Maternal protein restriction affects cardiovascular, but not respiratory response to L-glutamate microinjection into the NTS of conscious rats.

Purpose: Dysregulation of glutamatergic neurotransmission (GN) is linked to sympathetic-respiratory overactivity and hypertension. We investigated whether maternal protein restriction is able to alter GN into the nucleus of the solitary tract (NTS) in adult offspring.Methods: Wistar rat dams were fed with control (NP; 17% protein) or low-protein (LP; 8% protein) diet during pregnancy and lactation, and their offspring were evaluated at 70-90d old. Direct measurements of mean arterial pressure (MAP), heart rate (HR), respiratory frequency (RF) and respiratory (RV) and cardiac (CV) variabilities were assessed in consciousness. The evaluation of GN into NTS over cardiovascular system were assessed by microinjections of unilateral glutamate (L-glu 0.5 nmol/100nL) and bilateral kynurenic acid (Kyn 2.5 nmol/50nL). The NP and LP groups were compared using unpaired Student's t-test where p < 0.05 was considered significant.Results: The LP exhibited higher MAP at rest (p = 0.03) and after L-glu microinjection (p = 0.04), as well as an increase over HR after Kyn microinjection when compared to the NP (p = 0.049). In the RV, the LP group showed an increase of the component-standard deviation 1 (p = 0.037) at rest. In the CV, the LP presented an increase of the low frequency (LF) component of the pulse interval (PI) (p = 0.034), a decrease of high frequency (HF) of the PI (p = 0.034), beyond an increased LF/HF ratio of the PI (p = 0.027) when compared to the NP. The kynurenic acid microinjection did not produce changes in RV or CV (p > 0.05).Conclusions: Altered GN into the NTS may contribute to augmented blood pressure in protein-restricted offspring.

Increased sodium-calcium exchange current in right ventricular cell hypertrophy induced by simulated high altitude in adult rats.

Ventricular hypertrophy is associated with an increase in action potential (AP) duration which is potentially arrhythmogenic. The implication of the Na-Ca exchange current (I(Na-Ca)) in the lengthening of the AP is controversial. The role of this current in the increased duration of the low plateau of the AP in hypertrophied adult rat ventricular myocytes by simulated chronic high-altitude exposure ( approximately 4500 m) was evaluated. Electrophysiological experiments were carried out on isolated right ventricular myocytes from exposed and control rats with the perforated patch or the conventional whole-cell technique in current or in voltage clamp condition. With the two techniques, a significant increase of the low plateau duration was observed in hypertrophied myocytes as compared to controls. The low plateau in hypertrophied myocytes was depressed when Na was replaced by Li and was no longer recorded when intracellular Ca was buffered with EGTA. Inward tail currents, evoked either on repolarization to -80 mV following a depolarizing pulse to +10 mV or by interrupted AP technique, were greater in hypertrophied than in control myocytes and were abolished when Na was replaced by Li or when intracellular Ca was buffered with EGTA, indicating an increased Na-Ca exchange activity. The Li-sensitive current-voltage curves, obtained by a voltage clamp ramp protocol with an intracellular calcium buffered solution, were not significantly different in both hypertrophied and control myocytes, suggesting no modification in the density of the Na-Ca exchange protein. This was corroborated by the lack of difference in NCX1 mRNA levels between right ventricles from control and exposed rats. We conclude that increased duration of the low plateau of rat ventricular AP in altitude cardiac hypertrophy may be attributed to an increase of the inward I(Na-Ca). This augmented I(Na-Ca)may result from a modification in the intracellular Ca homeostasis.

Opposite effects of halothane on guinea-pig ventricular action potential duration.

Halothane protects the heart against the reperfusion injury observed after an ischemia. In ischemic or anoxic conditions, a large ATP-sensitive K(+) (K(ATP)) conductance is supposed to provide an endogenous protection to the myocardium. In this study, we tested the possibility that halothane acted by modulating this conductance. Isolated guinea-pig cardiomyocytes were successively studied in current clamp and in voltage-clamp conditions. Action potentials regulation by halothane was tested in control conditions and in situations where the K(ATP) channels were activated. In control conditions, halothane decreased action potential duration of myocytes but did not significantly alter the inward rectifying K(+) current. Conversely, halothane lengthened action potential of cells in which the K(ATP) conductance was activated, by inhibiting the K(ATP) current. In ischemic conditions, simultaneous shortening of long action potentials and lengthening of shortened ones would be expected to homogenize the absolute refractory period at the border between normoxic and anoxic zones. This effect, together with a decrease in calcium load, could protect the myocardium against re-entrant arrhythmias.

