Double-band sarcomeric SHG pattern induced by adult skeletal muscles alteration during myofibrils preparation.

To understand the reported difference between double band, sarcomeric second harmonic generation pattern of isolated myofibril and predominant single band pattern found in thick muscle tissues, we studied the effect of myofibril preparation on the second harmonic generation pattern. We found that double band sarcomeric second harmonic generation pattern usually observed in myofibrils (prepared from fresh tissue) is due to muscle alteration during the mixing and triton treatment processes. Single band sarcomeric second harmonic generation pattern could be observed in isolated myofibrils when this alteration is previously prevented using paraformaldehyd fixed tissue. We conclude that single band sarcomeric second harmonic generation pattern is a signature of adult muscle myofibrils in normal physiological condition, suggesting that sarcomeric second harmonic generation patterns could be used as a valuable diagnosis tool of muscle health.

Some aspects of the physiological role of ion channels in the nervous system.

Recent analyses of the genomes of several animal species, including man, have revealed that a large number of ion channels are present in the nervous system. Our understanding of the physiological role of these channels in the nervous system has followed the evolution of biophysical techniques during the last century. The observation and the quantification of the electrical events associated with the operation of the ionic channels has been, and still is, one of the best tools to analyse the various aspects of their contribution to nerve function. For this reason, we have chosen to use electrophysiological recordings to illustrate some of the main functions of these channels. The properties and the roles of Na+ and K+ channels in neuronal resting and action potentials are illustrated in the case of the giant axons of the squid and the cockroach. The nature and role of the calcium currents in the bursting behaviour of the neurons are illustrated for Aplysia giant neurons. The relationship between presynaptic calcium currents and synaptic transmission is shown for the squid giant synapse. The involvement of calcium channels in survival and neurite outgrowth of cultured neurons is exemplified using embryonic cockroach brain neurons. This same neuronal preparation is used to illustrate ion channel noise and single-channel events associated with the binding of agonists to nicotinic receptors. Some features of the synaptic activity in the central nervous system are shown, with examples from the cercal nerve giant-axon preparation of the cockroach. The interplay of different ion conductances involved in the oscillatory behaviour of the Xenopus spinal motoneurons is illustrated and discussed. The last part of this review deals with ionic homeostasis in the brain and the function of glial cells, with examples from Necturus and squids.

Properties and development of calcium currents in embryonic cockroach neurons.

In freshly dissociated neurons from embryonic cockroach (Periplaneta americana L.) brains, voltage-dependent calcium currents appear early in development (E14). Their intensity increases progressively during embryonic life until eclosion (E35). Their time course and voltage dependency are characteristic of high voltage activated (HVA) currents although a 10 mV shift of the I/V curve towards more negative potentials was observed between E18 and E23. Their sensitivity to omega-AgaTx-IVA and omega-CgTx-GVIA and insensitivity to both amiloride and isradipine indicate that the corresponding channels are of the P/Q and N types. These channels, as well as a small proportion of toxin-resistant (R) channels (about 20%), are blocked by mibefradil and verapamil. The physiological significance of these currents and their modifications during embryonic life is discussed.

Quantitative morphological analysis of embryonic cockroach (Periplaneta americana) brain neurons developing in vitro.

Neurons dissociated from the brain of embryonic cockroaches (Periplaneta americana) can be maintained in culture for several weeks. The survival as well as the progressive organization of the neurons into a complex network was studied during a 5-week period under different culture conditions. About 10% of the dissociated cells adhered to the culture dish. This figure remained constant throughout the culture. The cell diameter ranged from 10 to 20 microns and did not change significantly over time in culture. Whereas only a few cells exhibited neurites at the start of the culture, the number of cells exhibiting neurites increased to reach about 99% after 2 weeks. The different cells were then connected to each other, forming a network, which became more and more complex. The number of cells per cluster as well as the length and the diameter of the "connectives" that linked the different clusters were found to increase with time. The morphology of individual neurons within the network was visualized after intracellular injection of biocytin. Labeling with antibodies raised against serotonin or GABA indicated that neurons were able to differentiate and to acquire specific neurotransmitter fates. The serotonergic phenotype was found to appear progressively throughout the culture, in parallel with the formation of the network. Cell density, addition of fetal calf serum, and ecdysone were shown to influence the development of the network.

omega-AgaIVA-sensitive (P/Q-type) and -resistant (R-type) high-voltage-activated Ba(2+) currents in embryonic cockroach brain neurons.

