A voltage-sensitive persistent calcium conductance in neuronal somata of Helix.

1. An intracellular voltage clamp in conjunction with a patch pipette utilizing feed-back to monitor local current from the soma membrane were used to analyse transient and stationary currents in bursting pacemaker neurones in Helix pomatia and H. levantina. 2. A weak, net inward current flows during small (less than or equal 20 mV) depolarizations. This current exhibits slow activation kinetics, persistence during prolonged depolarization, and slow turning off at end of depolarization. Consequently, the steady-state current-voltage curve exhibits a region of negative resistance from about -55 to -35 mV. 3. The slow inward current and the negative resistance characteristic are rapidly and completely abolished by substitution of Co2+ or La3+ for Ca2+ and are partially blocked by the Ca-blocking drug D-600. Substitution of Tris or glucose for Na+ significantly reduces the inward current only after 15-20 min exposure, recovery being equally slow. 4. The inward current and the negative resistance characteristic of the I-V curve are greatly enhanced by Ba2+ substitution for Ca2+. This is ascribed in part to Ba2+ carrying current through the slow inward current channels and in part to a suppression of the late K+ current by Ba2+. 5. The inward current is also present in many non-bursting neurones but fails to appear as a net inward current due to short circuiting by a leakage current or by the delayed potassium current. In these cells the slow inward current contributes to inward going rectification. Replacement of Ca2+ with Ba2+ enhances the current so as to produce a net inward current during small depolarizations in these neurones. 6. It is concluded that the slow inward current is carried primarily by Ca2+ in the soma membrane of bursting pace-maker neurones and a number of non-bursting cells examined in the parietal ganglion of Helix. 7. The sensitivity to small depolarizations and persistence during prolonged depolarization suggests two roles for the Ca system in the generation of slow pace-maker oscillations. In this model the Ca system contributes to the slow depolarization which constitutes the onset of the pace-maker wave, and also contributes to the increment in [Ca] in which activates the Ca-sensitive K+ conductance responsible for repolarization. The inhibition of spontaneous bursting by Ca-blocking agents supports this model.     

High Activity K+ Channels in Rat Hippocampal Neurones Maintained in Culture

A channel was identified in cell-attached recordings in rat hippocampal neurones maintained in culture. This channel, which was highly active at the resting membrane potential, was present in most (73 %) patches studied. The channel was characterized by long duration openings and a high open probability (Po, mean value 0.73 at -70 mV) at negative patch potentials with mild voltage dependence over the range -40 to -120 mV. It showed inward rectification. There were up to five active channels in cell-attached recordings in experiments where the cells were bathed in sodium-containing Locke solution. The single channel conductances in cell-attached recordings with 140 or 40 mM K+ in the patch pipette were 26 and 12 pS, respectively. The channel was therefore selective for K+ over Na+. The channel was not permeable to Rb+ ions. The single channel conductance was 24 pS in excised inside-out patches bathed in symmetrical K+ (140 mM) solutions. Examination of the channel kinetics revealed that both the open and closed time distributions could be fitted by the sum of three exponentials, there being no pronounced voltage sensitivity between -60 and -120 mV. The 26 pS K+ channel was insensitive to extracellular TEA, apamin, 4-AP and dequalinium. Neither was it sensitive to intracellular Ca2+. Extracellular Ba2+ was effective in reversibly blocking the channel, the IC50 being 2.0 mM. There was no obvious effect of bath application of the K+ channel opener, lemakalim, or a cAMP analogue. This channel appears to contribute a significant proportion (at least 30 %) of the resting conductance in these neurones.     

Evidence for nonsynaptic neuronal interaction.

