The K-Cl cotransporter in the lobster stretch receptor neurone--a kinetic analysis.

Experiments were performed to define quantitatively the substrate (K(+) and Cl(-)) dependence of the transport function (production of equally large and oppositely directed K(+)and Cl(-) flows/currents) of an earlier (Theander et al., 1999) identified electroneutral K-Cl cotransporter in the slowly adapting stretch receptor neurone of the European lobster. The experiments were based on microelectrode techniques. This allowed us to perform steady-state measurements of the so-called "instantaneous" current-voltage relationships (around a holding voltage of -65 mV after a blockage of the cell's action potential and hyperpolarization-activated currents) and intracellular ion concentrations at various settings of the extracellular K(+) and Cl(-) concentrations. From the results, we could then define steady-state values of all of the cell's non-KCl cotransporter K(+) and Cl(-) currents. Finally, the negative sums of the inferred non-KCl cotransporter K(+) and Cl(-) currents could be taken as equivalents of the K-Cl cotransporter's K(+) and Cl(-) currents for the reason that, in steady state, all membrane currents add up to zero. For the cotransporter currents, thus inferred for a range from 2.5/410.5 to 40.0/448.0 mM external K(+)/Cl(-), we found that their absolute values increased in a nonlinear fashion from about 5 nA cell(-1) at the lowest, to about 20 nA cell(-1) at the highest external K(+)/Cl(-) concentrations. Formally, this relationship could be reproduced by a Hill function-based enzyme kinetic expression simulating inward and outward transmembrane electroneutral ion transports. Following insertion of this expression into a comprehensive model of electrical membrane functions and intracellular solute and solvent control in the lobster stretch receptor neurone, the model predictions suggested that the K-Cl cotransporter does play an important role in (a) keeping intracellular Cl(-) low for a proper function of the cell's inhibitory system, and (b) enabling rapid transmembrane K(+) shifts that provide for a stabilization of the cell's membrane voltage and membrane excitability in cases of varying extracellular K(+) concentrations. The model predictions gave, however, no clear evidence that the K-Cl cotransporter is critically involved in the cell's volume regulation in conditions of varying extracellular osmolalities.

Evidence that neuroepithelial endocrine cells control the spontaneous tone in guinea pig tracheal preparations.

The hypothesis that neuroepithelial endocrine (NEE) cells control spontaneous tone in isolated guinea pig tracheal preparations was examined. Epithelium-denuded preparations were unable to develop a normal oscillating tone in 12% oxygen (corresponding to systemic arterial oxygen levels) and, instead, developed a strong, smooth tone, similar to the "classic" tone in 94% oxygen. Inhibition of the hydrogen peroxide-producing NADPH oxidase in the NEE cells by 20 microM diphenyleneiodonium chloride transformed, in intact preparations in 94% oxygen, the tone from a strong, smooth type to an oscillating tone of considerably less force. Similar experiments in denuded preparations showed no change of tone and no oscillations. After pretreatment with the catalase inhibitor 3-amino-1,2, 4-triazole (1 mM), addition of 2 mM hydrogen peroxide to intact preparations displaying the oscillating tone caused a transformation to a strong, smooth type. These findings support the hypothesis that the spontaneous tone in this preparation is largely controlled by the oxygen-sensing NEE cells. For the first time, previous findings on isolated cells can be linked to effects in intact tissue preparations. The results also suggest that the regulation by the NEE cells involves the release of powerful relaxing and contracting factors from the epithelium.

Physiological oxygen concentration gives an oscillating spontaneous tone in guinea-pig tracheal preparations.

