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J Nargeot

Publications and source records attributed to J Nargeot.

At least 109 records · Page 6Linked to original sources
PubMed1987

Ca channels induced in Xenopus oocytes by rat brain mRNA.

RNA was isolated from brains of 16-d-old rats and poly(A) samples were injected into stage V and VI oocytes. After allowing 2-5 d for expression, most oocytes were exposed to medium in which the K had been replaced by Cs for 24 hr prior to recording. Ba currents were usually measured in Cl-free Ba-methanesulfonate saline. IBa in noninjected oocytes was often undetectable, but ranged up to 50 nA (22 +/- 4 nA, n = 21). In contrast, injected oocytes showed a peak IBa of 339 +/- 42 nA (n = 33). The threshold for activation of IBa was -40 mV, with peak currents at +10 to +20 mV. After a peak, currents decayed to a nearly steady level along a single-exponential time course (tau = 650 +/- 50 msec at +20 mV). The maintained current was 67 +/- 6% (n = 9) of the early peak amplitude. A prepulse duration of 5 sec was needed to examine the inactivation of barium currents in injected oocytes. The inward IBa could be observed in BaCl2 solutions at potentials positive to ECl and also in Na-free salines, indicating that neither Cl- nor Na+ was carrying the inward current. Although IBa displayed voltage-independent blockade by Cd (50% inhibition at 6 microM), the peptide Ca channel antagonist, omega-CgTX (1 microM), and the organic Ca channel-blocking agents (verapamil, compound W-7, and nifedipine) were uniformly ineffective. No effects were observed with the dihydropyridine antagonist nifedipine (even at 10 microM, or when cells were held at -40 mV) or agonist Bay K-8644. However, IBa was enhanced via activation of protein kinase C with 4-beta-phorbol dibutyrate (PBT2). In contrast, use of forskolin to activate protein kinase A did not alter IBa.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Direct evidence for the inhibition of platelet aggregation and release by intracellular cyclic AMP produced with a new photoactivatable derivative.

An increase in platelet cyclic AMP (cAMP) via stimulation of adenylate cyclase is thought to be the underlying mechanism by which potent prostaglandins i.e. PGD2, PGI2, inhibit platelet functions. We report here new and direct evidence for the inhibitory effects of cAMP on platelet aggregation and serotonin release. Washed platelets from rat were incubated with a new photoactivatable cAMP analogue (4,5-dimethoxy-2-nitrobenzyl ester); this compound is almost physiologically inert before irradiation and liberates free cAMP ("cAMP jumps") following light flashes. A single flash, delivered after 2 min incubation in 100-200 microM of the analogue, dramatically inhibited thrombin-induced aggregation, as compared with controls. Endogenous serotonin release, measured in the same samples by means of an electrochemically treated carbon electrode was undetectable after the cAMP jump. Pre-irradiated solutions added to platelets had no effect. The kinetics of the flash-induced effects were also studied. From these results we can conclude that: i) the photoactivatable cAMP derivative has to permeate through the platelet membrane; ii) the analogue remains photolabile; and, iii) intracellular cAMP, resulting from photolysis dramatically inhibits platelet aggregation and serotonin release. It is possible that cAMP exerts its effects by regulating cytoplasmic free calcium concentration and/or other actions affecting platelet activation.

Animals↗
PubMed1985

Photochemically produced intracellular concentration jumps of cAMP mimic the effects of catecholamines on excitation-contraction coupling in frog atrial fibers.

Previously, we reported that concentration jumps of cAMP produced by light flashes in the presence of a photosensitive analogue of cAMP increase the amplitude of the slow inward current (Isi) in isolated bullfrog atrial trabeculae (Nargeot et al. 1983). Here, using newly designed photolabile cyclic nucleotides (Nerbonne et al. 1984a), we have examined the effects of intracellular concentration jumps of cAMP and cGMP on excitation-contraction coupling in frog heart. Concentration jumps of cAMP increase the amplitude and the duration of action potentials, increase Isi and twitch tension. Following single flashes, maximum responses are observed in 10-30 s and recovery times are 30-120 s. The time courses of the cAMP-induced increases in Isi and phasic tension amplitudes are parallel, implying a direct correlation between Ca2+ influx through the slow channels and the development of phasic tension. Although the amplitudes are increased severalfold, cAMP jumps do not measurably alter the kinetics or voltage dependences of the current or tension. cAMP concentration jumps increase the delayed K+ current (IK) and decrease tonic tension; relaxation of contraction is not, however, influenced by cAMP jumps. Concentration jumps of cGMP, on the other hand, have no measurable effects on the action potential, Isi, IK or tension in this preparation.

