Effects of potassium channel openers on Na+ and K+ currents in rabbit sinus node and atrial myocytes.

The effects of the potassium channel openers (KCOs) Cromakalim and Lemakalim on rabbit sinoatrial and atrial myocytes were examined by means of the whole-cell patch-clamp technique. Lemakalin (up to 100 microM) had no effect on potassium current in sinoatrial cells. Both Lemakalim and Cromakalim (100 microM) displayed a two-fold action on atrial myocytes: (1) they increased an outwardly rectifying conductance at potentials positive to EK and, (2) they markedly decreased a TTX-sensitive Na+ current active in the voltage range -50/-30 mV. This novel action on TTX-sensitive currents is of particular interest since these two benzopyrans have been thought to specifically target potassium channels.

Hippocampal hypoglycaemia-activated K+ channels: single-channel analysis of glucose and voltage dependence.

The effect of glucose on kinetics and the voltage-dependent characteristics of glucose-sensitive channels in hippocampal neurons were examined with the cell-attached mode of the patch-clamp technique. Recordings of a 100-pS K+ channel in the presence or absence of glucose demonstrate that the increase in channel open state probability (Po) induced by glucose deprivation (40- to 400-times the control in high-glucose medium) was largely due to a decrease in the global amount of time spent by the channel in a long-lived closed state. The Po value of the same 100-pS channel was also found to increase (by approx. 80-times) following a depolarization of 40 mV from rest, the main factor responsible for this being a dramatic shortening of the long closed-times on depolarization. Another glucose-sensitive channel of smaller conductance (approx. 10 pS) showed a similar dependence of Po on glucose, but different dependence on voltage, with long openings at the same hyperpolarized potentials where the 100-pS channel was almost always closed. Our results indicate that the action of glucose on the kinetics of hippocampal channels closely resembles that of ATP-sensitive channels in pancreatic beta-cells. Furthermore, they indicate that the two types of glucose-sensitive channels found in hippocampal neurons, differing not only in their single-channel conductance but also in the dependence on voltage, could play different roles in the responses of these cells to modified energetic supply.

Glutamate uptake inhibition by oxygen free radicals in rat cortical astrocytes.

Formation of reactive oxygen species and disfunction of the excitatory amino acid (EAA) system are thought to be key events in the development of neuronal injury in several acute and long-term neurodegenerative diseases. Recent evidence suggests that the two phenomena may be interdependent. The present study is aimed at exploring possible molecular mechanisms underlying oxygen radical-EAA interaction. Exposure of cortical astrocytic cultures to either xanthine + xanthine oxidase (X/XO), a free radical-generating system, or hydrogen peroxide (H2O2) results in a marked decrease of high-affinity glutamate transport. Within 10 min of X/XO application, uptake falls to approximately 60% of its control value. In parallel no detectable release of lactate dehydrogenase occurs. X/XO effect is abolished in the presence of a mixture of scavenger enzymes (superoxide dismutase+catalase) or by the disulfide-reducing agents glutathione and dithiothreitol (DTT), but not by lipophilic antioxidants or ascorbate. The time course of inhibition shows an almost linear decline of glutamate transport during cell exposure to free radicals, while upon their inactivation the decline stops but established inhibition persists for at least 1 hr. In this situation, application of DTT significantly restores transport function. These data suggest that free radicals inhibit glutamate uptake primarily by long-lasting oxidation of protein sulfhydryl (SH) groups. Chemical modifiers of free SH groups, such as p-chloromercuribenzoate and N-ethylmaleimide, also induce uptake inhibition. Na+/K+ ATPase is a known target of oxygen radicals and may be involved in glutamate uptake inhibition. Indeed, ouabain, a blocker of the pump, reduces uptake in astrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

High sensitivity of glutamate uptake to extracellular free arachidonic acid levels in rat cortical synaptosomes and astrocytes.

By using both synaptosomes and cultured astrocytes from rat cerebral cortex, we have investigated the inhibitory action of arachidonic acid on the high-affinity glutamate uptake systems, focusing on the possible physiological significance of this mechanism. Application of arachidonic acid (1-100 microM) to either preparation leads to fast (within 30 s) and largely reversible reduction in the uptake rate. When either melittin (0.2-1 microgram/ml), a phospholipase A2 activator, or thimerosal (50-200 microM), which inhibits fatty acid reacylation in phospholipids, is applied to astrocytes, both an enhancement in extracellular free arachidonate and a reduction in glutamate uptake are seen. The two effects display similar dose dependency and time course. In particular, 10% uptake inhibition correlates with 30% elevation in free arachidonate, whereas inhibition greater than or equal to 60% is paralleled by threefold stimulation of arachidonate release. In the presence of albumin (1-10 mg/ml), a free fatty acid-binding protein, inhibition by either melittin, thimerosal, or arachidonic acid is prevented and an enhancement of glutamate uptake above the control levels is observed. Our data show that neuronal and glial glutamate transport systems are highly sensitive to changes in extracellular free arachidonate levels and suggest that uptake inhibition may be a relevant mechanism in the action of arachidonic acid at glutamatergic synapses.