By means of the whole cell patch-clamp technique, the biophysical and pharmacological properties of voltage-dependent Ba(2+) currents (I(Ba)) were characterized in embryonic cockroach brain neurons in primary culture. I(Ba) was characterized by a threshold of approximately -30 mV, a maximum at approximately 0 mV, and a reversal potential near +40 mV. Varying the holding potential from -100 to -40 mV did not modify these properties. The steady-state, voltage-dependent activation and inactivation properties of the current were determined by fitting the corresponding curves with the Boltzmann equation and yielded V(0.5) of -10 +/- 2 (SE) mV and -30 +/- 1 mV, respectively. I(Ba) was insensitive to the dihydropyridine (DHP) agonist BayK8644 (1 microM) and antagonist isradipine (10 microM) but was efficiently and reversibly blocked by the phenylalkylamine verapamil in a dose-dependent manner (IC(50) = 170 microM). The toxin omega-CgTxGVIA (1 microM) had no significant effect on I(Ba). Micromolar doses of omega-CmTxMVIIC were needed to reduce the current amplitude significantly, and the effect was slow. At 1 microM, 38% of the peak current was blocked after 1 h. In contrast, I(Ba) was potently and irreversibly blocked by nanomolar concentrations of omega-AgaTxIVA in approximately 81% of the neurons. Approximately 20% of the current was unaffected after treatment of the neurons with high concentrations of the toxin (0. 4-1 microM). The steady-state dose-response relationship was fitted with a Hill equation and yielded an IC(50) of 17 nM and a Hill coefficient (n) of 0.6. A better fit was obtained with a combination of two Hill equations corresponding to specific (IC(50) = 9 nM; n = 1) and nonspecific (IC(50) = 900 nM; n = 1) omega-AgaTxIVA-sensitive components. In the remaining 19% of the neurons, concentrations >/=100 nM omega-AgaTxIVA had no visible effect on I(Ba). On the basis of these results, it is concluded that embryonic cockroach brain neurons in primary culture express at least two types of voltage-dependent, high-voltage-activated (HVA) calcium channels: a specific omega-AgaTxIVA-sensitive component and DHP-, omega-CgTxGVIA-, and omega-AgaTxIVA-resistant component related respectively to the P/Q- and R-type voltage-dependent calcium channels.

Effects of nicotinic and muscarinic ligands on embryonic neurones of Periplaneta americana in primary culture: a whole cell clamp study.

The pharmacological properties of acetylcholine (ACh) receptors of cultured neurones from embryonic cockroach brains were studied using the whole-cell configuration of the patch-clamp technique. More than 90% of the studied neurones responded to ACh by a monophasic inward current, the intensity of which varied from cell to cell. The sequence of potency of the five tested agonists was ACh > nicotine=carbamylcholine > suberyldicholine=oxotremorine. The dose-response relationship was complex, suggesting the existence of two populations of receptors: high-affinity receptors (extrapolated K(d) around 10(-7) M) and low-affinity receptors (extrapolated K(d) around 5x10(-5) M). The current-voltage relationship of the induced current was linear between -80 and -40 mV and the extrapolated reversal potential was not significantly different from 0 mV. The sequence of decreasing potency of the antagonists of the ACh response was: methyllycaconitine > alpha-bungarotoxin > mecamylamine > curare > strychnine > bicuculline > atropine > picrotoxin. These results show: (1) that, in embryonic brain neurones, the response to ACh corresponds to the opening of non-selective cationic channels; and (2) that the pharmacology of the ACh receptors is mainly but not solely nicotinic. The nature of the single events which underlie this response, as well as the structure of the channels (homo or hetero-oligomeric) remain to be investigated.

VIP and secretin augment cardiac L-type calcium channel currents in isolated adult rat ventricular myocytes.

Vasoactive intestinal peptide (VIP) is colocalized in parasympathetic nerve terminals in the heart and coreleased from these nerve terminals with the "classical" neurotransmitter acetylcholine (Ach). VIP also exerts a positive inotropic effect on the intact heart and enhances adenylyl cyclase activity in isolated heart membranes. Using the whole-cell patch-clamp technique, we show here that VIP enhances Ca2+ and Ba2+ currents (IBa) through voltage-dependent L-type Ca2+ channels in adult rat ventricular myocytes. Neither the kinetics nor the voltage-dependent properties of the currents are affected. The effect of VIP on IBa is dose dependent with a half-maximal concentration of approximately 0.4 microM. The onset of the effect of VIP and the recovery phase are slow, suggesting the involvement of an intracellular second messenger. The effect of VIP on IBa is antagonized by a peptide analog of the growth hormone releasing factor ([Ac-Tyr1, D-Phe2]-GRF) which belongs to the same peptide family as VIP. Although VIP and the beta-adrenergic receptor agonist isoproterenol (ISO) enhance IBa peak amplitudes to approximately the same extent, the effect of VIP is not seen on all cells. Only approximately 50% of the isolated myocytes respond to 5 microM VIP, whereas 95% of the cells respond to ISO. Similar results were obtained using the amphotericin B perforated-patch whole-cell-recording technique, suggesting that the variable response to VIP does not reflect the loss of a pivotal intracellular regulator. The gastrointestinal hormone secretin, a peptide structurally related to VIP, also potentiates IBa in adult rat ventricular myocytes, although secretin is substantially more potent than VIP (half-maximal concentration for secretin is about 0.7 nM). Taken together, these results suggest that the VIP- (and secretin-) induced potentiation of IBa in adult rat ventricular myocytes is mediated through a non-VIP-preferring class of VIP receptors.

