Adenosine modulation of potassium currents in postganglionic neurones of cultured avian ciliary ganglia.

1. Potassium currents in cultured postganglionic neurones of avian ciliary ganglia were analysed under whole-cell voltage clamp and their modulation by adenosine determined. 2. In the presence of tetrodotoxin (200 nM), and with moderate holding potentials (Vh = -40 mV), the steady-state current-voltage (I/V) curve was N-shaped over the range from -70 mV to +155 mV. CsCl (1 M) blocked the current, indicating that it was carried by K+. If Ca2+ influx was blocked by CdCl2 (500 microM) then the outward current was reduced and the N-shaped I-V curve lost, indicating the presence of a calcium-activated potassium current (IK(Ca)); the remaining current, due to the delayed rectifier (IK), increased with depolarization up to about a conductance of 10 nS near + 50 mV. This IK was 50% activated at about +20 mV and 50% inactivated at about -50 mV. Adenosine (10 microM) had similar affects on the N-shaped I/V curve as did CdCl2, indicating that it blocked IK(Ca). However, adenosine had little affect on the steady-state current in the presence of CdCl, indicating that it did not much affect IK. 3. In the presence of tetrodotoxin (200 nM), a large inward current occurred for large hyperpolarizations from a Vh = -50 mV. This inward rectifying current (IIR) had a reversal potential near EK and showed 50% activation at about -130 mV. Adenosine (10 microM) reduced IIR, by as much as 50% at large hyperpolarizations beyond -80 mV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine modulation of calcium currents in postganglionic neurones of avian cultured ciliary ganglia.

1. Calcium currents in postganglionic neurones of cultured 7- to 10-day embryonic avian ciliary ganglia were analyzed under whole-cell voltage-clamp and their modulation by 2-chloroadenosine determined. 2. In the presence of tetrodotoxin (200 nM) in the medium to block the Na+ current and CsCl (105 mM) in the patch-clamp electrode to block the K+ current, two different components of the calcium currents (transient and sustained) were identified on the basis of their voltage-dependent kinetics as well as their sensitivity to the dihydropyridine agonist Bay K 8644 and antagonist nifedipine. 3. The sustained current inactivated very slowly (tau greater than 1000 ms; for test potentials from -20 mV to +40 mV) but was reactivated at a holding potential (Vh) of -40 mV. The current was increased on average over 50% by 1 microM of Bay K 8644 at a test potential of 0 mV and decreased over 35% by 1 microM of nifedipine. 4. The transient current inactivated slowly (tau less than 200 ms; for test potentials from -20 mV to +40 mV), and could be completely reactivated at a Vh of -80 mV. This current was unaffected by Bay K 8644 (1 microM) but reduced on average by 8% with nifedipine (1 microM). 5. The sustained and transient currents were decreased more than 70% by 5 microM of omega-conotoxin and decreased more than 50% by 250 microM verapamil. 6. 2-Chloroadenosine (1 microM) decreased the transient current by over 50% and the sustained current by less than 10%. In the presence of nifedipine (1 microM), 2-chloroadenosine decreased the transient current by over 30% and the remaining sustained current by 35%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitric oxide modulation of calcium-activated potassium channels in postganglionic neurones of avian cultured ciliary ganglia.

1. A study has been made of the modulation of calcium-activated potassium channels in cultured neurones of avian ciliary ganglia by sodium nitroprusside and L-arginine. 2. Sodium nitroprusside (100 microM) reduced the net outward current by 22 +/- 1% at 4.8 ms (mean +/- s.e. mean) and 25 +/- 1% at 350 ms during a test depolarization to +40 mV from a holding potential of -40 mV. The outward current remained reduced for the duration of the recording following a single application of sodium nitroprusside. These effects did not occur if the influx of calcium ions was first blocked with Cd2+ (500 microM). Application of ferrocyanide (100 microM) reduced the net outward current by only 6 +/- 3% at 350 ms during a test depolarization to +40 mV. 3. L-Arginine (270 microM) reduced the net outward current on average by 19 +/- 2% at 4.8 ms and 22 +/- 2% at 350 ms during a test depolarization to +40 mV. The current remained in this reduced state for the duration of the recording following a single application of L-arginine. These effects were reduced to 11 +/- 1% at 4.8 ms and 11 +/- 2% at 350 ms in the presence of N omega-nitro-L-arginine methyl ester (L-NAME, 100 microM). 4. In order to alleviate the dependence of calcium-activated potassium channels (Ik(Ca)) on the inward flux of calcium ions, the patch-clamp pipettes were filled with a solution containing 100 microM CaCl2, and the Ca2+ in the bathing solution was replaced with EGTA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitric oxide and arachidonic acid modulation of calcium currents in postganglionic neurones of avian cultured ciliary ganglia.