1. Evidence is presented that synaptic interactions within and between the statocyst and visual pathways of Hermissenda are eliminated after 0.5-4 min exposure to 20-40 mM Co2+. 2. Synaptic blockade was also produced by perfusion with low Ca2+ (5mM) plus 10-20 mM Co2+. 3. Depolarization of hair cells by impulses of type A photoreceptors remains after the same exposure to Co2+, or low Ca2+ plus Co2+. 4. The increased resistance previously observed during this depolarization of hair cells cannot be observed after exposure to Co2+. 5. The depolarization which remains after exposure to Co2+ did not change with different levels of membrane potential from -20 mV below to +10 mV above the resting level. 6. The time course of potassium accumulation, monitored by the amplitude of the type A impulse afterpotential, closely followed the time course of hair cell depolarization and also of changes in the amplitude of the hair cell afterpotential. 7. The depolarization of hair cells by type A impulses decreased with increased extracellular potassium, but was only slightly reduced by lowered extracellular potassium. 8. The amount of potassium accumulation following a type A impulse train could be estimated from the effects of changes in extracellular potassium in the perfusate on the type A impulse afterpotential. From this extimated increase of extracellular potassium it was possible to predict with some accuracy, the observed hair cell depolarization. 9. Although type A cells are not electrically coupled to ipsilateral hair cells, firing of these hair cells slightly depolarized the type A photoreceptor which excites them. 10. Strophanthidin (10-4 M) did not block the depolarization of hair cells by type A impulses. 11. The data are evidence for nonsynaptic excitation of hair cells by type A photoreceptor impulses. The data are also consistent with the interpretation that the excitation arises from potassium accumulation around the type A and hair cell axonal membranes.     

Whole-cell K+ currents in isolated rabbit corneal epithelial cells.

Membrane currents were recorded from enzymatically isolated cells from basal layers of rabbit corneal epithelium by the whole-cell clamp technique. Pipettes contained 140.4 mM KCl and extracellular K+ concentration was varied. The membrane currents on step voltage changes were rectangular currents with some fluctuations. The fluctuations disappeared near the zero-current potential. The reversal potential in normal Tyrode's solution with 5.4 mM K+ was -57.8 +/- 6.2 mV (mean +/- S.D., n = 10). Increasing [K+]o from 5.4 to 140.4 mM shifted the reversal potentials in the positive direction with a slope of 41.0 mV/decade. Concomitant depolarization of the resting potential was observed on increasing [K+]o. The whole-cell currents were blocked by Cs+ or Ba2+. These suggest that the major current component in the corneal epithelial cells in K+.     

Effects of guanidine on transmitter release and neuronal excitability.

1. Guanidine hydrochloride (CH5N3-HCl) was applied to frog neuromuscular junctions blocked by reduced external Ca2+, or increased external Mg2+ concentration, or by both. Guanidine produced a dose-dependent increase in the average number of quanta released by presynaptic action potentials, the threshold dose being 0-1-0-2 mM. No post-synaptic effects were observed. 2. Guanidine also increased the excitability of the motor nerve fibres, as evidenced by multiple firing to single electrical stimuli and finally by spontaneous action potentials. These effects were studied in greater detail in giant axons (Müller axons) in the spinal cord of lamprey. Exposure to guanidine produced in these axons a progressive increase in excitability, manifested by repetitive firing to a single electrical stimulus, spontaneous membrane potential oscillations and spontaneous bursts of action potentials. Guanidine had no effect on the resting potential. 3. The effect of guanidine on the excitability of Müller axons was mimicked in every detail simply by reducing the divalent cation concentration of the bathing solution. 4. Guanidine also produced dose-dependent increases in the duration of action potentials in Müller axons. This effect always preceded in time the appearance of the excitability effects and was not mimicked by reducing the divalent cation concentration. It is suggested that the broadening of the action potential is separate from the excitability effects and may reflect a decrease of delayed rectification. 5. Guanidine (0-3 mM) increased the frequency of miniature end-plate potentials (min. e.p.p.) in solutions containing 2-11 mM-K+ in such a way as to shift the relationship between min. e.p.p. frequency and extracellular K+ toward lower values of K+. This effect was interpreted to mean that guanidine produced a depolarization of the nerve terminal which summed with the depolarization produced by a given concentration of K+. The calculated depolarization produced by 0-3 mM guanidine was 5-7 mV. 6. The effects of guanidine on evoked transmitter release, excitability, and min. e.p.p. frequency are consistent with a hypothesis which states that guanidine binds at or near fixed negative changes on the outside of nerve membrane and reduces the screening effect of divalent cations.     

Do hippocampal CA1 neurones of the adult rat possess a slow inwardly-rectifying chloride conductance?

