the bathing solution; this inhibitor did not block the acinar hyperpolarization caused by nerve stimulation. Cyclic AMP stimulation of glands, bathed in chloride-free solution to prevent fluid secretion, produced a change in the gland cells which outlasted the period of exogenous cyclic AMP stimulation and expressed itself as a transient secretion upon return of the normal bathing solution. It was concluded that stimulus-secretion coupling in this gland involves a calcium-dependent second messenger system and that cyclic AMP is probably the second messenger. The evidence did not support the idea that cyclic AMP is also a second messenger for the acinar cell hyperpolarization evoked by nerve stimulation.

Inhibition of acetylcholine release from preganglionic frog nerves by ATP but not adenosine.

ATP is known to be released in association with acetylcholine at synapses in the vertebrate peripheral nervous system. Exogenously applied ATP and its derivatives have been shown to reduce the release of acetylcholine, so it has been postulated that ATP has a role in the modulation of transmitter secretion. More recent results have suggested, however, that specific adenosine receptors are responsible for the inhibitory effects of adenosine derivatives on transmitter release, and ATP, if released, must be hydrolysed to adenosine to produce inhibition. The original hypothesis that ATP itself might inhibit acetylcholine secretion would be strengthened if it were found that adenosine is very much less potent than ATP as an inhibitor of ACh secretion. We report here results which show this is the case in sympathetic ganglia.

The computation of simulated endplate potentials.

A method is described for computing simulated subthreshold responses (.e. endplate potentials) for the Falk-Fatt (1964) cable model of a muscle fibre due to a punctate change in ionic conductance. The method has been applied to a comparison of the Falk-Fatt and classical models. Comparison with experimental results suggests that the model is adequate to account for the response of the mouse muscle fibre, in which the endplate is highly localized. For frog muscle there are larger discrepancies, which may be due to the extended endplate in this species.

A calcium-readmission response recorded from Nauphoeta salivary gland acinar cells.

1. When cockroach salivary glands are exposed to bathing solutions without added calcium, reintroduction of calcium causes the acinar cells to hyperpolarize. The effect ('readmission response') may be very prolonged if the conditioning solution contains 5 mM-cobalt. 2. Evidence is presented against the possibility that the readmission response is mediated by transmitter released from the salivary nerves. 3. The readmission response is shown to reflect an increase in potassium conductance.

On the role of calcium in the electrical responses of cockroach salivary gland cells to dopamine.

1. The calcium dependence of the intracellularly recorded hyperpolarizing responses of salivary gland acinar cells of Nauphoeta cinerea (Olivier) to ionophoretically applied dopamine has been examined. The results of withdrawing calcium from the bathing solution were essentially the same whether or not other divalent cations were present. The effects of calcium withdrawal were rapidly reversed on replacement of calcium. 2. Small responses to dopamine were reduced or abolished by calcium withdrawal but could be restored by an increase in the amount of ejected dopamine. Calcium withdrawal did not have any consistent effect on the input resistance: a reduction in input resistance is therefore not the main cause of the reduction in the amplitude of the responses to dopamine. 3. In very low calcium solutions responses elicited by repeated prolonged dopamine applications progressively declined. 4. It is suggested that the hyperpolarizing responses to dopamine depend on an influx of calcium into the cytosol from a store which can be replenished only from the exterior.

A novel effect of cobalt treatment on calcium-dependent responses of the cockroach salivary gland.

After incubation in calcium-free solutions containing cobalt, the readmission of calcium caused prolonged but reversible hyperpolarization of acinar cells of cockroach salivary glands and prolonged fluid secretion. It is suggested that cobalt treatment increases the permeability of the acinar cell membrane to calcium.

On the effect of ionophoretically applied dopamine on salivary gland cells of Nauphoeta cinerea.

1. Responses to ionophoretically applied dopamine ('ionophoretic responses') have been recorded with an intracellular micro-electrode from acini of isolated salivary glands of the cockroach, Nauphoeta cinerea. They resembled responses to nerve stimulation ('neural responses') but they usually decayed more slowly and were sometimes more rapid in onset. 2. As already described for neural resonses, ionophoretic responses (i) included a secondary phase of depolarization (although more rarely than neural responses), (ii) increased in duration with increased amplitude and (iii) when small, added superlinearly. These features therefore reflect post-synaptic processes. By contrast, tachyphylaxis was much more marked for neural responses and is therefore mainly of presynaptic origin. 3. Ionophoretic and neural responses were affected similarly by increase in temperature. The times-to-peak of both decreased with values for Q10 of about 3. 4. The differences between the onset and decay of ionophoretic and neural responses can probably be explained by the inherently different conditions of application of the agonist. The similarities therfore provide further support for dopamine as the neurotransmitter.

On the time course of the electrical response of salivary gland cells of Nauphoeta cinerea to ionophoretically applied dopamine.