1. A study has been made of the modulation of high-voltage activated transient and sustained calcium currents in cultured neurones of avian ciliary ganglia by nitric oxide (NO) and arachidonic acid. 2. Sodium nitroprusside (100 microM) reduced the transient calcium current (ICa) on average by 31% and the sustained ICa by 32% during a test depolarization to +20 mV from a holding potential of -100 mV. This reduction was maintained for at least 30 min following a single application of sodium nitroprusside. 3. L-Arginine (270 microM) reduced the transient ICa on average by 28% and the sustained ICa by 22% and these effects were prevented by the presence of the NO-synthase competitive blocker NG-nitro-L-arginine methylester (L-NAME; 100 microM) in the bathing solution. 4. Arachidonic acid (50 microM) reduced the transient ICa on average by 28% and the sustained ICa by 33%. When added together, arachidonic acid (50 microM) and L-arginine (270 microM) produced the same effects as arachidonic acid alone. 5. Blocking the conversion of arachidonic acid to prostaglandins by addition of indomethacin (20 microM) to the bathing solution did not prevent the depression of either the transient or the sustained calcium current during application of arachidonic acid (50 microM). The effects of arachidonic acid were also not occluded by L-NAME (100 microM) when present in the bathing solution. 6. Inhibiting the biosynthesis of leukotrienes by applying L-663,536 (MK-886; 3 microM) to the bathing solution prevented the depression of both components of ICa during application of arachidonic acid (50 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cold potentiation of neuromuscular transmission in the avian biventer cervicis muscle

The neuromuscular response to a reduction of temperature from 34 to 26°C was examined in vitro in the avian biventer cervicis preparation. Cooling over this range potentiated neuromuscular transmission 104% although there was a 35% depression of direct muscle contractility. This potentiation was reduced 53% when the stimulation frequency was increased from 6/min to 24/min, suggesting the involvement of prejunctional processes in the cold response. When the safety margin of neuromuscular transmission was narrowed either prejunctionally with Mg²⁺ or postjunctionally with d-tubocurarine, cooling potentiated transmission to 127 and 212% respectively. After cholinesterase (ChE) inhibition by eserine the cold potentiation was not observed but this does not indicate that the neuromuscular response to cooling involves substantial inhibition of ChE since the potentiation in muscles treated with both eserine and tubocurarine was restored to within 20% of the response in untreated muscles. Postjuctional sensitivity, which was studied with the tonic response of the muscle to acetylcholine, did not change substancially at 26°C. These results indicate that the potentiation of neuromuscular transmission that occurs with cooling is due primarily to an increased release of neurotransmitter rather than ChE inhibition or an increased postjunctional sensitivity.     

Facilitative effect of carbamazepine on previously induced hippocampal long-term potentiation.

The effects of carbamazepine (CBZ) on previously induced hippocampal long-term potentiation (LTP) were examined. Acute experiments were performed on 33 adult, male rabbits. Field potentials in the dentate gyrus were elicited by single shocks to the perforant path, and LTP was induced by tetanic stimulation to the pathway without induction of seizure discharge. At a CBZ serum level of about 5 micrograms/ml (value +/- SD = 5.40 +/- 1.28 micrograms/ml), the previously induced LTP in population spikes (PSs) and population excitatory postsynaptic potentials (EPSPs) was facilitated. At a CBZ serum level of about 15 micrograms/ml (value +/- SD = 14.28 +/- 1.29 micrograms/ml), the LTP in PS alone was decreased. The effects of carbamazepine on synaptic inhibition were examined by the paired-pulse test. The inhibition was enhanced with induction of LTP. After administration of CBZ, at a CBZ serum level of about 5 micrograms/ml the inhibition was further enhanced, while it was attenuated at a CBZ serum level of about 15 micrograms/ml. These results suggest that CBZ has a facilitative effect on previously induced LTP.     