The presence of an inwardly-rectifying Cl- current was studied in hippocampal CA1 neurones using sharp intracellular microelectrodes. Following pharmacological block of the hyperpolarization-activated Ih current, a slow depolarizing sag of hyperpolarizing electrotonic potentials appeared when the microelectrode contained KCI or CsCl, but was absent with K acetate; the sag threshold was approximately 10 mV negative to rest. Under voltage clamp, slow inward current relaxations were observed on stepping to potentials between -80 and -130 mV; the activation time constant decreased with increasing hyperpolarization (>1 s at -130 mV). This conductance (termed G(Cl,slow)) was partially depressed by 100 microM Zn2+. We propose that G(Cl,slow) can contribute significantly to the resting conductance of adult hippocampal CA1 neurones only when they are loaded with Cl- via the recording electrode.     

Cell electrical potentials during enhanced sodium extrusion in guinea-pig kidney cortex slices.

1. Experiments were performed on outermost slices of the guinea-pig kidney which are mainly made up of proximal tubular cells. 2. Kidney cells loaded with Na+ by chilling at 0.6 degrees C for 2.5 hr, when subsequently rewarmed to 25 degrees C in a medium containing 16 mM-K+ extrude Na+ at enhanced speed for about 10 min. This Na+ movement is accompanied by efflux of Cl and influx of K+. 3. Measurements of cell potential during enhanced Na+ extrusion show that cells hyperpolarize to values about 30 mV more negative than the K+ equilibrium potential. 4. This hyperpolarization is only partly inhibited by 1 mM ouabain or by 2 mM ethacrynic acid but both agents added together suppress it completely. 5. With 16 mM-Rb instead of 16 mM-K the hyperpolarization is smaller. 6. A diminished extracellular K+ concentration outside of the cells, within the slice, can account for only a small part of the hyperpolarization. 7. The hyperpolarization is proportional to the rate of Na+ pumping. 8. Cl- seems to shunt the hyperpolarization to a greater extent than K+. 9. It is concluded that Na+ extrusion is capable of transferring electric charge across the membrane.     

Chemical hypoxia in hippocampal pyramidal cells affects membrane potential differentially depending on resting potential

The aim of the present study was to analyze the effect of chemical hypoxia (cyanide) on the membrane potential of hippocampal CA1 neurons and to elucidate the reason for previously found differences in the reaction to hypoxia in these cells. Recordings were performed in brain slices from 8-19-day-old rats with whole-cell patch clamp on cells identified with near-infrared video microscopy. Cyanide (0.1-2.0 mM) caused different responses depending on the resting potential of the cells: hyperpolarization (or an initial depolarization followed by hyperpolarization) was generally seen in cells with less negative resting potential (-56+/-6.1 mV), and depolarization in cells with more negative resting potential (-62+/-3.4 mV). After 10 min in cyanide the membrane potential in all cells had reached approximately the same level (-62+/-5.8 mV), the direction and size of the voltage response having an inverse linear relation to the resting potential (k=-0.98, r=0.71). The direction of the cyanide response was not reversed by current injection (depolarization by 12 mV) in cells with more negative resting potential (-60+/-2.8 mV). Wash out of cyanide caused hyperpolarization in 70% of the cells. Presence of ouabain (2 microM) resulted in pronounced depolarization during cyanide perfusion, and potentiated the hyperpolarization during wash out indicating that this part of the effect is not dependent on a reactivation of the Na/K pump. In conclusion, chemical hypoxia with cyanide changes the membrane potential in CA1 cells in size and direction depending on the original resting potential of the cells. The present findings suggested that cyanide activated not only K+ channels but in addition increased a Na+ current which has a more positive equilibrium potential.     

Temperature-sensitive activation of G-protein regulating the resting membrane conductance of Aplysia neurons

Raising the temperature from 22 to 32 degrees C induced a marked hyperpolarization (15-30 mV) associated with an increase in membrane conductance of Aplysia neurons, whereas lowering the temperature from 22 to 12 degrees C caused a significant depolarization (10-20 mV) with a decrease in conductance. These temperature effects were far greater than those expected from the Nernst equation. The reversal potentials of these temperature responses corresponded with the equilibrium potential of K+, suggesting these responses were produced by opening or closing of K+ channels. Ouabain (5 x 10(-4) M) did not affect these temperature responses though it depolarized all cells examined (5-25 mV). Intracellularly injected guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) selectively depressed the response to warming without affecting the response to cooling. Intracellular application of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) produced a gradual increase in K+ conductance of the resting membrane and apparently depressed the response to warming while it markedly augmented the response to cooling. These results suggest that GTP binding protein can be activated thermally to open K+ channels without receptor stimulation. It is significant that the resting membrane potential of the neuron in the central nervous system may be regulated not only by Na+ pump but also by spontaneous activation of a certain GTP-binding protein, at least in Aplysia.     