The spontaneous tone in isolated six-segment preparations of guinea-pig trachea was examined. In 12% oxygen (corresponding to normal systemic arterial oxygen pressure) the preparations developed a spontaneous tone with regular oscillations (6.6 min(-1)), usually grouped in so-called complexes (7.5 h(-1)). The average tone during an entire complex amounted to 12% of a maximum KCl-induced contraction. The complex tone was highly stable during observation periods of at least 4 h, and was reversibly transformed to the 'classical', smooth type when exposed to 94% oxygen. Stretch of preparations in low oxygen resulted in a fast, stable change of tone, while preparations in traditionally high oxygen reacted slower, and lost 40% of the active tension during the hour following stretch. Indomethacin (10 microM) did not eliminate the oscillating behaviour, but reduced the average size of the tone by 44%. Exposure to the C-fibre blocking agent capsaicin (50 microM) and the local anaesthetic lidocaine (1 mM) completely eliminated the oscillations and complexes, although the preparations retained a smooth tone. Atropine, propranolol and tetrodotoxin did not affect the complex tone. This study demonstrates for the first time that guinea-pig tracheal preparations that are exposed to near-physiological oxygen concentrations develop a new type of oscillating spontaneous tone, which is largely prostaglandin-independent, but appears to require transmitter release from sensory C-fibres. We argue that the complex tone is physiological, and that traditionally high oxygen (95%) probably results in non-physiological hyperoxic changes in this preparation.

Cl- transport in the lobster stretch receptor neurone.

Experiments were performed to identify mechanisms underlying non-leakage and non-H+/HCO3--linked transmembrane Cl- transports in the slowly adapting stretch receptor neurone of the European lobster, using intracellular microelectrode and pharmacological techniques. In methodological tests, it was established that direct estimates of intracellular Cl- with ion-sensitive microelectrodes are statistically identical with indirect estimates by means of a GABA method, where 1-2 mM GABA is transforming the cell's membrane voltage into its Cl- equilibrium voltage from which the Cl- concentration is inferred by the Nernst equation. From experiments using sodium orthovanadate and ethacrynic acid, supposed to block primary Cl- pumps, and bumetanide, supposed to block Na-K-Cl co-transporters, it appeared that neither of the two Cl- transport systems exists in the stretch receptor neurone. It could be shown, however, that the cell is equipped with an electroneutral K-Cl co-transporter that (a) is blockable by furosemide in high (Km approximately 350 microM), by 4-acetamido-4'-isothiocyanato-stilbene-2,2-disulphonic acid (SITS) in medium-high (Km approximately 35 microM), and by 4, 4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) in low (Km approximately 15 microM) doses, (b) is (transiently) activatable by (1 mM) n-ethylmaleimide, (c) is not suppressed by extracellular Rb+ or NH4+, and (d) is not directly coupled to any transmembrane transports of Na+, H+ or HCO3-. From functional tests, with varying transmembrane K+ and Cl- gradients, evidence obtained that the K-Cl co-transporter is able to reverse its transport direction and to adjust its transport rate in a considerable range. As a whole, the results speak in favour of the K-Cl co-transporter being responsible (a) for normally keeping the intracellular Cl- concentration at low levels, for an optimization of the cell's inhibitory system, and (b) for achieving fast transmembrane shifts of K+ (and Cl-), as a means of stabilizing the cell's membrane excitability in conditions of varying extracellular K+ concentrations.

Capsaicin can abolish spontaneous tone in guinea-pig trachealis.

The properties of spontaneous tone in isolated preparations of guinea-pig tracheal smooth muscle were examined. Experiments with control preparations revealed that 5-15 min after stretching the muscle with 0.15 mN, the spontaneous tone assumed a plateau value from which it declined gradually during the following hour. During the plateau, the force amounted to approximately 35% and 1 h later to approximately 20% of a maximum KC1 contraction. The tone was independent of tetrodotoxin, atropine and propranolol. Indomethacin quickly and completely relaxed the tone in 15 of 21 preparations. However, four preparations retained some tone even after 1 h of treatment. Exposure to the C-fibre influencing drug capsaicin resulted in a dose-dependent, reversible suppression of spontaneous tone, normally preceded by a transient increase in force. No spontaneous tone at all remained after 1 h of 10 microM capsaicin. This effect was also found in preparations pretreated with tetrodotoxin, atropine and propranolol. Preparations, deprived of spontaneous tone by capsaicin-treatment, contracted distinctly when exposed to 10 microM arachidonic acid. This contraction was almost completely abolished by indomethacin, which indicates that the prostaglandin synthesis is functioning after capsaicin treatment and, thus, that inhibition of this synthesis is not responsible for the capsaicin effect. Exposure to phosphoramidon increased the spontaneous tone almost threefold. Addition of 3 nM neurokinin A in the permanent presence of capsaicin gave weaker contractions in preparations where prostaglandin synthesis had been abolished by indomethacin, as compared to contractions in preparations with intact prostaglandin synthesis. The data indicate that a continuous release of tachykinins from sensory C-fibres is essential for the generation of spontaneous tone and that a combination of tachykinins and prostaglandins determine the size of the tone in this preparation.

