Molecular response of a sub-antarctic population of the blue mussel (Mytilus edulis platensis) to a moderate thermal stress.

The Kerguelen Islands (49°26'S, 69°50'E) represent a unique environment due to their geographical isolation, which protects them from anthropogenic pollution. The ability of the endemic mussel, part of the Mytilus complex, to cope with moderate heat stress was explored using omic tools. Transcripts involved in six major metabolic functions were selected and the qRT-PCR data indicated mainly changes in aerobic and anaerobic energy metabolism and stress response. Proteomic comparisons revealed a typical stress response pattern with cytoskeleton modifications and elements suggesting increased energy metabolism. Results also suggest conservation of protein homeostasis by the long-lasting presence of HSP while a general decrease in transcription is observed. The overall findings are consistent with an adaptive response to moderate stresses in mussels in good physiological condition, i.e. living in a low-impact site, and with the literature concerning this model species. Therefore, local blue mussels could be advantageously integrated into biomonitoring strategies, especially in the context of Global Change.

A Model for Transfer of P-Glycoproteins in MCF-7 Breast Cancer Cell Line with Multiple Transfer Rules.

In this paper, we consider a direct protein transfer process between cells in co-culture. Assuming that cells continually encounter each other, and from some hypotheses on cell-to-cell rules of transfer, we derive discrete and continuous Boltzmann-like integro-differential equations. The novelty of this model is to take into account multiple transfer rules. This new transfer model is used to fit the experimental data of cell-to-cell protein transfer in breast cancer.

Contribution of changes in the chloride driving force to the fading of I(GABA) in frog melanotrophs.

Chloride redistribution during type A gamma-aminobutyric acid (GABA(A)) currents (I(GABA)) has been investigated in cultured frog pituitary melanotrophs with imposed intracellular chloride concentration ([Cl(-)](i)) in the whole cell configuration or with unaltered [Cl(-)](i) using the gramicidin-perforated patch approach. Prolonged GABA exposures elicited reproducible decaying currents. The decay of I(GABA) was associated with both a transient fall of conductance (g(GABA)) and shift of current reversal potential (E(GABA)). The shift of E(GABA) appeared to be time and driving force dependent. In the gramicidin-perforated patch configuration, repeated GABA exposures induced currents that gradually vanished. The fading of I(GABA) was due to persistent shifts of E(GABA) as a result of g(GABA) recovering from one GABA application to another. In cells alternatively clamped at potentials closely flanking resting potential and submitted to a train of brief GABA pulses, a reversal of I(GABA) was observed after 150 s recording. It is demonstrated that, in intact frog melanotrophs, shifts of E(GABA) combine with genuine receptor desensitization to depress I(GABA). These findings strongly suggest that shifts of E(GABA) may act as a negative feedback, reducing the bioelectrical and secretory responses induced by an intense release of GABA in vivo.

Regulation of GABAA receptor by protein tyrosine kinases in frog pituitary melanotrophs.

The effects of protein tyrosine kinase (PTK) and PTK inhibitors on the GABAA receptor function were studied in cultured frog pituitary melanotrophs by using the patch-clamp technique. Extracellular application of the PTK inhibitors genistein (10-9 to 10-5 M) or lavendustin A (10-12 to 10-7 M) provoked a bell-shaped potentiation of the whole-cell current induced by GABA (3x10-6 M). In contrast, at high concentrations, genistein (10-4 M) and lavendustin A (10-5 M) reversibly reduced the GABA-evoked current. Daidzein and lavendustin B, the inactive analogs of genistein and lavendustin A, respectively, did not modify the current induced by GABA. In the inside-out configuration, bath application of the recombinant PTK pp60c-src (75 U/ml) inhibited the GABA-activated chloride current, and the inhibitory effect of pp60c-src was prevented by genistein (10-7 M). Immunoblotting revealed that genistein, at doses of 10-7 M or 10-4 M, markedly inhibited tyrosine phosphorylation of the beta2/beta3 subunits of the GABAA receptor. Extracellular application of the PKA activator Bt2cAMP (10-3 M), the PKA/PKC inhibitor H7 (10-5 M) and the Cam KII inhibitor W7 (10-5 M) reversibly diminished the whole-cell GABA-induced current. Internal application of H7 and W7 (10-4 M) did not modify the dose-dependent effects of genistein. Internal application of sodium orthovanadate (10-4 M), a protein tyrosine phosphatase inhibitor, decreased the GABA-evoked current and markedly reduced the potentiating effect of genistein. The present study provides the first evidence that, in frog pituitary melanotrophs, the GABAA receptor is phosphorylated at least on its beta2/beta3 subunits by an endogenous PTK. Our data also demonstrate that tyrosine phosphorylation exerts an inhibitory effect on GABAA receptor function.