Identification of Calcium-Activated Potassium Channels in Cultured Equine Sweat Gland Epithelial Cells

The patch-clamp recording technique was used to examine the properties of the K+ channels in cultured equine sweat gland epithelial cells. With symmetric K+ solutions (140 mM), a single population of K+ channels was identified with a slope conductance of 187 pS and a reversal potential of around 0 mV. The channel was selective for K+ over Na+. Channel activity was increased by membrane depolarization. A 10-fold increase in [Ca2+]i produced an approximate 60 mV negative shift in the open state probability (Popen)-voltage curve. Externally applied tetraethylammonium ions (TEA+) caused a rapid and flickery block of the channel and reduced the unitary current amplitude. TEA+ bound to the blocking site with stoichiometry of 1:1 and with a dissociation constant (Kd) of 186 +/- 27 microM at +40 mV. A weak voltage dependence of Kd was observed. Iberiotoxin (100 nM) reduced Popen but had no effect on single-channel conductance. Neither glibenclamide (10 microM) nor intracellular adenosine 5'-triphosphate (ATP, 1 mM) altered channel activity. In addition, ATP, when applied extracellularly, transiently activated the channel by increasing Popen. Channel activity was low around the resting membrane potential in the intact epithelia, indicating that these channels might not contribute to the resting K+ conductance. However, the channel could be activated in a regulated manner. The K+ channels may play a role in transepithelial fluid secretion in sweat gland.     

FMRFamide effects on membrane properties of heart cells isolated from the leech, Hirudo medicinalis.

1. The effects of the cardioactive peptide FMRFamide were tested on enzymatically dissociated muscle cells isolated from hearts of the leech. These cells were normally quiescent, with resting potentials near -60 mV. 2. Superfusion of FMRFamide induced a strong depolarization in isolated heart cells (e.g., greater than 40 mV with 10(-6) M FMRFamide). The depolarization was maintained in the continued presence of peptide and persisted long after its removal. Less frequently, FMRFamide superfusion elicited an episodic polarization rhythm. 3. The response of isolated heart cells to bath-applied FMRFamide showed a 1- to 2-min latency. The latency decreased with repeated applications of FMRFamide. 4. The FMRFamide response was diminished by Na+ replacement but persisted with Ca2+ channel blockade. 5. In voltage-clamped heart cells (-60 mv), superfusion of FMRFamide elicited a slow inward current with a transient and a sustained component. 6. Current-voltage (I-V) curves during FMRFamide superfusion in normal leech saline showed that FMRFamide also enhanced voltage-dependent outward currents activated at depolarized levels. 7. Under conditions in which K+ currents were substantially blocked, the FMRFamide-dependent I-V curve was net inward from -90 to +50 mV. A voltage-dependent component was blocked by Co2+ and a linear component by Na+ replacement. 8. We conclude that FMRFamide elicits a persistent inward current with a Na+ component and in addition modulates both voltage-dependent Ca2+ and K+ currents that may contribute to the normal myogenic activity of leech heart muscle cells.     

Mechanisms underlying excitatory effects of thyrotropin-releasing hormone on rat hypoglossal motoneurons in vitro.