Properties of electrolyte-filled glass microelectrodes: an experimental study.

The electrochemical and electrical properties of geometrically defined electrolyte-filled microelectrodes were studied at various transelectrode current passages, using radiotracer (38Cl and 42K) and electrical techniques. Geometrically, the electrodes were defined by their tip properties that, for standard (single-barrelled, 3.0 M KCl-filled, approximately 10 M[ohm]) electrodes implied a tip opening radius of 0.135 microm and a tip taper of 0.0215 microm/microm in the most distal (0-150 microm), and of 0.0105 microm/microm in the next most distal (150-1000 microm) tip regions. From the radiotracer studies it followed that (a) in the absence of transelectrode current passage, K+ and Cl- are leaking from the electrode tip in amounts corresponding to currents of +/- 3.8 nA, and (b) in the presence of transelectrode current passage, the flow of K+ and Cl- through the electrode tip changes with the transelectrode current in a statistically linear fashion so that K+ carries about 80% and Cl- about 20% of any electrode-injected current. From the electrical measurements it appeared that the standard electrodes are characterized by (a) a tip potential of -2.6 mV, and (b) a resistance that changes from an instantaneous, non-rectifying type to a steady state, outwardly rectifying type, within tenths of a second of constant current flow. The outward current rectification was seen to be reduced by raising [KCl] in the immersing solution, or by lowering it in the filling solution. Together, the observed electrode properties are consistent with the electrode electrolyte's solute and solvent turnover being governed by electro-osmotic as well as by electrodiffusion laws.

Properties of electrolyte-filled glass microelectrodes: a model analysis.

A novel dynamic mathematical microelectrode model (a model of solvent and solute kinetics in electrolyte-filled microelectrodes) was deduced from experimental observations made on standard (single-barrelled, 3.0 M KCl-filled, approximately 10 M[ohm]) electrodes using (a) electrodiffusion, electro-osmosis, and continuity equations that were placed into the constraints of electrode geometry, and (b) handbook/textbook parameter values, only. The model proved to be able to faithfully reproduce all observed electrochemical and electrical electrode properties, i.e. even those that constituted no part of the model's experimental basis. In theoretical tests, the model shows, for the standard electrode that (a) inside the electrode, any profiles in electrical potential and electrolyte concentration are occurring at the most distal part (approximately 50 microm) of the tip region, (b) asymmetrical shifts in electrolyte concentration just inside the electrode tip opening are the true cause of the electrode's current rectification, and (c) strong transelectrode currents are producing water flows across the electrode orifice that may affect the volume of smaller and medium-sized cells. In further tests, the model shows, among other things, for non-standard electrodes that (a) decreasing the electrode electrolyte concentration will give rise to marked decreases in electrolyte leakage from the electrode, but only very minor changes in tip potential, and (b) increasing the surface charge of the electrode glass (increases in zeta potential) and/or decreasing the electrode electrolyte concentration will produce increases in electro-osmotic water transport, which may be desirable for the intracellular injection of water-soluble (electro-neutral) substances.

Analysis of leak current properties in the lobster stretch receptor neurone.