The sigma-ligand (+)-pentazocine depresses M current and enhances calcium conductances in frog melanotrophs.

Gramicidin-perforated patch clamp experiments and microfluorimetric measurements were performed to study the ionic mechanisms involved in the sigma-receptor-mediated stimulation of frog (Rana ridibunda) pituitary melanotrophs. The sigma-ligand (+)-pentazocine (50 microM) depressed a sustained outward K(+) current. The kinetic properties of this K(+) component, investigated by analyzing tail currents, were reminiscent of those of the M current (I(M)), with an activation threshold close to -60 mV, a -21-mV half-maximal activation potential, and two-component exponential deactivation kinetics at -90 mV. (+)-Pentazocine (20 microM) produced a 12-mV rightward shift of the activation curve and accelerated the deactivation rate of the tail current. It is also demonstrated that (+)-pentazocine (20 microM) reversibly increased both voltage-dependent calcium conductances and internal calcium level. Altogether, these results suggest that the sigma-receptor-induced modulation of I(M) and calcium currents likely underlies the increase of intracellular [Ca(2+)].

Gramicidin-perforated patch revealed depolarizing effect of GABA in cultured frog melanotrophs.

1. In frog pituitary melanotrophs, GABA induces a transient stimulation followed by prolonged inhibition of hormone secretion. This biphasic effect is inconsistent with the elevation of cytosolic calcium and the inhibition of electrical activity also provoked by GABA in single melanotrophs. In the present study, standard patch-clamp configurations and gramicidin-perforated patches were used to investigate the physiological GABAA receptor-mediated response and intracellular chloride concentration ([Cl-]i) in cultured frog melanotrophs. 2. In the gramicidin-perforated patch configuration, 1 microM GABA caused a depolarization associated with an action potential discharge and a slight fall of membrane resistance. In contrast, at a higher concentration (10 microM) GABA elicited a depolarization accompanied by a transient volley of action potentials, followed by a sustained inhibitory plateau and a marked fall of membrane resistance. Isoguvacine mimicked the GABA-evoked responses, indicating a mediation by GABAA receptors. 3. In gramicidin-perforated cells, the depolarizing excitatory effect of 1 microM GABA was converted into a depolarizing inhibitory action when 0.4 microM allopregnanolone was added to the bath solution. 4. After gaining the whole-cell configuration, the amplitude and/or direction of the GABA-evoked current (IGABA) rapidly changed before stabilizing. After stabilization, the reversal potential of IGABA followed the values predicted by the Nernst equation for chloride ions when [Cl-]i was varied. 5. In gramicidin-perforated cells, the steady-state I-V relationships of 10 microM GABA- or isoguvacine-evoked currents yielded reversal potentials of -37.5 +/- 1.6 (n = 17) and -38.6 +/- 2.0 mV (n = 8), respectively. These values were close to those obtained by using a voltage-ramp protocol in the presence of Na+, K+ and Ca2+ channel blockers. The current evoked by 1 microM GABA also reversed at these potentials. 6. We conclude that, in frog pituitary melanotrophs, chloride is the exclusive charge carrier of IGABA. In intact cells, the reversal potential of IGABA is positive to the resting potential because of a relatively high [Cl-]i (26.5 mM). Under these conditions, GABA induces a chloride efflux responsible for a depolarization triggering action potentials. However, GABA at a high concentration or in the presence of the potentiating steroid allopregnanolone exerts a concomitant shunting effect leading to a rapid inhibition of the spontaneous firing.

Multiple modulatory effects of the neuroactive steroid pregnanolone on GABAA receptor in frog pituitary melanotrophs.