Action Potentials↗
PubMed1985

Calcium channels are 'unblocked' within a few milliseconds after photoremoval of nifedipine.

The organic Ca2+ antagonists are potent inhibitors of Ca2+ influx in cardiac and smooth muscle and are widely used clinically in the treatment of various cardiovascular disorders. It appears that Ca2+ antagonist binding prevents the normal movement of ions through Ca2+ channels, perhaps via an open-channel blockade mechanism. Although this concept is generally accepted, questions do remain about the detailed relationship between binding and blockade in the case of the structurally diverse organic Ca2+ channel blockers; e.g., (1) do they bind preferentially to open, closed and/or inactivated channels; (2) are there multiple binding sites; (3) do they act at extracellular and/or intracellular sites; and (4) does blocking or unblocking depend on membrane potential or its history? The dihydropyridine Ca2+ antagonist, nifedipine, contains an o-nitrobenzyl moiety and is photolabile; irradiation yields a molecule devoid of channel blocking activity and the photoconversion reactions are complete within 100 microseconds. Taking advantage of these properties to study the mechanistic details of nifedipine blockade of Ca2+ channels, we examined the waveform of the slow inward Ca2+ current (Isi) in atrial fibers before and following flash-induced removal of nifedipine. After flashes, we find that nifedipine blockade is reversed within at most a few milliseconds and that the rate of Isi reactivation parallels the normal, voltage-dependent activation rate. Our results imply that nifedipine binds to and stabilizes resting, closed Ca2+ channels and are not in agreement with the recent conclusions of Morad and coworkers that photoconversion of nifedipine must be followed by membrane repolarization in order to effect recovery of Isi and tension.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

[Mechanism of action of sympathetic and parasympathetic neuromediators at the level of the heart cell: use of photosensitive molecules].

We have shown in the first part of this work, how pharmacological technics can be associated to electrophysiological measurements, to study the action of neurotransmitters on the excitation-contraction coupling in cardiac cells. However, to better understand their mechanism of action, it is necessary to have informations about the link between the activation of the receptor and the conductance changes. Kinetics measurements have been very useful to investigate this problem at the nicotinic cholinergic receptor. This study was performed with technics such as iontophoresis, voltage relaxations, noise analysis and recently patch clamp measurements. Nicotinic responses are very fast (in the order of the msec) and are however the best known. The binding of the agonist to the receptor would lead to the opening of a channel only by molecular conformation change. By comparison, beta-adrenergic and muscarinic responses in heart or smooth muscle can be considered as "slow responses" because they are thought to be mediated by intracellular biochemical steps. We have developed a new technique using photosensitive compounds, which allows to jump the extra- or intracellular concentration of some active molecules. The experiments have been performed on frog atrial fibers, in current or voltage clamp conditions using a double sucrose gap technique. Various photochemical reactions have been exploited. The photoisomerization of an azobenzene derivative (Bis-Q) allows to study the muscarinic-induced potassium conductance. Bis-Q binds to muscarinic receptors and blocks the effect of agonists. These drugs have the additional property that their cis and trans configuration have different potencies. The photoisomerization cis----trans or trans----cis is obtained a few msec after a 1 msec duration light flash (respectively visible or UV), and then induces a concentration jump of antagonist near the receptor. The results show that the potassium conductance change has a slow relaxation time constant (tau = 500 msec.) and this suggest rather successive intracellular steps than the opening of a channel directly linked to the receptor. Cyclic nucleotides are considered as the intracellular messengers for various neurotransmitters or hormones. Our photochemical technique allows to jump the intracellular concentration of cAMP and cGMP in a few msec. cAMP (or cGMP) orthonitrobenzyl derivatives are able to penetrate cell membrane without injection and have no effect up to 100 microM. A UV flash (1 msec duration) induces a photolysis of the derivative and generates instantaneously cAMP (or cGMP) inside the cell.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed1983

Time course of the increase in the myocardial slow inward current after a photochemically generated concentration jump of intracellular cAMP.