Hypoglycemia-activated K+ channels in hippocampal neurons.

Channels linking the electrical and metabolic activities of cells (KATP channels) have been described in various tissues, including some brain areas (hypothalamus, cerebral cortex and substantia nigra). Here we report the existence in hippocampal neurons of K+ permeant channels whose activity is regulated by extracellular glucose. They are open at the cell resting potential and respond to transient hypoglycemia with a reversible increase in activity. The one type so far characterized has a conductance of approximately 100 pS in isotonic K+, is inhibited by the sulphonylurea glibenclamide (1 microM), and is activated by the potassium channel opener lemakalim (0.1-1 microM). These data provide a direct demonstration of the presence, in hippocampal neurons, of glucose-sensitive channels that could belong to the KATP family.

A role for the arachidonic acid cascade in fast synaptic modulation: ion channels and transmitter uptake systems as target proteins.

Recent evidence indicates that arachidonic acid (AA) and its metabolites play a fast messenger role in synaptic modulation in the CNS. 12-Lipoxygenase derivatives are released by Aplysia sensory neurons in response to inhibitory transmitters and directly target a class of K+ channels, increasing the probability of their opening. In this way, hyperpolarization is achieved and action potentials are shortened, leading to synaptic depression. Other types of K+ channels in vertebrate excitable cells have been found to be sensitive to arachidonic acid, lipoxygenase products, and polyunsaturated fatty acids (PUFA). In the mammalian CNS, arachidonic acid is released upon stimulation of N-methyl-D-aspartate (NMDA)-type glutamate receptors. We found that arachidonic acid inhibits the rate of glutamate uptake in both neuronal synaptic terminals and astrocytes. Neither biotransformation nor membrane incorporation are required for arachidonic acid to exert this effect. The phenomenon, which is rapid and evident at low microM concentrations of AA, may involve a direct interaction with the glutamate transporter or its lipidic microenvironment on the outer side of the cell membrane. Polyunsaturated fatty acids mimic arachidonate with a rank of potency parallel to the degree of unsaturation. Since the effect of glutamate on the synapses is terminated by diffusion and uptake, a slowing of the termination process may potentiate glutamate synaptic efficacy. However, excessive extracellular accumulation of glutamate may lead to neurotoxicity.

Effects of protein kinase inhibitors on canine Purkinje fibre pacemaker depolarization and the pacemaker current i(f).

1. The effects of the protein kinase inhibitors H-7 and H-8 were investigated on diastolic depolarization of the action potential with microelectrodes and on the pacemaker current if with the two-microelectrode voltage clamp in canine cardiac Purkinje fibres. 2. Both 200 microM-H-7 and 100 microM-H-8 had no significant effect on the slope of diastolic depolarization but eliminated the actions of isoprenaline (1 microM). 3. We examined the actions of H-7 and H-8 on if in the presence and absence of isoprenaline. H-7 (200 microM) shifted the pacemaker current if in the negative direction on the voltage axis, whereas 100 microM-H-8 had no significant effect by itself. Both 200 microM-H-7 and 100 microM-H-8 can reverse or prevent the actions of isoprenaline (1-5 microM) on if. 4. We applied activators of the cyclic AMP cascade down-stream to the beta-receptor, to further evaluate where H-7 and H-8 might be exerting their effects. When exposing Purkinje fibres to an adenylyl cyclase activator (forskolin, 10-50 microM), a phosphodiesterase inhibitor (IBMX, 100 microM) and a permeable cyclic AMP analogue (8-chlorophenylthio-cyclic AMP, 200 microM-1 mM), the amplitude of if was increased. H-7 and H-8 at 100-200 microM eliminated each of these actions. 5. These results suggest that a phosphorylation process is involved in the modulation of the pacemaker current, if, in Purkinje fibres. The different actions of H-7 and H-8 on basal if suggest the hypothesis that other protein kinases, possibly protein kinase C, might also be involved in regulating basal phosphorylation of if in Purkinje fibres.

Sodium current block caused by group IIb cations in calf Purkinje fibres and in guinea-pig ventricular myocytes.