Inhibitory effect of adenosine on transmission in sympathetic ganglia

Summary The effect of bath-applied adenosine on transmission in the isolated superior cervical ganglion of the rat was investigated. The compound post ganglionic action potential was recorded as an index of ganglionic transmission. Adenosine and 2-chloroadenosine were equipotent in producing a dose-dependent inhibition of the amplitude of the compound action potential. At the highest concentration tested (1 mM) adenosine and 2-chloroadenosine produced about 30% decrease in the amplitude of the compound action potential. This inhibitory effect was antagonized by theophylline (1 and 100 M) which by itself had no significant effect on ganglionic transmission. The adenosine uptake blocker dipyridamole (1 and 100 M) failed to potentiate the inhibitory action of adenosine. Both 4-aminopyridine (20 M) and high frequencies of stimulation (3, 10 and 20 Hz) were effective in nearly completely abolishing the inhibitory effect of adenosine on ganglionic transmission.The results suggest that the inhibitory effect of adenosine on ganglionic transmission may be the result of activation of presynaptic adenosine receptors in the ganglion.     

The role of lipid-derived second messengers in cell growth and transformation

Recent information on the structure of growth factor receptors and oncogenes has advanced our understanding of the biochemical mechanism of growth control. Several peptide growth factor receptors have now been purified and, with a few notable exceptions, they fall into a family of homologous proteins with growth factor-stimulated protein-tyrosine kinase (PTK) activity. Mutations in the genes encoding for both the growth factors and the growth factor receptors of this family have been shown to cause cell transformation. Thus, PTKs have been implicated both in normal growth control and in cell transformation. The critical targets for these PTKs have not been determined. Several but not all growth factors stimulate production of the phosphatidylinositol (PI)-derived second messengers, diacylglycerol and inositol-1,4,5-trisphosphate. By purifying and characterizing the enzymes involved in PI turnover we are attempting to determine how this system is regulated by growth factors and oncogene products. We have found that fibroblasts contain two distinct PI kinases, and that one of these enzymes can be precipitated with antibodies against phosphotyrosine only after addition of platelet-derived growth factor. The relevance of the PI turnover pathway in propagation of growth signals is discussed.     

The effect of nitric oxide on the efficacy of synaptic transmission through the chick ciliary ganglion.

1. The effect of nitric oxide on the efficacy of synaptic transmission in the chick ciliary ganglion of post-hatched birds has been determined by use of the size of the postganglionic compound action potential resulting from chemical transmission through the ganglion as a measure of synaptic efficacy. 2. Sodium nitroprusside (100 microM) increased the synaptic efficacy by an average 26%. This is likely to be due to its ability to release nitric oxide, as potassium ferricyanide (100 microM) did not cause a potentiation. Sodium azide (100 microM), shown in sympathetic ganglia to stimulate production of cyclic GMP, did not modulate synaptic efficacy significantly. 3. 8-Br-cyclic-GMP (100 microM) increased synaptic efficacy by an average 61%. The addition of 8-Br-cyclic-AMP (100 microM) had less effect, increasing transmission by on average 46%. 4. The nitric oxide synthase blocker, NG-nitro-L-arginine methyl ester (L-NAME, 100 microM) was added prior to the tetanic stimulation of the preganglionic nerves at 30 Hz for 20 s, a procedure known to produce both post-tetanic potentiation and long-term potentiation of synaptic transmission through the ganglion. L-NAME reduced the long-term potentiation by an average of 47% but did not significantly change the post-tetanic potentiation. 5. Following the brief application of 8-Br-cyclic AMP, 8-Br-cyclic GMP and sodium nitroprusside there was an enhancement of the efficacy of synaptic transmission that persisted after the withdrawal of the drugs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Actions of capsaicin on peripheral nociceptors of the neonatal rat spinal cord-tail in vitro: dependence of extracellular ions and independence of second messengers.