1. The hypoglossal motor nucleus contains binding sites for the neuropeptide thyrotropin-releasing hormone (TRH) and is innervated by TRH-containing fibers. Although excitatory effects of TRH on hypoglossal motoneurons (HMs) have been described, the ionic mechanisms by which TRH exerts such effects have not been fully elucidated. Therefore, we investigated the effects of TRH on HMs in transverse slices of rat brainstem with intracellular recording techniques. 2. TRH was applied by perfusion (0.1-10 microM) or by pressure ejection (1.0 microM), while HMs were recorded in current or voltage clamp. In all cells tested, TRH caused a depolarization and/or the development of an inward current. These effects were fully reversible, dose dependent, and showed only modest desensitization with long applications. In addition, although TRH increased synaptic activity in many cells, the depolarizing response to TRH was maintained in tetrodotoxin (0.5-1.0 microM)-containing or in a nominally Ca(2+)-free perfusate containing 2 mM Mn2+. Thus TRH acts directly on HMs to cause the depolarization. 3. Hyperpolarizing current (or voltage) steps superimposed on the TRH-induced depolarization (or inward current) revealed a decreased input conductance. Extrapolated instantaneous current-voltage relationships obtained before and at the peak of the response to TRH intersected (i.e., reversed) at -101 mV, negative to the expected K+ equilibrium potential (EK). When extracellular [K+] was raised from 3 to 12 mM, the reversal potential was shifted in the depolarizing direction and the magnitude of the TRH-induced depolarization was diminished. Moreover, the TRH response was enhanced in size from depolarized potentials (i.e., further from EK). Taken together, these results indicate that TRH depolarizes HMs, in part, by decreasing a resting K+ conductance. 4. Similar to TRH, bath-application of 2 mM Ba2+ caused a depolarization associated with decreased conductance, suggesting that Ba2+ also blocks a resting K+ conductance. The Ba(2+)-sensitive and TRH-sensitive resting K+ conductances are apparently identical; in the presence of Ba2+, the customary TRH-induced decrease in conductance was occluded. 5. It is noteworthy that the TRH-induced inward current (ITRH), although diminished, was not entirely blocked by Ba2+. This second Ba(2+)-insensitive component of ITRH was not associated with a measurable change in input conductance. It was especially evident during current-clamp recordings, when the diminutive TRH-induced current was still capable of causing a substantial depolarization. The ionic basis of the residual TRH-induced inward current remains to be determined. 6. We investigated the functional consequences of these mechanisms of action of TRH on spike firing behavior of HMs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ionic basis of resting membrane potential in frog taste cells

Intracellular recording of the resting membrane potential was made from taste cells of the bullfrog by replacing the interstitial fluid surrounding the cells by various physiological saline solutions. The resting potential of the taste cell was -28 +/- 4 mV (mean +/- S.E.) after replacement of the interstitial fluid by normal Ringer solution. The resting potential was very much dependent on the interstitial K+ concentration ([K+]). Tenfold increase in [K+]o decreased the resting potential by 18 mV. Total removal of interstitial Na+ increased the resting potential by 40%. Ouabain in a concentration of 0.5 nM decreased the resting potential by 36% possibly due to inhibition of the Na+-K+ exchange pump. Neither total removal nor fourfold increase of interstitial Ca2+ changed the resting potential. Complete elimination of interstitial Cl- did not change the resting potential. The mean permeability ratio PNa/PK was calculated to be 0.41. It is concluded that the resting potential of a frog taste cell consists of ionic and metabolic components, and that the ionic component is due to the permeability of the cell membrane to K+ and Na+.     

Acetylcholine-induced electrical responses in neuroblastoma cells

The response to iontophoretic application of acetylcholine in the mouse neuroblastoma cell line N1E-115 was composed of three phases. The initial fast depolarizing phase was blocked by 10 microM d-tubocurarine, but not by 0.1 microM atropine. This phase was followed by a transient hyperpolarization which in turn was followed by a secondary slow depolarization. Both the hyperpolarization and slow depolarization were blocked by atropine (0.1 microM), but not by d-tubocurarine (10 microM). The hyperpolarization and slow depolarization were also evoked by iontophoretic application of the muscarinic agonist methacholine. Under voltage-clamp conditions, an initial fast inward current, a transient outward current, and a secondary slow inward current were recorded in response to acetylcholine application. These three phases of current correspond to the three phases of the membrane potential response. The initial fast inward current increased in amplitude by hyperpolarization of the membrane, and decreased by depolarization. The mean reversal potential was estimated to be -1 mV. The outward current increased in amplitude by depolarization, decreased by hyperpolarization, and reversed its polarity at -67 mV. Alteration of external K+ concentration shifted the reversal potential in the manner expected for an increase in potassium permeability. The slow inward current increased in amplitude by hyperpolarization, decreased by depolarization, and reversed its polarity at +20 mV. It is concluded that the initial fast inward current is mediated by a nicotinic receptor similar to that in muscle end-plate membranes and in postsynaptic membranes of the sympathetic ganglia. Both the outward current and the slow inward current are mediated by muscarinic receptors. The outward current results from an increase in the membrane permeability to K+, and the slow current appears to be carried, at least in part, by Na+.     