Functional expression of the L-type calcium channel in mice skeletal muscle during prenatal myogenesis.

The densities of skeletal muscle intramembrane charge movement and macroscopic L-type Ca(2+) current have been shown to increase during prenatal development. In the present work, the electrophysiological characteristics of L-type Ca(2+) channels were analyzed over the embryonic period E14 to E19 using the whole-cell and cell-attached procedures. At the macroscopic level, the whole-cell L-type Ca(2+) conductance increased 100% between E14 and E19. This enhancement was accompanied by a small negative shift of the voltage dependence and a marked acceleration of the inactivation kinetics. At the single-channel level, the unitary conductance decreased significantly from 13.2 +/- 0.1 pS (n = 8) at E14 to 10.7 +/- 0.3 pS (n = 7) at E18 and the open probability was multiplied by 2. No significant change of the density of functional channels was observed during the same period. In contrast to the density of intramembrane charge movement, which, under the same conditions, has been shown to increase between 16 and 19 days, L-type Ca(2+) channels properties change mostly between 14 and 16 days. Taken together, these results suggest that the two functions of the dihydropyridine receptor are carried by two different proteins which could be differentially regulated by subunit composition and/or degree of phosphorylation.

Intracellular calcium signals measured with indo-1 in isolated skeletal muscle fibres from control and mdx mice.

1. Intracellular free calcium concentration ([Ca2+]i) was measured with the fluorescent indicator indo-1 in single skeletal fibres enzymatically isolated from the flexor digitorum brevis and interosseus muscles of control and dystrophic mdx C57BL/10 mice. Measurements were taken from a portion of fibre that was voltage clamped to allow detection of depolarization-induced changes in [Ca2+]i. 2. The mean (+/- s.e.m.) initial resting [Ca2+]i from all control and mdx fibres tested was 56 +/- 5 nM (n = 72) and 48 +/- 7 nM (n = 57), respectively, indicating no significant overall difference between the two groups. However, when comparing a batch of control and mdx fibres obtained from mice older than approximately 35 weeks, resting [Ca2+]i was significantly lower in mdx (16 +/- 4 nM, n = 11) than in control fibres (71 +/- 10 nM, n = 14). 3. Changes in [Ca2+]i elicited by short (5-35 ms) depolarizing pulses from -80 to 0 mV showed similar properties in control and mdx fibres. After a 5 ms duration pulse the mean time constant of [Ca2+]i decay was, however, significantly elevated in mdx as compared to control fibres, by a factor of 1.5-2. For longer pulses, no significant difference could be detected. 4. In response to 50 ms duration depolarizing pulses of various amplitudes the threshold for detection of an [Ca2+]i change and the peak [Ca2+]i reached for a given potential were similar in control and mdx fibres. 5. Overall results show that mdx skeletal muscle fibres are quite capable of handling [Ca2+]i at rest and in response to membrane depolarizations.

SR47063, a potent channel opener, activates KATP and a time-dependent current likely due to potassium accumulation.

(i) We studied the effects of a new cromakalim analogue, SR47063, in guinea-pig ventricular cells. The experiments were carried out in whole-cell patch clamp with internal and external solutions supposedly similar to the physiological ones. (ii) SR47063 reversibly activated a time-independent current reversing near the potassium equilibrium potential, and a time-dependent current reversing at a more positive potential. Both currents were blocked by application of glibenclamide. (iii) The time-independent and the time-dependent currents were activating for the same concentration of agonist in every cell, this concentration being very different from cell to cell. (iv) The amplitude of the time-dependent current was shown to depend directly neither on agonist concentration nor on potential, but rather on the amplitude of the current flowing during the prepulse before the test pulse. (v) We conclude that SR47063 is a potent KATP channel opener acting at concentrations lower than one micromolar, and that the time-dependent current is likely due to accumulation and depletion of potassium in restricted areas of the cells.

Reversible activation of the ATP-dependent potassium current with dialysis of frog atrial cells by micromolar concentrations of GDP.