Regulation of the frequency-dependent facilitation of L-type Ca2+ currents in rat ventricular myocytes.

1. An increase in the rate of stimulation induces an augmentation of L-type Ca2+ currents (ICa) and concomitant slowing of current decay in rat ventricular cells. This facilitation is quasi immediate (1-3 s), graded with the rate of stimulation, and occurs only from negative holding potentials. We investigated this effect using trains of stimulation at 1 Hz and the whole-cell patch-clamp technique (18-22 degrees C). 2. The decay of ICa is normally bi-exponential and comprises fast and slow current components (ICa,fc and ICa,sc, respectively). Facilitation of ICa was observed only when ICa,fc was predominant. 3. Facilitation developed during the run-up of ICa with the interconversion of ICa,sc into ICa,fc, and vanished during the run-down of ICa with the loss of ICa,fc.Ni2+ (300 microM) and nifedipine (1 microM) suppressed facilitation owing to the preferential inhibition of ICa,fc. 4. Facilitation of ICa was not altered (when present) or favoured (when absent) by the cAMP-dependent phosphorylation of Ca2+ channels promoted by isoprenaline or by intracellular application of cAMP or of the catalytic subunit of protein kinase A (C-sub). A similar effect was observed when the dihydropyridine agonist Bay K 8644 was applied. In both cases, facilitation was linked to a preferential increase of ICa,fc. 5. Following intracellular application of inhibitors of protein kinase A in combination with a non-hydrolysable ATP analogue, ICa consisted predominantly of ICa,sc and no facilitation was observed. The calmodulin antagonist naphthalenesulphonamide had no effect on facilitation. 6. When Bay K 8644 was applied in combination with isoprenaline, cAMP or C-sub, the decay of ICa was slowed with the predominant development of ICa,sc, and facilitation of ICa was nearly abolished. Facilitation also depended on extracellular Ca2+, and was suppressed when Ba2+ replaced Ca2+ as the permeating ion. 7. When no EGTA was included in the patch pipette, facilitation was not further enhanced but a use-dependent decrease of ICa frequently occurred. When BAPTA was used in place of EGTA, the rate of inactivation of ICa was reduced and facilitation was abolished. 8. In conclusion, the facilitation of ICa that reflects a voltage-driven interconversion of ICa,fc into ICa,sc is also regulated by Ca2+ and by cAMP-dependent phosphorylation. The presence of the gating pattern typified by ICa,fc is required. Ca2+ may exert its effect near the inner pore of the Ca2+ channel protein and control the distribution between the closed states of the two gating pathways.

Repriming of L-type calcium currents revealed during early whole-cell patch-clamp recordings in rat ventricular cells.

1. The establishment of the whole-cell patch-clamp recording configuration (WCR) revealed a type of inhibition to which L-type Ca2+ channels were subject in static rat ventricular myocytes before obtaining the WCR. 2. Immediately after membrane disruption (< 10 s), the Ca2+ current (ICa) was absent but gradually increased in amplitude to reach its final waveform (amplitude and kinetics) 2-3 min after the WCR was reached. 3. Three distinct phases (P) were identified. First, no inward but an outward current, blocked (1-2 min) by Cs+ dialysing from the patch pipette (P1), was recorded. Second, overlapping with (P1), ICa increased dramatically to reach a maximum peak amplitude within 2-3 min (P2). Concomitantly, its rate of decay, initially monoexponential and slow, became biexponential owing to the appearance of a fast component of inactivation (P3). Complete interconversion between slow and fast components sometimes occurred. 4. Changes in current waveform were not related to voltage loss or series resistance variation, and suppression of an outward current (P1) was unlikely to account for P2 and P3. 5. The run-up of ICa was independent of the nature of the permeating ions, the membrane holding potential, depolarization, rate of stimulation, the intracellular Ca2+, ATP, Mg2+, Cs+ and the pH of the pipette solution. Since large Ca2+ currents were recorded using the perforated patch technique, the run-up of ICa is not explained by the wash-out of an inhibitory endogenous macromolecule during cell-pipette exchanges. 6. Pharmacological manipulations, including the use of Ca(2+)-Ba(2+)-EGTA and exposure of the cells to isoprenaline and/or Bay K 8644 prior to recording, did not alter the mechanism primarily responsible for build-up. Unrepriming of channel activity was required before these modulations could be effective. 7. Currents could however be instantly augmented when cells were extracellularly superfused during the run-up step. The wash-out of an inhibitory agent originating in the cell itself (such as H+, NH4+ and lactate) and accumulating in the extracellular microenvironment of the cells seems unlikely. Rather, we suggest that pressure-induced mechanostimulation may be involved in the restoration of Ca2+ channel activity.