The action of group IIb cations [Cadmium (Cd2+), Zinc (Zn2+), Mercury (Hg2+)] on the cardiac fast sodium current (INa) was investigated in calf Purkinje fibres and in ventricular cells isolated from guinea-pig hearts. In calf Purkinje fibres, INa was depressed by submillimolar concentrations of Zn2+ and Hg2+. With both cations, the current reduction occurred at all voltages in the range of current activation and the voltage dependence of peak current was unchanged. The degree of peak current inhibition depended on the cation concentration but not on voltage. The position of the inactivation curve on the voltage axis was unaltered at cation concentrations giving substantial current inhibition, and moved to the right only with concentration exceeding 1-1.5 mM. These effects can be interpreted as due to INa channel blockade. The action of Zn2+ and Hg2+ was similar to that described earlier of Cd2+ on Purkinje fibres (DiFrancesco et al. 1985b). INa was also inhibited by group IIb cations in isolated guinea-pig ventricular cells. Depression of INa by Cd2+, Zn2+ and Hg2+ was essentially voltage-independent, in agreement with its being caused by channel block. The dependence of INa block by Cd2+ upon external Na concentration [Na+0] was investigated in ventricular myocytes. The fraction of INa block by 0.1 mM CdCl2 was 0.50 at 140 mM, 0.81 at 70 mM and 0.83 at 35 mM [Na+]0. A similar increase of block efficiency at low [Na+0] was observed with 0.05 mM CdCl2. In both the Purkinje fibre and the ventricular cell, the order of potency of INa block by group IIb cations was Hg2+ greater than Zn2+ greater than Cd2+. Manganese (Mn2+, 2-5 mM), an ion of group VIIa, also depressed the INa in Purkinje fibres and ventricular myocytes. This effect was however due mainly to a positive shift on the voltage dependence of current kinetics rather than to a reduction of the conductance of the channel (GNa), and can be accounted for by an ion-screening action of Mn2+ on the external membrane surface. The block by group IIb cations is a typical property of cardiac Na+ channels and characterizes the cardiac as opposed to other types of Na+ channel.

A novel action of isoproterenol to inactivate a cardiac K+ current is not blocked by beta and alpha adrenergic blockers.

The K+ current iKl sets the resting potential in cardiac cells. Here we report that isoproterenol (ISO), a prototypical beta agonist, increases inactivation of iKl. This action of ISO on iKl is mimicked by permeant analogues of cAMP but is not blocked by the beta blockers propranolol and pindolol or the alpha blockers prazosin or yohimbine. We suggest that this novel action of ISO may contribute to pacemaker activity in the Purkinje strand and be mediated through a class of receptors different from classical beta's or alpha's.

Acetylcholine reverses effects of beta-agonists on pacemaker current in canine cardiac Purkinje fibers but has no direct action. A difference between primary and secondary pacemakers.

We have investigated the actions of acetylcholine in the absence and presence of the beta-agonist isoproterenol in cardiac Purkinje fibers. beta-Agonists, like isoproterenol, increase the magnitude of the pacemaker current (If) in cardiac myocytes by shifting its activation voltage more positive on the voltage axis. We find that acetylcholine has no effect on If in the absence of isoproterenol. However, if If is first increased by beta-agonist stimulation, acetylcholine can then return If to control levels. This effect on If is exerted through muscarinic receptors since atropine prevents this action of acetylcholine. Functionally, this action of acetylcholine can guarantee the maintenance of ventricular pacemakers when there is high parasympathetic tone but can also prevent extra ventricular beats when sympathetic and parasympathetic tone are both high.

Muscarinic control of the hyperpolarization-activated current (if) in rabbit sino-atrial node myocytes.

1. The mechanism by which acetylcholine (ACh), by stimulation of muscarinic receptors, acts to inhibit activation of the hyperpolarization-activated 'pacemaker' current, if was investigated in isolated rabbit sino-atrial (SA) node myocytes. 2. Intracellular loading with GTP gamma S, a non-hydrolysable analogue of GTP, did not impair the ACh action on if, but made it irreversible. On the other hand, the ACh action on if disappeared after a few minutes of cell loading with GDP beta S, a GDP analogue known to bind to G-proteins and prevent their receptor-stimulated action. Furthermore, incubation of cells in a solution containing pertussis toxin (PTX) led to abolition of the if response to ACh. These results indicate that the inhibitory effect of ACh on if is mediated by G-proteins activated by muscarinic receptors. 3. Intracellular loading with phosphodiesterase (PDE) increased the rate of if current run-down, but did not abolish the inhibitory action of ACh on if. 4. Extracellular perfusion with isobutylmethylxanthine (IBMX), a PDE inhibitor, increased if activation by shifting the current activation range to more positive voltages, as inferred by a three-pulse protocol analysis; in the presence of IBMX, the inhibition of if by ACh was not abolished. 5. The ACh-induced if depression persisted also in cells loaded with cyclic GMP. In these cells, as in those loaded with PDE, the if run-down was fast. 6. Oxotremorine, a muscarinic agonist coupled to adenylate cyclase but not to phosphoinositide turnover in cardiac cells, simulated ACh in its inhibitory action on if. The above results rule against the ACh action being mediated by PDE or by phosphoinositide turnover. 7. To investigate the possible involvement of cyclic AMP as a second messenger in the ACh action on if, we loaded cells with cyclic AMP and IBMX; under these conditions the action of ACh disappeared within a few minutes of whole-cell recording. 8. In cells where the slow inward Ca2+ current (isi) was measured together with if, ACh was seen to depress both currents. 9. In cells superfused with forskolin, the if amplitude on stepping to the half-activation voltage range was enhanced as a consequence of a depolarizing shift of the activation curve; ACh was not effective on if following stimulation by forskolin, but strongly depressed in the same cell the if current stimulated to a similar degree by isoprenaline.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of the hyperpolarization-activated current (if) induced by acetylcholine in rabbit sino-atrial node myocytes.