Voltage-clamped atrial trabeculae from bullfrog hearts were exposed to membrane-permeant photolyzable o-nitrobenzyl esters of cAMP and cGMP. UV flashes produced intracellular concentration jumps of cAMP or cGMP. With the cAMP derivative, flashes resulted in an increased slow inward current (Isi), producing a broadened action potential. The Isi reached a maximum 10-30 sec after the flash and decreased over the next 60-300 sec. The first increases were observable within 150 msec; this value is an upper limit imposed by the instrumentation. Responses to flashes lasted longer at higher drug concentrations and in the presence of the phosphodiesterase inhibitor papaverine; effects of flashes developed and decreased faster at higher temperature. Although the amplitude of the Isi was increased, its waveform and voltage sensitivity were not affected. Intracellular concentration jumps of cAMP failed to affect the muscarinic K+ conductance. There were no observable effects of cGMP concentration jumps. The data confirm (i) that cAMP regulates the Isi and (ii) that the 5- to 10-sec delay between application of beta-agonists and the onset of positive inotropic effects, observed in previous studies, has been correctly ascribed to events prior to the interaction between cAMP and protein kinase.

Animals↗
PubMed1982

A photoisomerizable muscarinic antagonist. Studies of binding and of conductance relaxations in frog heart.

These experiments employ the photoisomerizable compound, 3,3'-bis-[alpha-(trimethylammonium)methyl]azobenzene (Bis-Q), to study the response to muscarinic agents in frog myocardium. In homogenates from the heart, trans-Bis-Q blocks the binding of [3H]-N-methylscopolamine to muscarinic receptors. In voltage-clamped atrial trabeculae, trans-Bis-Q blocks the agonist-induced potassium conductance. The equilibrium dose-response curve for carbachol is shifted to the right, suggesting competitive blockade. Both the biochemical and electrophysiological data yield a dissociation constant of 4-5 microM for trans-Bis-Q; the cis configuration is severalfold less potent as a muscarinic blocker. Voltage-clamped preparations were exposed simultaneously to carbachol and Bis-Q and were subjected to appropriately filtered flashes (less than 1 ms duration) from a xenon flashlamp. Trans leads to cis and cis leads to trans photoisomerizations cause small (less than 20%) increases and decreases, respectively, in the agonist-induced current. The relaxation follows an S-shaped time course, including an initial delay or period of zero slope. The entire waveform is described by [1 - exp(-kt)]n. At 23 degrees C, k is approximately 3 s-1 and n is 2. Neither k nor n is affected when: (a) [Bis-Q] is varied between 5 and 100 microM; (b) [carbachol] is varied between 1 and 50 microM; (c) carbachol is replaced by other agonists (muscarine, acetylcholine, or acetyl-beta-methylcholine); or (d) the voltage is varied between the normal resting potential and a depolarization of 80 mV. However, in the range of 13-30 degrees C, k increases with temperature; the Q10 is between 2 and 2.5. In the same range, n does not change significantly. Like other investigators, we conclude that the activation kinetics of the muscarinic K+ conductance are not determined by ligand-receptor binding, but rather by a subsequent sequence of two (or more) steps with a high activation energy.

Animals↗
PubMed1981

Analysis of the negative inotropic effect of acetylcholine on frog atrial fibres.