Experiments were performed to characterize the so-called leak current of the slowly adapting stretch receptor neurone of the European lobster with respect to its ionic basis, its kinetics and its pharmacology. Estimates of the leak current were obtained by subtraction of a Na-K pump current and of an unspecific impalement current from a non-dynamic ('instantaneous') current, recorded in a voltage range from approximately -120 to approximately -30 mV, after blockage of spike-generating currents and a hyperpolarization-activated inwardly rectifying current (Q-current). The leak current, estimated in this way, was seen to reverse direction at the cell's K+ equilibrium voltage, thus indicating that it is carried by K+ passing through channels which, also, proved to be permeable to Rb+ and NH4+, but not permeable to Na+ or Cl- to any significant extent. Kinetically, the leak current was found to be characterized by being enhanced by increases in extracellular K+ and by being subject to outward rectification, most distinctly at elevated extracellular [K+]. In quantitative terms, these kinetic properties could be accounted for by a mathematical model comprising (1) a one-site two-barrier Eyring formulation describing ion permeation through membrane channels and (2) an ordinary dose-response relationship describing the channel-opening effect of K+ at an extracellular regulatory site. Pharmacologically, the leak current proved to be distinguished by being reversibly blockable, in a non-voltage dependent manner, by CO2+ (Kd = 0.9 mM, Hill coefficient 1.1) and procaine, but not by Ba2+, Gd3+, bupivacaine (a local anesthetic), or other K+ channel blockers such as TEA, 4-AP and Cs+. It is concluded that, in native unimpaled cells, the K+ carried leak current (1) is setting the resting voltage together with the (mainly) Na(+)-carried Q-current and the Na-K pump current, (2) is determining the cell's firing threshold, together with the spike generating currents, and (3) is also stabilizing the cell's membrane excitability in conditions of varying extracellular [K+], by virtue of its K+ sensitivity.

Functional effects of a hyperpolarization-activated membrane current in the lobster stretch receptor neurone.

The functional effects of a hyperpolarization-activated membrane current (IQ) in the slowly adapting lobster stretch receptor neurone were investigated. From comparisons between changes in membrane excitability due to blockage of IQ by Cs+, in normally impaled and native unimpaled (Edman et al. 1987 b) cells, it could be concluded that the resting voltage of native cells is distinctly more negative than -65 mV (average membrane voltage of impaled cells) and, therefore, under the control of an activated IQ. Starting from this conclusion, impaled cells were polarized to holding (resting) voltages around -75 mV and their polarization and excitability properties studied after tetanic impulse activity and variation of various external influences (K+, pH, temperature), both in control conditions and after blockage of IQ by 2 mM Cs+. It was found that an unblocked IQ (a) greatly accelerates the initial (90%) decay of post-tetanic hyperpolarization, and (b) depresses distinctly any polarization and excitability alterations due to increases in extracellular K+ concentration (from 2.5 to 10 mM), variations in extracellular pH (between 6.4 and 8.6), and changes in temperature (between 14 and 24 degrees C). It was inferred that in well polarized cells, IQ plays a role as a stabilizer of membrane polarization and excitability in conditions of varying external influences. From a model study of IQ it could be concluded that, with its slow dynamic responses, the current is well adapted to its functional purposes and to the rather slow homeostatic effects of the cell's Na-K pump.

Spontaneous vasomotion in hamster cheek pouch arterioles in varying experimental conditions.

The spontaneous rhythmic motor activity (vasomotion) of arterioles was studied in vivo in the hamster cheek pouch by means of intravital microscopy. In control conditions, arteriolar vasomotion was regularly present in healthy preparations, independent of anesthesia (pentobarbital sodium or alpha-chloralose), composition of the superfusate [tris(hydroxymethyl)aminomethane-buffered or N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid-supported HCO3(-)-buffered saline solutions) or a combined nerve, alpha- and beta- receptor blockade. In arterioles with internal diameters between 13 and 52 microns, the vasomotion frequency (3-15 cycles/min) and amplitude (2-10 microns) were not significantly correlated to vessel size. The frequency and amplitude of the spontaneous arteriolar vasomotion could be modified by changes in the physical and chemical environment of the preparation. Thus, addition of pinacidil, a K+ channel activator, to the superfusate dose dependently decreased and finally suppressed vasomotion, in combination with an increase of the vessel diameter. These effects could be counteracted if glibenclamide (10(-6) M), a K+ channel blocker, was added to the superfusing solution. In the absence of any major changes in vessel diameter, vasomotion was also abolished by increasing the PO2 (from approximately 15 to 30 mmHg), varying the pH (from 7.40 to 7.22 or 7.65), and lowering the temperature to 15 degrees C in the superfusion solution. The abolition of vasomotion observed because of increased PO2 and changes in pH could be reversed by addition of glibenclamide (10(-6) M) or tetraethylammonium chloride (TEA, 5 x 10(-3) M), another K+ channel blocker, to the superfusion solution; in the case of lowered temperature only glibenclamide was effective.(ABSTRACT TRUNCATED AT 250 WORDS)

Ion (H+, Ca2+, Co2+) and temperature effects on a hyperpolarization-activated membrane current in the lobster stretch receptor neurone.