1. We have tested the hypothesis that capsaicin-induced activation, desensitization and impairment of peripheral nociceptor function is mediated by separate mechanisms. This was investigated by use of an in vitro preparation of the neonatal rat spinal cord with the functionally attached tail in which the cord and tail were separately superfused with physiological solution. Activation of peripheral fibres by noxious (capsaicin, bradykinin, 5-hydroxytrptamine, heat, pinch) and innocuous (light brush) stimuli was assessed by recording the depolarization of a spinal ventral root (L3-L5). 2. Brief administration of capsaicin produced dose-related depolarizing responses (EC50 = 280 nM). These responses could be reproduced for many hours following the repeated application of capsaicin at a submaximal concentration. Prolonged application of 0.5-2.0 microM capsaicin induced a selective desensitization to subsequent brief administrations of capsaicin. Prolonged administration at 20-50 microM produced an additional non-selective reduction in responses to all noxious stimuli without changing innocuous brush responses. 3. Removal of extracellular calcium from the tail superfusate did not reduce the response to capsaicin or prevent capsaicin-induced desensitization. However, high concentrations of capsaicin no longer induced a non-specific reduction of responses to other noxious stimuli. The response to a brief administration of capsaicin was unaffected by calcium channel blocking drugs including nifedipine, cadmium or omega-conotoxin. On the other hand high extracellular calcium increased the incidence of the non-selective reduction of responses to all noxious stimuli produced by high concentrations of capsaicin. 4. Replacement of extracellular sodium with choline blocked peripheral nerve conduction but did not prevent the desensitization produced by capsaicin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Time-dependent effect of glutamate on long-term potentiation in the suprachiasmatic nucleus of rats

The effect of glutamate on optic nerve stimulation-evoked field potentials in rat suprachiasmatic nucleus (SCN) was examined in vitro. Glutamate application for 20 min induced long-term potentiation (LTP) of the field potentials in the SCN at nighttime, whereas that induced a weak LTP at daytime. On the other hand, application for 40 min induced LTP in the SCN during the daytime, whereas it induced a weak one at nighttime. These results indicate that the effect of glutamate is dependent on the application time and that the effect is influenced by the duration of glutamate exposure.     

The use of the human neuroblastoma SH-SY5Y to study the effect of second messengers on noradrenaline release

• 1.1. Recent data suggesting that the human neuroblastoma SH-SY5Y is a suitable cell line in which to study the effect of second messengers on NA release are discussed in the context of current views on exocytosis.
• 2.2. Release of NA is evoked by depolarization, as well as activation of muscarinic (M₃) and bradykinin (B₂) receptors in SH-SY5Y cells which have not been differentiated by the addition of growth factors.
• 3.3. Evoked release is enhanced by activation of protein kinase C.
• 4.4. Activation of protein kinase C decreases the changes in intracellular calcium evoked by carbachol, bradykinin and 100 mM K⁺.
• 5.5. SH-SY5Y express N-type and L-type voltage sensitive Ca²⁺ channels. L-Type Ca²⁺-channels are coupled to NA release under conditions of weak depolarization. However with strong depolarization (100 mM K⁺) both L-type and N-type channels are involved.
• 6.6. Muscarinic- and neuropeptide Y receptors are coupled to the inhibition of Ca²⁺ channel activity.

     

Actions of ketamine on synaptic transmission in frog sympathetic ganglia.

The effects of ketamine, a clinically used general anaesthetic, on isolated paravertebral sympathetic ganglia were investigated with extracellular and intracellular techniques. Ketamine, in concentrations of 10^?5M–10^?3M, reversibly blocked orthodromically induced action potentials. In concentrations which completely abolished the nicotinic response, ketamine did not entirely depress the muscarinic and noncholinergic components of ganglionic transmission. Ketamine depressed the postsynaptic membrane sensitivity to acetylcholine which was determined by comparing the responses to iontophoretically applied acetylcholine before and after ketamine. Concentrations of ketamine less than 10^?3M, which effectively blocked ganglionic transmission, did not significantly alter the passive membrane properties of the postsynaptic membrane. At 10^?3M, ketamine acted to simultaneously depolarize the postsynaptic membrane and increase the effective membrane resistance by decreasing the membrane conductance to potassium. In concentrations greater than 10^?3M, ketamine acted non-selectively to block reversibly the nicotinic, muscarinic and non-cholinergic ganglionic responses. These results demonstrate that ketamine. in addition to its strong central action, also has a direct depressant action upon peripheral synaptic transmission.     

The effects of baclofen and two GABAB-receptor antagonists on long-term potentiation

Summary The aim of the present study was to investigate whether activation or inhibition of GABA_B receptors in hippocampal slices of rats has an impact on the synaptic plasticity in the CA1 area. Long-term potentiation (LTP) was induced by tetanic stimulation of the Schaffer collateral/commissural fiber tract and the responses of CA1 pyramidal neurons were recorded extracellularly. The increase in population spike amplitude after tetanic stimulation was taken as a measure of LTP.The selective GABA_B receptor blockers phaclofen (1 mM) and CGP 35348 (100 M) facilitated the induction of LTP. Although baclofen (1 M) reduced the population spike amplitude, it did not affect LTP. If, however, the stimulation voltage was increased to compensate for the baclofen-induced decline in population spike amplitude, LTP was facilitated. Under these conditions the induction of LTP was accompanied by the appearance of additional population spikes.In conclusion, GABA_B receptors appear to exert a modulatory action on LTP.Send offprint requests to H. R. Olpe at the above address     

The mode of action of fluoride ions on neuromuscular transmission in frogs.