1. The action of acetylcholine (ACh) on the hyperpolarization-activated ('pacemaker') current if was studied in single myocytes from the sino-atrial (SA) node region of the rabbit heart, where low doses of ACh slow spontaneous activity by prolonging the diastolic depolarization phase. 2. Besides activating an outward component at voltages positive to the K+ equilibrium potential (iK,ACh), ACh depressed the current if activated on hyperpolarization at concentrations in the range 0.03-1 microM. 3. The ACh-dependent if depression was dissected from modifications of iK,ACh by blocking iK,ACh with barium and was studied under conditions that minimized the interference of other current changes caused by ACh. 4. The study of if modification by ACh with three-pulse protocols and the measurement of fully activated I-V relations of if with and without ACh revealed that ACh acted on if by shifting the current activation range to more negative voltages, with no obvious alteration of the fully activated current amplitude. 5. The action of ACh on if was opposite to that caused by catecholamines. The presence of isoprenaline (IP) did not prevent ACh inhibition of if, nor did the presence of ACh prevent the if stimulation caused by IP. The effects of IP and ACh on if were additive. 6. The ACh-induced inhibition of if was reversed by addition of atropine and could be mimicked by muscarine, indicating that muscarinic receptors mediate it. The implications of these findings on the regulation of pacemaker activity by ACh is discussed.

Acetylcholine inhibits activation of the cardiac hyperpolarizing-activated current, if.

Acetylcholine (ACh) in low doses (0.1-1 microM) reversibly inhibits voltage-dependent activation of the "pacemaker" current, if, in isolated sino-atrial node cells. This action is brought about by a negatively-directed shift of the current activation curve, opposite to that due to catecholamines on the same current. The if inhibition is antagonized by atropine, indicating the involvement of muscarinic receptors. In cells incubated in pertussis toxin-containing solutions, if does not respond to ACh, suggesting that G-proteins mediate the ACh-induced if depression. Further, ACh can inhibit if following catecholamine-induced stimulation, but has a negligible effect on if stimulated by forskolin, a direct activator of adenylate-cyclase. Our results indicate that ACh acts on if by inhibiting basal adenylate-cyclase activity.

Properties of the hyperpolarizing-activated current (if) in cells isolated from the rabbit sino-atrial node.

Individual cells were isolated from the sino-atrial node area of the rabbit heart using an enzyme medium containing collagenase and elastase. After enzymatic treatment the cells were placed in normal Tyrode solution, where beating resumed in a fraction of them. Isolated cells were studied in the whole cell configuration. Action potentials as well as membrane currents under voltage-clamp conditions were similar to those in multicellular preparations. Pulses to voltages more negative than about -50 mV caused activation of the hyperpolarizing-activated current, if. Investigation of the properties of this current was carried out under conditions that limited the influence of other current systems during voltage clamp. The if current activation range usually extended approximately from -50 to -100 mV, but varied from cell to cell. In several cases, pulsing to the region of -40 mV elicited a sizeable if. Both current activation and deactivation during voltage steps had S-shaped time courses. A high variability was however observed in the sigmoidal behaviour of if kinetics. Plots of the fully-activated current-voltage (I-V) relation in different extracellular Na and K concentrations showed that both ions carry the current if. While changes in the external Na concentration caused the current I-V relation to undergo simple shifts along the voltage axis, changes in extracellular K concentration were also associated with changes in its slope. Again, a large variability was observed in the increase of I-V slope on raising the external K concentration. The current if was strongly depressed by Cs, and the block induced by 5 mM-Cs was markedly voltage dependent. Adrenaline (1-5 microM) and noradrenaline (1 microM) increased the current if around the half-activation voltage range and accelerated its activation at more negative voltages. Often, however, drug application failed to elicit any modification of if. Current run-down was observed in nearly all cells, although at a highly variable rate. It was accelerated by raising the extracellular K concentration but did not show a marked use dependence. Both the if activation curve and the fully activated I-V relation were affected by run-down, the former being shifted to more negative values along the voltage axis and the latter being depressed with no apparent change of the if reversal potential.(ABSTRACT TRUNCATED AT 400 WORDS)