The effects of some ions (H+, Ca2+, Co2+) and temperature on the Q-channel (a channel which conducts a hyperpolarization-activated membrane current, IQ) was investigated in the slowly adapting lobster stretch receptor neurone. It was found: (1) that increases in pH cause an increase in the maximum channel conductance; (2) that Co2+ (1 mM) and increases in extracellular Ca2+ produce a positive shift of the relationship between the membrane voltage and the steady-state channel activation along the voltage scale; (3) that Co2+ and decreases in extracellular pH cause a decrease in rate of the voltage-dependent step of the channel's gating reaction; (4) that none of the ions has an effect on the rate of the voltage-independent step of the gating reaction; and (5) that Co2+ and K+ do not interfere with each other's actions on the channel's gating reaction and maximum ion conductivity. Increases in temperature from 13 to 23 degrees C were found: (1) to cause an increase in the maximum Q-channel conductance by a factor of 1.7; (2) to have no effect on the voltage dependence of the channel's gating reaction; but (3) to lead to an increase in rate of the gating reaction by a factor of about five for the voltage-dependent, and by a factor of about three for the voltage-independent gating step. The temperature sensitivity of the IQ mechanism proved to be mathematically reproducible by a previously developed, and slightly modified Q-channel model (Edman et al. 1987, Edman & Grampp 1989), and to be of no significance in the control of membrane excitability in a thermally unstable environment.

Effects of hypertonic NaCl solution on microvascular haemodynamics in normo- and hypovolaemia.

The aims of this study were to investigate possible resuscitation effects of a single, 10-min, 350-microliters intravenous infusion of 7.5% NaCl in hamsters in hemorrhagic shock and to compare the effects of such infusion with an identical one of 0.9% NaCl on the hamster cheek pouch microcirculation during normovolaemia and after acute bleeding to a hypotension level of about 40 mmHg. No significant differences could be detected between the effects of either infusion given to normovolaemic normotensive hamsters. In the animals subjected to haemorrhage, upon bleeding, arterioles larger than 40 microns constricted, arterioles smaller than 40 microns dilated and venular diameter did not change, while blood flow decreased in all vessels. The main differences between the infusions after haemorrhage were a significant increase in mean arterial pressure and arteriolar blood flow, venoconstriction and a tendency for the smaller arterioles to remain more dilated and the larger ones more constricted after the hypertonic infusion. Central nervous and/or reflex excitation of the sympathetic nervous system could account for the constriction of venules and larger arterioles, while a direct effect of hyperosmolarity could explain the dilatation of the smaller arterioles. The study can therefore help to explain some of the mechanisms underlying the reported resuscitation effect of 7.5% NaCl infusion in animals during severe haemorrhagic hypovolaemia.

Ion permeation through hyperpolarization-activated membrane channels (Q-channels) in the lobster stretch receptor neurone.

In the lobster stretch receptor neurone it is possible to demonstrate a hyperpolarization-activated membrane current, IQ, which appears to be carried by Na+ and K+ in combination. The ion permeability of the membrane channel conducting this current (Q-channel) was investigated using conventional electrophysiological techniques including intracellular ion concentration measurements. It was found that none of the ions choline, protonated Tris, Rb+, NH4+, Li+, and protonated hydroxylamine was able to pass through the Q-channel which, thus, appears to be permeable to Na+ and K+ only. With increasing extracellular Na+ concentrations, IQ was increased up to a saturation level. This behaviour could be described by a one-site-two-barriers version of the Eyring rate theory, assuming that the permeant ions are turned over at specific saturable channel sites which 'sense' 70% of the transmembrane potential difference. With increasing extracellular K+ concentrations, IQ was increased in accordance with a simple first-order dose-response relationship. This finding can be accounted for by assuming that K+ increases all rates of turn-over of the permeant ions at their specific sites by similar relative amounts. Changes in extracellular Na+ and K+ concentrations were found to have no effect on the gating properties of the Q-channel.