Voltage-dependent regulation of L-type cardiac Ca channels by isoproterenol.

The beta-adrenergic cascade is important for the regulation of voltage-dependent Ca channels by phosphorylation. Here we report that isoproterenol (ISO) profoundly alters the voltage-dependent properties of L-type Ca channels studied in rat ventricular cells. ISO (1 microM) shifted both threshold and maximal activation of Ba current (IBa) towards more negative potentials (approx. 10 mV). An equivalent shift was observed in the steady-state voltage-dependent inactivation curve. As a consequence, the potentiation induced by ISO on IBa was greater for weak depolarizations and from negative holding potentials (Vh). We have excluded that the contribution of minor uncompensated series resistances, the activation of Cl currents or changes in junction potential during the experiments account for these effects. In addition, ISO had a dual effect on IBa decay depending on the voltage step (acceleration below, slowing above -10 mV). In conclusion, it is postulated that the voltage dependence of the potentiating effects of ISO on Ca channels activity may ensure a selective regulation among heart tissues with different membrane resting potentials.

Modulation of Ca currents in isolated frog atrial cells studied with photosensitive probes. Regulation by cAMP and Ca2+: a common pathway?

We have studied the regulation of cardiac Ca current by intracellular cyclic AMP (cAMP) and Ca2+, using photosensitive, caged compounds and the whole-cell, patch-clamp technique in isolated frog atrial cells. Although both low voltage activated (LVA) and high voltage activated (HVA) Ca channels were found to be present in these cells, only the HVA Ca currents were sensitive to modulation by isoproterenol or dihydropyridines (DHPs). The application of extracellular isoproterenol, as well as the photorelease of intracellular cAMP or Ca2+ at micromolar and submicromolar concentrations, respectively, had no effect on LVA Ca currents. In contrast, these agents: (i) increased the amplitude of currents through HVA channels, carried by either Ca2+ or Ba2+ with a similar time-course, (ii) slowed the decay of the current when Ba2+ was the permeating ion, and (iii) modulated the agonist effect of the DHP Bay-K 8644. The strong similarities between the effects of cAMP and Ca2+ suggest that both of these intracellular messengers might eventually lead to the phosphorylation of HVA Ca channels. It is possible that Ca-dependent phosphorylation of the channels may account for the potentiation of Ca current induced by repetitive stimulation.

Characterization of voltage-dependent calcium channels expressed in Xenopus oocytes injected with mRNA from rat heart.

1. The properties of voltage dependent cardiac Ca channels expressed in Xenopus laevis oocytes after injection of mRNA from rat heart were investigated using the double-microelectrode voltage-clamp technique. 2. Endogenous Ba current (IBa,E) and expressed cardiac Ba current (IBa,C) were studied at various external concentrations of barium (Ba2+). These two entities could be distinguished by their amplitude and their pharmacology. IBa,C was more sensitive to the inorganic Ca channel blocker manganese (Mn2+). The contaminant IBa,E presented properties of voltage dependence identical to IBa,C, but was negligible in the presence of a low external Ba2+ concentration (2 mM). 3. In 2 mM-Ba2+, IBa,C activated at -35 mV, peaked at -14 mV, and reversed at +26 mV. Steady-state inactivation properties, in consideration of the half-inactivation potential of -35 mV, were also typical of L-type Ba currents. However, the decay of IBa,C was very slow (time constant of inactivation near 600 ms). No evidence for the expression of cardiac transient Ca channels (T-type) was found. 4. IBa,C was enhanced after exposure to the 1,4-dihydropyridine (DHP) agonist Bay K 8644. The enhancement of IBa,C was voltage dependent (maximum at -30 +/- 5 mV) and associated with a slowing in current decay. Current-voltage and concentration-response curves obtained for various Ba2+ concentrations revealed an antagonism between external Ba2+ and the 1,4-DHP agonist Bay K 8644. Similar results were found using the (-)Bay K 8644 pure agonist isomer. 5. We conclude that oocytes injected with mRNA from rat heart expressed only the high threshold, long-lasting or L-type Ca channels. The availability of expressed L-type Ca channels for quantitative pharmacological studies using low Ba2+ concentration has been demonstrated.