We studied the effects of internal and external solutions on potassium currents in frog atrial cells. Experiments were carried out in whole cell recording in the presence of tetrodotoxin and cobalt in the bath to suppress the inward currents. In the absence of pyruvate and glucose in the external solution, a time-independent current increased progressively in a few minutes till the death of the cell. This current had the properties of the ATP-sensitive potassium current IK(ATP) in mammalian cells. In the presence of pyruvate and glucose in the external solution, the membrane current stayed low for 30 min. Addition of guanosine monophosphate (GMP, 40 microM), guanosine triphosphate (GTP, 40 to 1000 microM), adenosine diphosphate (ADP, 40 microM) or adenosine triphosphate (ATP, 3000 microM) to the internal solution had no major effect on the current amplitude. In contrast, addition of GDP (20 or 40 microM) produced a loss of rectification in a few minutes. The current activated by GDP was time independent as was the current observed in the absence of glucose and pyruvate. It was sensitive to cesium and barium, it was blocked when ATP was added to GDP in the internal solution, and it was suppressed by the sulphonylurea glibenclamide (1 microM). We suggest that GDP produced a local depletion of ATP, by displacement of the equilibrium between ATP, GDP, ADP and GTP. This hypothesis is supported by the fact that the current activated by GDP was rapidly suppressed when adding GTP in excess to the internal solution.

Action of cromakalim on potassium membrane conductance in isolated heart myocytes of frog.

1. The effects of cromakalim on membrane currents were studied at 20 degrees C in frog atrial and ventricular cells in patch clamp recording using the whole cell configuration. 2. When cromakalim (1 microM) was applied in the external medium, a time-independent current was activated in a few minutes. Cromakalim induced a weak increase of inward membrane currents recorded during hyperpolarization and a large increase of outward membrane currents recorded during depolarization. 3. The current voltage relationship of the cromakalim-induced current reversed near EK and rectified in the outward direction. 4. The cromakalim-activated current was inhibited by external application of cesium (20 mM), barium (1.8 mM), tolbutamide (1 mM) and glibenclamide (1 microM). 5. The effects of cromakalim were insensitive to a cytosolic increase in adenosine 5'-triphosphate (ATP, 3-5 mM). Cromakalim had no effects when applied in the cell. 6. Our results confirm that cromakalim activates an IK(ATP)-like conductance and suggest that the effects of the drug are due to an action on the external side of the membrane and are independent of the ATP cell content.

Activation properties of the inward-rectifying potassium channel on mammalian heart cells.

The early phase of activation of the inward-rectifying potassium channel is studied on single cells from guinea-pig heart. The current is quasi-instantaneous when it is outward, but activates with time when it is inward. This relaxation is exponential and its time-constant decreases with hyperpolarization. The I/V curve reflects a strong inward rectification and has a negative slope conductance on depolarization. Similar results were recorded in the absence of sodium, calcium, chloride ions and in isotonic potassium. Cesium slows down the phase of activation, and eventually appears to block the channels by suppression of the activation. Barium, conversely, does not affect the activation, but promotes an 'inactivation' of this current, which blocks it. These results are independent on the cells' dissociation method. They suggest that this current is the inward rectifier, called IK1 on heart. Its activation curve suggests that the inward and outward currents are flowing through the same channels. The inward rectifier is time- and voltage-dependent on heart as on other tissues. The effects of cesium and barium are also similar. The importance of its negative slope conductance is discussed.

Action potential-like responses due to the inward rectifying potassium channel.

This paper describes experiments carried out in the absence of sodium and calcium in the external solution. Frog atrial trabeculae were stimulated in current clamp with the double sucrose gap technique. The voltage responses looked like slow action potentials with a clear threshold. These responses were not suppressed in the presence of EGTA, in the presence of sodium or calcium channel blockers, or when sulfate ions replaced chloride. Guinea pig isolated ventricular myocytes were studied in whole cell clamp mode with a patch pipette. Under current clamp, they displayed also voltage responses with a threshold. These responses were resistant to cadmium (5 mM), and were suppressed by barium (0.5 mM). A negative slope conductance is required to take into account these results. The membrane current in current clamp can be estimated by plotting the response in the phase plane. This analysis shows that on both types of preparations, the current responsible for the negative slope is not time dependent. This current is suppressed by barium. It can be concluded that it is the outward current flowing through the inward rectifying potassium channels. To confirm this hypothesis, data obtained in voltage clamp on the same preparations were introduced into a computer model to predict the response in current clamp. The results were in agreement with the experiments. Similar responses could be recorded and analyzed on skeletal muscle in isotonic potassium solution. These results show that the inward rectifier can induce by itself properties looking like excitability on different preparations. The physiological significance of this effect in normal conditions is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of the Na+-Ca2+ exchange in the calcium paradox in frog auricular trabeculae.