Effects of hypertonic NaCl solution on the hamster cheek pouch microcirculation in normo- and hypovolemia.

The aim of this study was to evaluate the effects of a single, 10 min, intravenous infusion of a hypertonic NaCl solution [2400 mOsm/l; infused volume 0.35 +/- 0.03 ml (SEM)] on the hamster cheek pouch microcirculation during normovolemia and after acute bleeding to a hypotension level of about 40 mmHg. Upon bleeding, the arterial pressure dropped to 39 mu 1 mmHg, arterioles greater than 40 microns constricted 12 +/- 3% (from their control value), arterioles less than 40 microns dilated 6 +/- 2%, venules stayed largely unchanged, while RBC velocity and volume flow decreased 57 +/- 7% in all vessels. During the subsequent hypertonic NaCl infusion, the arterial pressure increased rapidly to a new steady state level of 66 +/- 3 mmHg. After the infusion, the large arterioles stayed constricted 11 +/- 1% and the small arterioles dilated 7 +/- 1% for a 1-h observation period. The venules constricted initially by 6 +/- 2% and returned to control diameter in 30-40 min. RBC velocity and calculated volume flow returned to the pre-hemorrhage control values in about 10 min for the arterioles and in 40 min for the venules. An identical hypertonic infusion given to normovolemic hamsters caused no significant alterations of the measured variables.

Effects of hypertonic NaCl solution on the hamster cheek pouch microcirculation in normo- and hypovolemia

The aim of this study was evaluate the effects of a single, 10 min, intravenous infusion of a hyperonic NaCl solution [2.400 mOsm/l; infused volume 0.35 ñ 0.03 ml (SEM) on the hamster cheek pouch microcirculation during normovolemia and after acute bleeding to a hypotension level of about 40 mmHg. Upon bleeding, the arterial pressure dropped to 39 µ 1 mmHg, arterioles > 40 -m constricted 12 ñ 3% (from their control value), arterioles < 40 µm dilated 6 ñ 2%, venules stayed largely unchanged, while RBC velocity and volume flow decreased 57 ñ 7% in all vessels. Durign the subsequent hypertonic NaCl infusion, the arterial pressure increased rapidly to a new steady state level of 66 ñ 3 mmHg. After the infusion, the large arterioles stayed constricted 11 ñ 1% and the small arterioles dilated 7 ñ 1% for a 1-h observation period. The venules constricted iniatially by 6 ñ 2% and returned to control diameter in 30-40 min. RBC velocity and calculated volume flow returned to the pre-hemorrhage cotrol values in about 10 min for the arterioles and in 40 min for the venules. An identical hypertonic infusion given to normovolemic hamsters caused no significant alterations of the measured variables

Analysis of gated membrane currents and mechanisms of firing control in the rapidly adapting lobster stretch receptor neurone.

1. The gated membrane currents (a tetrodotoxin-sensitive Na+ current and a tetraethylammonium- and 4-aminopyridine-sensitive K+ current) of the rapidly adapting stretch receptor neurone of lobster were investigated with respect to their kinetic properties using electrophysiological, pharmacological, and mathematical techniques. 2. The currents were found to be controlled by slow inactivations as well as by fast Hodgkin-Huxley (1952) gating processes. They could be described by kinetic expressions which differed from those inferred for the slowly adapting receptor (Gestrelius & Grampp, 1983a; Gestrelius, Grampp & Sjölin, 1983) only with respect to some of the parameter values. 3. With these expressions, and additional equations for the cell's pump and leak current components (Edman, Gestrelius & Grampp, 1986), a mathematical receptor model was formulated which accurately predicts the impulse activity of the living preparation in different functional circumstances and which, therefore, was adopted as an appropriate theory of firing regulation. 4. From a model analysis it appeared (a) that the 'rapid' adaptation of the receptor's impulse activity is mainly an effect of slow Na+ current inactivation starting a regenerative process of accommodation which, basically, is due to a small ratio of subthreshold Na+ to K+ currents; (b) that, because of the transmembrane Na+ influx being limited by accommodation, impulse firing is only little affected by a Na+-dependent pump current activation; and (c) that the phenomenon of increased firing frequency initially during prolonged stimulation ('negative adaptation') is an effect of the slow K+ current inactivation being faster than the slow Na+ current inactivation at comparable degrees of membrane polarization. 5. From further model studies it also appeared that, during depolarizations between successive action potentials evoked by constant stimulation, the membrane behaves like a high-resistance constant-current generator feeding into a short-circuiting capacitor. In consequence, the cell's stimulus sensitivity (change in firing frequency with stimulation strength) is, at functionally relevant stimulation intensities, mainly determined by the membrane capacitance and by the amplitude of the interspike membrane depolarization while, at higher stimulation intensities and firing frequencies, it becomes more and more a function of the spike duration itself.