1. An attempt has been made to account for the latency and slow time course of the electrical responses of salivary gland cells of Nauphoeta cinerea (Olivier) to nerve stimulation and ionophoretically applied dopamine from a pipette placed as close as possible to the acinar surface. 2. The effects have been investigated on the time course of the ionophoretic response of changes in the distance of the pipette from the acinar surface and of the amount of dopamine ejected. 3. The observed changes were smaller than those predicted by models in which the rate limiting step was assumed to be diffusion or slow receptor kinetics. Indirect evidence suggests that the time course of the response is determined by processes subsequent to receptor activation.

On the receptors which mediate the hyperpolarization of salivary gland cells of Nauphoeta cinerea Olivier.

The actions of sympathomimetics and of catecholamine antagonists have been investigated on the membrane potential and responses to nerve stimulation of acinar cells of the salivary gland of Nauphoeta cinerea Olivier. 2. Hyperpolarizations such as those evoked by nerve stimulation and by low concentrations of adrenaline, noradrenaline and dopamine were not produced by the alpha-agonists amidephrine and methoxamine. Isoprenaline was active, but only in concentrations above 100 muM. 3. Tyramine, an indirectly acting sympathomimetic, and high concentrations of methoxamine caused an increase in the rate of the small transient hyperpolarizations sometimes seen in the absence of stimulation. 4. The response to nerve stimulation was unaffected by propranolol (20 muM) an alpha-adrenergic antagonist. Phentolamine, an alpha-adrenergic antagonist, reduced matching responses to nerve stimulation and to dopamine to about the same extent. 5. As on other systems responsive to low concentrations of dopamine, apomorphine was active, although only in high concentration, and ergometrine and methysergide were antagonistic both to nerve stimulation and to dopamine.

The responses to nerve stimulation of the salivary gland of Nauphoeta cinerea Olivier.

A study has been made with intracellular electrodes of the responses of acini of the salivary gland of Nauphoeta cinerea Olivier to salivary duct nerve stimulation. The gland is a paired structure and offers the possibility of investigating the interaction between ipsi- and contralateral nerve stimulation. 2. The characteristics of the responses are as previously described for field stimulation (House, 1973). The latency is of the order of 1 sec and almost independent of the amplitude of the response which may attain a hyperpolarization of about 80 mV. The depolarization which sometimes follows can be disassociated from the preceding hyperpolarization and is presumably an independent response. 3. The stimulus-response relationship shows that acini are multiply innervated. Those close to the mid line recieve axons from both ipsi- and contralateral salivary duct nerves. The response to a test stimulus T can be augmented by an immediately preceding conditioning stimulus C, the joint response being greater than the sum of the separate responses. This effect occurs even when C and T are delivered to different nerves. For longer intervals between C and T, the response to T is depressed.

On the actions of compounds related to dopamine at a neurosecretory synapse.

1 The effects of a number of substances related to dopamine, including all its methylated derivatives, were investigated on the membrane potential and response to nerve stimulation of cockroach salivary gland cells. 2 Only N-methyldopamine (epinine), N,N-dimethyldopamine and N,N-dimethylnoradrenaline, all with unsubstituted hydroxyl groups, directly resembled dopamine in producing a hyperpolarization which could be as large as that caused by maximal nerve stimulation. During the continued presence of these substances the hyperpolarization waned and responses to nerve stimulation declined. 3 Many of the compounds caused one or both of two other effects, namely an increase in the rate of 'spontaneous miniature hyperpolarizations' and an enhancement of the submaximal responses to single nerve stimuli. There were no obvious structural requirements for these effects.

Voltage-current relationship of a carbachol-induced potassium-ion pathway in Aplysia neurones.

1. The electrical characteristics of a potassium ion selective pathway produced by the action of carbachol on Aplysia neurones (Kehoe, 1972b) has been studied. 2. The relationship between current and voltage has been found to be non-linear, the conductance increasing with depolarization and decreasing with hyperpolarization. The degree of rectification was reduced when the external potassium was raised to 50 mM from its normal value of 10 mM. 3. The direction of the rectification and the effect of increased potassium are as predicted by the 'constant field' theory, but the degree of rectification is somewhat larger.

Conductance changes associated with the secretory potential in the cockroach salivary gland.

1. Conductance changes in the acini of the cockroach salivary gland have been examined during nerve stimulation by means of two intracellular electrodes placed in the same acinus, the first electrode being used for recording membrane potential and the second for current injection.2. The transient hyperpolarization (secretory potential) in the acinus evoked by nerve stimuli is accompanied by a rise in membrane conductance. The conductance, however, remains high for a longer period than that of the response.3. Applying the analysis of Trautwein & Dudel (1958) to the secretory potentials recorded in the acinus (assumed to behave electrically like a single cell) gives estimates of the ;transmitter equilibrium potential'. The values indicate that the neurotransmitter increases the membrane potassium conductance.4. The hyperpolarization of the acinus evoked by 10(-6)M dopamine in the bathing fluid is also associated with an increase in membrane potassium conductance.