This investigation deals with the effects of fluoride ion (F^?) on the frog sciatic nerve-sartorius muscle preparation; intracellular recording and voltage clamp techniques were employed. The resting and action potentials and the membrane resistance of the muscle fibre were not appreciably affected by F^? (5mM). The amplitude and falling phase of both the endplate potentials (EPPs) and miniature endplate potentials (MEPPs) were augmented by F^? (5 mM), while the frequency of MEPP's and the quantal content of the EPP remained unchanged. The amplitude of potentials evoked by iontophoretic application of acetylcholine was enhanced and the decay prolonged by F^? (5mM); these effects were still seen following the inhibition of cholinesterase activity by pretreatment with anticholinesterase drugs. Both the amplitude and falling phase of endplate current (EPC) of glycerol-treated preparations were augmented after application of F^? (5mM), whereas EPC equilibrium potential and the voltage sensitivity of its half decay time were unaffected. These results suggest that F^? potentiates the response of the endplate membrane to acetylcholine mainly by increasing the sensitivity of the cholinergic receptor, probably through its stabilizing action on the receptor in its activated form.     

Unique effect of tetrapentylammonium ions on sympathetic transmission in rat vas deferens

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Imipramine counteracts corticosterone-induced enhancement of glutamatergic transmission and impairment of long-term potentiation in the rat frontal cortex

The effects of corticosterone administration lasting for 7 and 21 days were studied ex vivo in rat frontal cortex slices prepared 48 h after the last dose of the hormone. In slices originating from corticosterone-treated animals, the amplitude of extracellular field potentials recorded in cortical layer II/III was increased. Corticosterone administration also resulted in an increase of the mean frequency, but not the mean amplitude, of spontaneous excitatory postsynaptic currents (sEPSCs) in layer II/III pyramidal neurons. These effects were accompanied by a reduced magnitude of long-term potentiation (LTP) of field potentials. In a separate set of experiments, rats were treated with corticosterone for 21 days and additionally with a tricyclic antidepressant, imipramine, beginning on the eighth day of corticosterone administration. In this experimental group, the amplitude of field potentials, the mean frequency of sEPSCs and the magnitude of LTP were not different from the control, indicating that corticosterone-induced modifications of basal glutamatergic transmission and synaptic plasticity were reversed by the antidepressant.     

Cyclic GMP modulates the intensity of post-tetanic potentiation in bullfrog sympathetic ganglia.

Post-tetanic potentiation of fast excitatory postsynaptic potential in curarized sympathetic ganglia of bullfrog was recorded by sucrose-gap technique. Diazepam inhibited the post-tetanic potentiation in a dose-dependent manner. Dibutyryl 3',5'-guanosine monosphosphate (dibutyryl cyclic GMP; 10^?6M) and a phosphodiesterase inhibitor, 1-methyl, 3-isobutyl xanthine (SC 2964; 10^?6M) mimicked the action of diazepam. Cyclic 3',5'-adenosine monophosphate or its dibutyryl derivative (up to 10^?5M) had no effect on this response. The inhibition obtained with diazepam could be potentiated in the presence of dibutyryl cyclic GMP. Since post-tetanic potentiation is a consequence of events that occur presynaptically, it was inferred that diazepam exerts its action on the presynaptic terminals and endogenous cyclic GMP may participate in diazepam's action. When the resting membrane potential from the presynaptic terminals was recorded, dibutyryl cyclic GMP depolarized these terminals without altering the potential of interganglionic nerve axons. Diazepam and γ-aminobutyric acid (GABA) had similar effects. The axon terminal depolarization elicited by both drugs could be blocked by picrotoxin, a specific GABA receptor antagonist. The uptake of [³H]GABA into the ganglion failed to be changed by dibutyryl cyclic GMP but the release of [³H]GABA from the ganglion was increased by dibutyryl cyclic GMP (10^?6 M). Since a GABA store is located in glial cells of sympathetic ganglia, the release of [³H]GABA elicited by dibutyryl cyclic GMP may mediate the depolarization of axon terminals elicited by this drug.