Cyclic-AMP-dependent phosphorylation modulates the stereospecific activation of cardiac Ca channels by Bay K 8644.

Voltage-gated Ca channels have been reported to be regulated by membrane potential, phosphorylation and binding of specific agonists or antagonists such as dihydropyridines. We report here evidence that cyclic AMP (cAMP) modulates the activation of Ca-channel current by the dihydropyridine agonist Bay K 8644. Bay K 8644 (racemate) alone induces a primary voltage-dependent, potentiating effect on peak current amplitude and accelerates the current decay. In contrast, in the presence of cAMP activators, we observed a striking slowing of the decay in addition to the increase in peak current. The agonist (-)-Bay K 8644, but not the antagonist (+)-Bay K 8644, when applied in combination with cAMP, forskolin or isoproterenol, mimics the effect of the racemate. We have interpreted the results presented here in respect of a cAMP-dependent modulation of Bay K 8644 effects on cardiac Ca-channel currents. It may open the new perspective that dephosphorylated and phosphorylated Ca channels have distinct pharmacology.

Two pathways for Ca2+ channel gating differentially modulated by physiological stimuli.

In cardiac muscle, Ca2+ entry through voltage-gated Ca2+ channels plays an important role in the generation of action potentials and in the development of tension. Although it had been assumed that there was a single type of cardiac Ca2+ channel, recent studies reveal that multiple Ca2+ channel types coexist in some myocardial cells. Here, we report that macroscopic Ca2+ current (ICa) waveforms in isolated adult rat ventricular myocytes comprise two kinetically distinct components; these are referred to here as ICa (fc) and ICa (sc) to denote the fast and slow components, respectively, of ICa decay. In contrast to findings in other cells, the properties of ICa (fc) and ICa (sc) suggest the presence of two pathways for gating of a single type of high-threshold Ca2+ channel rather than two distinct Ca2+ channel types. In addition, gating via ICa (fc) and ICa (sc) is regulated by changes in membrane potential and stimulation frequency. Hyperpolarized potentials and low stimulation frequencies reveal preferential activation via ICa (fc); depolarized potentials and high stimulation frequencies, in contrast, favor activation via ICa (sc). After exposure to beta-adrenergic agonists or the Ca2+ agonist BAY K 8644, peak ICa amplitudes increase owing to the preferential augmentation of ICa (fc); beta-agonists and BAY K 8644 also increase ICa (sc), albeit to a smaller extent than ICa (fc). Thus, in addition to voltage- and frequency-dependent regulation, the two pathways for Ca2+ channel gating are modulated differentially by beta-adrenergic and Ca2+ channel agonists.

Electrophysiological expression of endothelin and angiotensin receptors in Xenopus oocytes injected with rat heart mRNA.

Functional endothelin and angiotensin receptors have been expressed in Xenopus oocyte following the microinjection of rat heart mRNA. Under voltage clamp conditions, application of these peptides clearly induced oscillatory Ca2+-activated chloride currents in a dose-dependent manner. In addition, no direct modulation of expressed or native cardiac Ca channels was observed.

Effects of the Ca-antagonist nicardipine on K+ currents and Na+-Ca2+ exchange in frog atrial fibres.

This paper concerns the specificity of nicardipine, a calcium antagonist from the dihydropyridine class which is used in the treatment of cardiovascular disorders. It is well established that in cardiac cells dihydropyridines inhibit the calcium current (Isi) and the correlated phasic tension. In this study we demonstrate that nicardipine, in the concentration range which blocks Isi (KD = 1 microM) also decreases the amplitude of the potassium-delayed current (KD = 3 microM) in frog atrial fibres. Moreover, tonic tension, which has been reported to be directly dependent on the Na+-Ca2+ exchange, was also reduced by nicardipine and the time course of the onset of both contraction and relaxation was significantly slowed. These results indicate that on depolarized membranes, dihydropyridines probably affect voltage-dependent mechanisms with a high threshold of activation that are unrelated to Ca2+ channels, such as potassium-delayed current and Na+-Ca2+ exchange.