After a Ca2+-free ouabain perfusion of 8-10 min duration, reperfusion of isolated stimulated frog auricular trabeculae with Ca2+ ouabain containing medium resulted in a large and transient contracture. The contracture was weaker in a quiescent preparation or in the absence of ouabain. In sodium-free (Li+ substitute) ouabain containing medium, the amplitude of the contracture was largely decreased while it disappeared completely in Na+-free medium without ouabain. Moreover this contracture was suppressed by 15 mM Mn2+. Although the approach was only indirect, these results suggest that the contracture is due to an entry of Ca2+ ions through the Na+-Ca2+ exchange mechanism and that this could be the unique route of calcium entry during calcium repletion.

Inhibition by Ba of background current and of its modifications by carbachol in frog atrium.

In previous work on frog atrium [11], we have shown that the carbachol-induced K current (iCCh) and the inward rectifying K channel (iK1) are both inhibited by cesium ions. A more detailed study [1, 2] indicates that the dose-response and the voltage dependence of the inhibition by Cs of iK1 and iCCh are similar. These results are in favor of a modulation by acetylcholine of the iK1 channels. Such a conclusion has been put forward by [3] and by [9]. However, Cs ions can also block the pacemaker currents iK2 [8] and if [5]. An 'if like' component seems to be present in frog atrium (Bonvallet and Ojeda, unpublished results). For these reasons, we have studied the action of barium which can be considered as a more specific inhibitor of the inward rectifying K channels in skeletal muscle [13], [14] and in heart muscle [4]. We report here results obtained on two types of preparations: one in which carbachol increases the background conductance (typical effect) and another one (four fibres from the same frog heart) where carbachol decreases the background conductance (atypical effect) as observed by Carmeliet and Ramon (1980) in sheep Purkinje fibres. In both cases, the results show that, in presence of 5 mM barium, carbachol has no effect on the background current.

Identification and properties of phencyclidine-binding sites in nervous tissues.

[3H]Phencyclidine (PCP) binds to a single class of noninteracting binding sites in rat brain membranes with an affinity Kd of 0.25 microM and a maximal binding capacity BM of 2.4 pmol/mg of membrane protein. PCP derivatives also interact with the muscarinic and mu-opiate receptors in rat brain membranes with affinities that are one or two orders of magnitude lower than those observed for the [3H]PCP-binding sites. Activities of 25 PCP derivatives in the rotarod assay are closely correlated to affinities of these molecules for the [3H]PCP-binding sites, but not for the muscarinic or mu-opiate receptors. Monohydroxylation of PCP generally decreases the affinity of PCP for the [3H]PCP- and muscarinic-binding sites and does not change the affinity for the mu-opiate receptor. The metaphenolic derivative of PCP does not follow these general rules; the affinities of this derivative for the [3H]PCP- and mu-opiate-binding sites are 8 and 430 times higher, respectively, than those of PCP itself. Voltage-clamp experiments with N1E 115 neuroblastoma cells show that PCP is an efficient blocker of both the K+ channel (EC50 = 2.6 microM) and the Na+ channel (EC50 = 9.2 microM).

Block by Cs of K current iK1 and of carbachol induced K current iCch in frog atrium.

1. In frog atrium, Cs ions block both the inward rectifier iK1 and the carbachol induced K current iCch. 2. Both iK1 and iCch display a high affinity for Cs with a K0.5 of 4 X 10(-5) M for iK1 and of 8 X 10(-5) M for iCch at V = -50 mV. 3. Block of both iK1 and iCch is strongly voltage dependent. When fitted by the block model of Woodhull (1973), delta is greater than 1 for the two currents. 4. From these similarities, action of Cch on frog atrium K permeability could be interpreted as a modification of iK1.

Effects of Cs on acetylcholine induced current. Is ik1 increased by acetylcholine in frog atrium?

1.20 mM Cs+ ions reduce background current and decrease drastically the K+ depletion process, probably as a consequence of the reduction of iK1. The background current-voltage relationship becomes linear. 2. 20 mM Cs+ ions completely abolish the current induced by acetylcholine. 3. The possibility that the K+ current induced by acetylcholine is due to an increase of iK1 is discussed.