Effect of sodium and sodium-substitutes on the active ion transport and on the membrane potential of smooth muscle cells

1. The changes of the ion content and of the membrane potential of taenia coli cells have been studied during prolonged exposure to Na-deficient solutions containing either Li or choline.2. A K-free solution containing either 71 mM-Na-71 mM-Li or 71 mM-Na-71 mM choline causes a slower loss of cellular K than a 142 mM-Na solution. In both these Na-deficient solutions the membrane hyperpolarizes to about -100 mV for periods up to 6 hr. This hyperpolarization is partially abolished by 2 x 10(-5)M ouabain.3. Replacing all extracellular Na by Li and maintaining 5.9 mM-K causes a fast loss of all Na and a progressive replacement of K by Li. These changes of the intracellular ion content are accompanied by a depolarization of the cells, suggesting that intracellular Li cannot substitute for Na in activating the ion pump.4. Exposing K-depleted cells to a K-free 71 mM-Na-71 mM-Li solution results in a ouabain sensitive transport of Na and Li against their electro-chemical gradient.5. The K-uptake by K-depleted cells from a solution containing 0.59 mM-K is increased by reducing [Na](o) to half of its normal value. This finding indicates that external Na inhibits the active Na-K exchange.6. In Na-enriched tissues half of the Na efflux is due to a ouabain insensitive Na-exchange diffusion. If Li is used as a Na substitute, the Na-Li exchange compensates for the diminution of the Na-exchange diffusion unless ouabain is added.     

Properties of a facilitating calcium current in pace-maker neurones of the snail, Helix pomatia.

1. Simultaneous measurements of local voltage clamp currents from patches of soma membrane and K activity at the soma surface were used to analyse the time and voltage dependence of the slow inward current in bursting pace-maker neurones of the snail (Helix pomatia). 2. At low levels of depolarization (less than or equal to mV) a net inward current is recorded simultaneously with an efflux of K ions from the cell. 3. With larger depolarizations (20-170 mV from holding potential of -50 mV) the deficit in net outward charge transfer compared with K efflux and the appearance of inward-going tail currents following repolarization, reveal a persistent inward-going current also under these conditions. This inward current is carried primarily by Ca ions, as demonstrated by its voltage dependence (a minimum at about + 115 mV) and its disappearance in Co-Ringer. It is identified with the slow inward Ca current Iin slow (Eckert & Lux, 1976). 4. The inward current predicted from comparisons of current trajectories reaches a maximum at 15-20 msec (for depolarizations from -50 to 0 mV) and gradually declines with sustained depolarization. 5. Partial inactivation is removed by repolarization to -50 mV and the Ca dependent deficit is greater in the sum of repeated voltage clamp pulses than during sustained depolarization. It is largest for pulses of 25-100 msec duration, decreasing as pulse duration increases. 6. Responses to repeated activation with 100 msec pulses with different repolarization intervals reveal a minimum Iin slow at short intervals (e.g. 20 msec) due to failure to remove partial inactivation. At intermediate intervals (e.g. 200-400 msec) Iin slow shows facilitation. This is revealed in calculations of the net charge transfer and current deficits and is also shown in the tail currents following repolarization. The deficit increases progressively with repetitive stimulation. With longer intervals (e.g. 800-1000 msec) defacilitation during repeated stimulation after the first two pulses is revealed in calculations of deficits, current trajectories and in the tail currents. 7. Although facilitation depends on duration of repolarization between pulses, increasing intermediate hyperpolarizations from the holding potential of -50 mV are usually ineffective in increasing Iin slow. Strong preceding hyperpolarization can even decrease the magnitude of Iin slow and prevent its facilitation with repetitive stimulation,whereas preceding depolarizing pulses can increase Iin slow without preventing its facilitation with repetitive stimulation. 8. The properties of Iin slow are contrasted with previously described membrane conductances and compared with properties attributed to Ca fluxes in other systems.     

Graded activation of myofibrils and the effect of diameter on tension development during contractures in isolated skeletal muscle fibres.