Voltage-clamp experiments have been performed on frog atrial preparations in order to study the mechanism of the inotropic effect of acetylcholine (ACh) at various concentrations. The amplitude of the slow inward current (Is) is reduced even at low ACh concentrations; such low concentrations have little or no effect on potassium permeability. Dose-effect relationships for Is inhibition (Is/Is max) by ACh show a half amplitude dose (K0.5 around 8 X 10(-8) M ACh. The reduction of Is is attributed largely to a decrease of the maximal conductance of the slow channel (gs). Steady-state activation and inactivation parameters are not affected by ACh. Experiments in a Na-free solution (Na replaced by Li ions) or in a Ca-free solution (with EGTA) indicate that the "slow sodium current" is more sensitive to ACh than the "slow Ca current", although these two currents both seem to flow through the slow channel. The decrease of the phasic component of contraction observed in the presence of ACh is very well correlated with the decrease of Is (K0.5 = 8 X 10(-8) M ACh), while the increase of the tonic tension may be related to the outward potassium current induced by high concentrations of ACh. The significant difference between the half amplitude dose (K0.5) observed in the dose effect curves with ACh for Is inhibition (K0.5 = 8 X 10(-8) M) and for ACh-induced extra-current (K0.5 - 10(-6) M) may indicate the presence of two muscarinic receptors.

Acetylcholine↗
PubMed1978

The action of acetylcholine on background conductance in frog atrial trabeculae.

The action of acetylcholine (ACh) on membrane potential and currents in frog atrial muscle has been studied with a double sucrose gap technique. The results show the following. 1. ACh induces the development of an extra current, outward at the resting potential, which is dependent on the ACh concentration. 2. The preparation does not show any sign of desensitization. 3. The reversal potential of the current induced by ACh is between 0 and 20 mV more negative than the resting potential and behaves as a K electrode. 4. The mechanism of ACh-induced K conductance presents inward going rectification properties. 5. The delayed outward current is not affected by ACh. However the evolution of its tail current seems to indicate a process of K accumulation related to the ACh-induced current.

Acetylcholine↗
PubMed1976

[Electrophysiologic study of the cholinergic receptor of the myocardial membrane].

A double sucrose gap voltage clamp technic has been used to study the extra-current induced by acetylcholine (Iach) on the myocardial membrane on frog atrial trabeculae. I) No desensitization of the Iach current is noted for repeated perfusions of Ach. II) The Iach current is suppressed by atropine. III) The reversal potential Each is more negative than the resting potential --20 mV less than or equal to Each less than or equal to OmV.IV) The relationship Iach/Holding potential for various [K+]o shows a) That Each behaved as a potassium electrode, b) an inward going rectification. These results indicate that the cholinergic receptor might be related with the gk1 channel.

Acetylcholine↗
PubMed1976

Current clamp and voltage clamp study of the inhibitory action of DNP on membrane electrical properties of frog auricular heart muscle.

Current clamp studies showed that after 10 minutes under DNP 10(-4) M the membrane potential does not change significantly while an important shortening of the action potential duration and a diminished amplitude are observed. Voltage clamp studies have been performed on the slow inward and delayed outward currents. DNP 10(-4) M induced a marked decrease of the slow inward current related to the reduction in both conductance and driving force, and a decrease in the amplitude of the delayed current. The decrease of the slow inward current seems to be mainly responsible for the suppression of the plateau of the action potential during metabolic inhibition.

Action Potentials↗
PubMedDate not supplied

New photoactivatable cyclic nucleotides produce intracellular jumps in cyclic AMP and cyclic GMP concentrations.

The cyclic nucleotides cyclic AMP and cyclic GMP are important intracellular messengers mediating the responses to neurotransmitters and neurohormones and regulating cellular function over a wide range of time scales. Despite the widespread acceptance of this second messenger mechanism in many systems, much remains unknown about their mechanism of action, except that such events are associated with increases or decreases in intracellular cyclic nucleotides. Quantitative descriptions of cyclic nucleotide-dependent processes are hampered by the absence of a means by which intracellular cyclic nucleotide concentrations can be accurately controlled. We have now designed, synthesized and characterized new, substituted photolabile cyclic nucleotide analogues, the 4,5-dimethoxy-2-nitrobenzyl esters of cyclic AMP and cyclic GMP (Fig. 1), which are physiologically inert before irradiation and which liberate free cyclic AMP or cyclic GMP on absorption of a photon. The thermal properties and photolysis rates and efficiencies of light-induced release of cyclic nucleotides from these analogues are more favourable than for the simple o-nitrobenzyl derivatives used previously. These molecules should permit intracellular "concentration jumps' of cyclic AMP or cyclic GMP to be produced in cells under physiological investigation with spatial and temporal resolution unmatched by conventional techniques.

Chemical Phenomena↗