Current activation by membrane hyperpolarization in the slowly adapting lobster stretch receptor neurone.

1. A polarization-induced membrane current, IQ, was investigated in the slowly adapting lobster stretch receptor neurone using conventional electrophysiological techniques including intracellular ion measurements. 2. The current was readily blocked by Cs+ in a voltage-dependent manner, but proved to be unaffected by tetrodotoxin, tetraethylammonium and 4-aminopyridine. 3. From an analysis of the ionic basis of IQ, it appeared that the current is carried by both Na+ and K+ through a membrane channel whose permeability for K+ is about six times larger than that for Na+ in a normal ionic environment. In the presence of reduced external Na+ concentration the Q-channel increases its permeability for both Na+ and K+, but more so for Na+ than for K+. 4. Kinetically, IQ was found to be characterized by a steep sigmoidal relationship between membrane voltage and steady-state current activation, and by a bell-shaped relationship between membrane voltage and the time constant of the exponential phase of current activation or deactivation. A significant feature of the latter relationship is a tendency to level off at finite time-constant values in both strongly hyperpolarizing and strongly depolarizing voltage regions. 5. From the experiments a mathematical IQ model was inferred. This model was based on constant-field and channel-gating kinetics involving a voltage-dependent reaction step in series with a voltage-independent reaction step. The model was found to successfully reproduce IQ behaviour in the living preparation. 6. Functionally, the activation of IQ was found to play a role in setting the cell's resting polarization and membrane excitability. This function was inferred from experiments on unimpaled cells in which it was possible to demonstrate some overlap between the voltage ranges of IQ activation and impulse initiation. In addition, in impaled cells the activation of IQ was found to cause some shortening of post-tetanic membrane hyperpolarization and to accelerate, thereby, the post-tetanic restoration of membrane excitability to control levels.

Transmembrane ion balance in slowly and rapidly adapting lobster stretch receptor neurones.

The transmembrane exchange of Na+, K+, and Cl- in slowly and rapidly adapting lobster stretch receptor neurones was studied using ion-sensitive microelectrodes in combination with conventional electrophysiological techniques. The investigation was founded on the assumption that the transmembrane ion exchange is accomplished by active and passive transports which add up to zero in steady state for each ion involved. The active transports are assumed to include Na+ and K+ transports driven by an electrogenic Na-K pump. To these transports are also added equimolar fluxes of K+ and Cl- leaking from the impaling micro-electrode. The passive transports are assumed to pass through membrane channels in accordance with constant field kinetics. For a quantitative evaluation of the transmembrane ion exchange in resting conditions measurements were made of the resting concentrations of Na+, K+ and Cl-; the voltage dependence of the ungated leak current; and ouabain-induced changes in resting membrane current and intracellular ion concentrations. From the results it follows that both the resting pump current and the leak permeabilities for the ions investigated have values which do not seem to differ between slowly and rapidly adapting receptor neurones. For a quantitative evaluation of the relation between internal Na+ and pump current production, measurements were made of the outward membrane current as a function of internal Na+ and K+ following a shift of these ions by means of prolonged repetitive impulse activation. It was found that the investigated relation is compatible with Garay-Garrahan kinetics (Garay & Garrahan, 1973) in both receptor neurones, but the results imply a larger maximum Na+-extrusion capacity in slowly than in rapidly adapting cells. From recordings of the time course of post-tetanic normalization of both the membrane current and intracellular Na+ concentration, cell volume values could be deduced which were closely similar in slowly and rapidly adapting receptors. A corresponding similarity was also found for the cell area which was derived from membrane capacitance measurements.