If the space constant of the T-system (lambdaT) its not large in comparison with the radius (a) of a muscle fibre, different levels of depolarization should activate different proportions of the cross-section. This possibility was tested in isolated muscle fibres with isotonic and isometric K contractures. 2. During isonic contractures with more than 40 mM-K, wavy myofibrils appeared in the centre of the fibre. The sarcomere spacings (s) of the wavy myofibrils, measured parallel to the long axis of the myofibrils, were 1-9-1-95 mum. However, the superficial myofibrils could shorten to or below s=1-5 mum without becoming wavy. 3. In the same muscle fibre where myofibrils became wavy during K contractures, no waviness appeared during repetitive electric stimulation in normal Ringer (50 shocks/sec, 12 degrees C), although s decreased below 1-5 mum. Wavy myofibrils were interpreted as not activated. 4. With isometric contractures it was found that the amount of depolarization needed to obtain maximal tension was smaller for fibres of shorter radius. The degree of depolarization for producing maximal tension is related to a by 6 mV/10mum. 5. These results strongly suggest that in K contractures lambdaT is not large in comparison with a.     

Voltage-sensitive calcium flux into bovine chromaffin cells occurs through dihydropyridine-sensitive and dihydropyridine- and omega-conotoxin-insensitive pathways

The fluorescent Ca2+ indicator FURA-2 was used to characterize the depolarization-related intracellular Ca2+ signalling process in bovine adrenal chromaffin cells. Depolarization with high K+ (10-65 mM) gave rise to a very rapid increase in intracellular free Ca2+ concentration, which subsequently decayed slowly towards a "plateau". The size of this initial increase varied sigmoidally with the calculated membrane potential, the relationship being described well by a Boltzmann distribution function for a transition between two states (transition potential, -23 mV). A dihydropyridine calcium channel agonist [(+)202-791, 1 microM] raised intracellular free Ca2+ concentration further in the presence of 30 mM K+, and it enhanced the initial intracellular Ca2+ response to depolarization. Voltage-sensitive calcium channels in chromaffin cells are believed to include the L-type. Several dihydropyridine calcium channel antagonists [(-)202-791, nifedipine, nitrendipine; 1-5 microM], known to be active on L-type channels, caused only modest inhibition of K+ -induced increase in intracellular free Ca2+ concentration: c. 50% (at 30 mM K+) and 25% (at 40-70 mM K+). In addition, omega-conotoxin GVIA (1-10 microM), a blocker of neuronal N- and L-type calcium channels, reduced the initial increase in intracellular free Ca2+ concentration only slightly at 55 mM K+. Further, the dihydropyridine-insensitive component of the intracellular Ca2+ signal was also insensitive to omega-conotoxin, which was otherwise quite active in a central nervous rat in vivo preparation Gd3+ (40 microM), a potent calcium antagonist in the chromaffin cell, blocked the intracellular Ca2+ response to depolarization. When added at different times after K+ stimulation, however, Gd3+ reduced intracellular free Ca2+ concentration to control levels along a slow time course of several minutes. Similar results were obtained when EGTA was added to reduce extracellular Ca2+ concentration to sub-nanomolar levels, in the presence of high K+. We conclude that bovine chromaffin cells are equipped with at least two different classes of voltage-dependent calcium channels, only one of which is likely to be the L-type channel. We also propose that depolarization, in addition to stimulating Ca2+ influx, may also lead to enhancement of Ca2+ release from an intracellular store.     

Serotonergic stretch receptors induce plateau properties in a crustacean motor neuron by a dual-conductance mechanism.