1. The effects of the neuroactive steroid pregnanolone (5 beta-pregnan-3 alpha-ol-20-one) on the electrical response to GABA were investigated in cultured frog pituitary melanotrophs using the patch-clamp technique. 2. Low concentrations of pregnanolone (0.01-1 microM) in the extracellular solution enhanced the current evoked by submaximal concentrations of GABAA receptor agonists and prolonged the GABA-induced inhibition of the spontaneous action potentials in a dose-dependent manner. 3. Pregnanolone augmented the opening probability of the single GABA-activated channels but did not modify the conductance levels. 4. Pregnanolone (1 microM) shifted the GABA dose-response curve towards the low GABA concentrations, reducing the EC50 from 4.2 to 1.8 microM. 5. Internal cell dialysis with pregnanolone (1 or 10 microM) did not alter the GABA-evoked current. 6. Pregnanolone accelerated the desensitization of both the current and conductance increases caused by GABA. 7. High concentrations of pregnanolone (30 microM) markedly and reversibly diminished the current evoked by 10 microM GABA. 8. At high concentrations (10-30 microM), pregnanolone induced an outward current which reversed at the chloride equilibrium potential. 9. It is concluded that, in frog pituitary melanotrophs, pregnanolone exerts a dual inverse modulation and a direct activation of the GABAA receptor-channel depending on the concentrations of both GABA and steroid. Pregnanolone acts on an extracellular site on the GABAA receptor inducing conformational changes of the receptor-channel complex, resulting in a desensitized less-conducting state.

Electrophysiological effects of various neuroactive steroids on the GABA(A) receptor in pituitary melanotrope cells.

The action of steroids on the bioelectrical response to gamma-aminobutyric acid (GABA) has never been studied in pituitary cells. In the present study, we have thus investigated the effects of a series of neuroactive steroids on the GABA-activated current in frog melanotrope cells in primary culture, using the patch-clamp technique in the whole-cell configuration. Bath perfusion of 3alpha-isomers of pregnanolone or tetrahydrodeoxycorticosterone (1 microM) significantly enhanced the current evoked by short pulses of GABA (3 microM) and accelerated its desensitization. In contrast, the 3beta-isomers (30 microM) had no effect on the GABA-activated current. Addition to the bath solution of dehydroepiandrosterone or dehydroepiandrosterone sulfate (10 microM) inhibited the GABA-activated current without modifying its kinetics while pregnenolone sulfate (10 microM) both inhibited the GABA-activated current and accelerated its decay rate. The effects of pregnane steroids were not impaired by the central-type benzodiazepine receptor antagonist flumazenil (10 microM). In conclusion, the present study reveals that neuroactive steroids may exert multiple modulatory activities on the GABA(A) receptor borne by melanotrope cells. The effect of steroids on the current evoked by GABA is rapid, reversible, stereospecific and not mediated through the benzodiazepine binding site of the GABA(A) receptor.

Adenosine inhibits L- and N-type calcium channels in pituitary melanotrophs. Evidence for the involvement of a G protein in calcium channel gating.

It has been previously demonstrated that activation of A1 adenosine receptors in frog melanotrophs causes inhibition of spontaneous action potential discharges and alpha-melanocyte-stimulating hormone secretion. In the present study, we have investigated the effect of adenosine on high-voltage-activated (HVA) calcium currents in cultured melanotrophs, using the whole-cell variant of the patch-clamp technique with barium as a charge carrier. Adenosine and the specific A1 adenosine receptor agonist R-PIA (50 microM each) produced a decrease of the amplitude of the barium current, while the selective A2 adenosine receptor agonist CGS 21680 did not affect the current. The inhibitory effect of R-PIA was observed throughout the activation range of the current, with stronger responses at more positive potentials. R-PIA inhibited both the L- and N-type components of the current, the effect on the N-component being two-fold higher than on the L-component. The inhibitory effect of R-PIA was rendered irreversible by addition of GTP gamma S (100 microM) to the intracellular solution. Pre-treatment of the cells with pertussis toxin (1 microgram/ml; 12 h) totally abolished the effect of R-PIA on the HVA calcium channels. Conversely, addition of a high concentration of cAMP (100 microM) together with the phosphodiesterase inhibitor IBMX (100 microM) to the intracellular solution did not modify the effect of R-PIA on the current. It is concluded that, in frog melanotrophs, adenosine induces inhibition of L- and N-calcium currents and that this effect is mediated by a pertussis toxin-sensitive G protein. Our data also indicate that the inhibitory effect of adenosine on the calcium currents is not mediated by inhibition of adenylyl cyclase.