1. The mechanisms for induction of bistable plateau potential properties by a set of serotonergic/cholinergic peripheral stretch receptor cells [gastropyloric receptor (GPR) cells] were examined in the crab stomatogastric ganglion (STG) with the use of intracellular recording techniques. 2. GPR cell stimulation evoked nicotinic excitatory postsynaptic potentials (EPSPs) and induced plateau potential capability in the dorsal gastric (DG) motor neuron. The plateau potential could be triggered during a GPR train either by the summating nicotinic EPSPs or by brief intracellular current injection. After pharmacological blockade of nicotinic and muscarinic receptors, a slow depolarization in response to GPR stimulation was revealed. Prolonged plateau potentials could still be evoked after this treatment. Local application of serotonin (5-HT; 10 microM to 1 mM) mimicked the noncholinergic plateau inducing effects of GPR stimulation in the DG motor neuron. 3. The synergistic action of acetylcholine (ACh) and 5-HT was examined by stimulating the GPR cells at different frequencies (1-20 Hz). The plateau induction was present down to 2 Hz. The time to onset for triggering a plateau during a GPR train was determined by the co-released ACh. 4. The 5-HT-evoked slow depolarization persisted in tetrodotoxin (TTX; 0.1-1 microM), and the DG motor neuron could still produce a plateau potential on brief depolarization in the absence of the spike-generating mechanism. 5. In normal TTX-containing saline, the 5-HT-evoked depolarization was accompanied by a weak and variable decrease in apparent input conductance. After substituting one-half of the extracellular sodium with either Trisma-HCl or choline, the decrease in apparent input conductance became more pronounced. This decrease was converted to an increase in apparent input conductance when extracellular Ca2+ was replaced with Mg2+. 6. Under voltage-clamp conditions, local application of 5-HT caused a slow inward current of prolonged duration in DG. The current versus voltage relationship had an inverted U-shape with no apparent reversal potential in the entire voltage range investigated (-90 to -5 mV). The 5-HT-induced changes in input conductance showed a complex voltage dependence, with a conductance decrease from moderately depolarized voltages. 7. Extracellular Cs+ (2-4 mM) caused the DG to hyperpolarize 2-4 mV from rest, whereas lowering extracellular Ca2+ caused it to depolarize 7-15 mV. The combined action of low extracellular Ca2+ and 2-4 mM Cs+ caused an almost complete block of the slow 5-HT-evoked depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation and characterization of slow, depolarizing responses of cardiac ganglion neurons in the crab, Portunus sanguinolentus.

1. Tetrodotoxin-resistant, active responses to depolarization of the large cardiac ganglion cells were studied in semi-isolated preparations from the crab, Portunus sanguinolentus. Impulse activity was monitored with extracellular electrodes, simultaneous recordings from two or three large cells were made with intracellular electrodes, and current was passed via a bridge or second intracellular electrode. Preparations were continuously perfused with saline containing 3 x 10(-7) M tetrodotoxin (TTX). 2. About 20 min after introduction of TTX, small-cell impulses and resultant EPSPs in large cells cease, while rhythmic, spontaneous bursting of large cells continues. A pacemaker depolarization between bursts and slow depolarizations underlying the impulse bursts are prominent at this time. Shortly after, spontaneous burst rate slows, and at ca. 25 min, the ganglion becomes electrically quiescent. 3. In the quiescent, TTX-perfused ganglion, injection of depolarizing current into any one of the large cells results in active responses. At current strengths of sufficient intensity and duration (e.g., 20 nA, 20 ms; 5 nA, 500 ms) to depolarize a large cell by ca. 10 mV from resting potential (-53 mV, avg), the graded responses become regenerative and of constant form, provided the stimulation rate is less thna 0.15/s. Such responses have been termed "driver potentials." At more rapid rates, thresholds are increased and responses reduced. 4. Driver potentials of anterior large cells reach peak amplitudes of ca. 20 mV (to -32 mV), have maximum rates of rise of 0.45 V/s and of fall of 0.2 V/s, and a duration of ca. 250 ms. They are followed by hyperpolarizing afterpotentials, a rapidly decaying one (1 s) to -58 mV, followed by a slowly decaying one (7.5 s), -55 mV. Responses of posterior large cells are smaller (16 mV) and slower; the site of active response may be at a distance from the soma. 5. The ability of elicit near-synchronous responses and the identity of amplitude and form of responses among anterior cells and of posterior cells, regardless of which cell receives depolarizing current, indicates that all cells undergo active responses and are stimulated by electrotonic spread of depolarization. 6. The responses involve a conductance increase since memses during a driver potential are much reduced. 7. Depolarization by steady current increases the absolute threshold, decreases the maximum depolarization of the peak, and slows rates of rise and fall. Hyperpolarization increases rates of rise and fall; the absolute value reached by the peak depolarization is unchanged. Hyperpolarization reduces the amplitude of the rapid after-potential relative to the displaced resting potential. 8. Hyperpolarizing current pulses imposed during the rise and peak of driver-potential responses are followed by redevelopment of a complete response. Sufficiently strong hyperpolarization can terminate a response. The current strength needed to terminate a response decreases the later during the response the pulse is given...