Fig 1.
A) Extraction of the hemolymph from the posterior adductor muscle of Mytilus galloprovincialis. B) In a few seconds the hemocytes attached to the bottom of the Petri dish and assumed a very thin and flat shape. The granulocyte lysosomal compartment is easily identifiable in this magnification view (represented in false colours) of a hemocyte firmly attached to the bottom of the recording chamber in Modified Artificial Sea Water (MASW). Note the faint peripheral area comprised between the granule compartment and the cytoplasmic membrane. C) The panel illustrates the progressive flattening and adhesion of granulocytes adhering to the glass bottom of the recording chamber. The process is typically complete in a few minutes. Note the different diameter of the same cell in the first and last frame.
Fig 2.
The time-dependent currents in Mytilus galloprovincialis hemocytes are modulated by the osmotic pressure.
A) Schematic representation of the osmotic pressure of the bath solutions vs time adopted to investigate the response of hemocytes to different osmotic pressures. The osmolality profile shows that the experiment started in the hyposmotic solution (∏ = 897 mosmol/kg), then, at t = 0 s, the bath was perfused with the hyperosmotic solution (∏ = 1178 mosmol/kg) and finally (at t≈1850 s) the bath was again perfused with the hyposmotic solution. B) The mean values of the steady-state sequential currents, elicited every 10 s (see typical currents in panel C), are plotted as a function of time. It can be observed that the increase of the osmolality causes a drastic decrease of the current. The traces in panel C) represent three typical time-dependent currents elicited by voltage pulses to -100 mV (replicated every 10 s) in hyposmotic (1st and 3rd trace, on the left and the right, respectively) and hyperosmotic bath solutions (middle trace). The arrow emerging from each trace points to the correspondent data point in B). D) The hyperosmotic bath solution determines a decrease of the steady-state current elicited by V = -100 mV (left bar) without altering the capacitance of the cells (right bar). Current bar represents the increase of the mean steady-state current in hyposmotic condition with respect to hyperosmotic solution (Ihypo/Ihyper at the left axis) ± SEM from 7 different experiments, while the cell capacitance (Chypo/Chyper at the right axis) remained almost unaltered, thus indicating that the current increase is not due to an increase of the cell surface (and to a consequent increase of the number of available channels) in hyposmotic conditions.
Fig 3.
Characteristics of the inwardly-rectifying time-dependent currents in mytilus hemocytes.
A) Voltage protocol applied to M. galloprovincialis hemocytes eliciting time-dependent currents. The duration of the main pulse was 5 s and the applied voltages ranged from +50 mV up to –120 mV in –10 mV decrements. B) Typical whole-cell inwardly rectifying time-dependent currents that activate slowly on the application of the hyperpolarizing pulses represented in A and deactivate in a fraction of seconds after the voltage repolarization to positive values. Holding and tail membrane potentials were +40 mV and -50 mV, respectively. Experiments performed in the hyposmotic bath solution with osmolality equal to 897 mosmol/kg. C) Values of the normalized currents (INorm) obtained mediating the final segment of each steady-state current from at least 4 different experiments. The data were normalized with respect to the absolute value of the current at V = -110 mV and plotted as a function of the applied transmembrane potential. D) Filled symbols represent GNorm in hyposmotic solutions plotted as a function of the membrane potential; the solid line represents the best fit of the experimental data by the Boltzmann equation. Interestingly, in the same set of cells, a comparison of the Boltzmann distributions was left-shifted towards more negative membrane potentials by as much as -44.9 ± 4.4 mV when the hyposmotic solution (filled circles) bathing the cell was replaced by an identical hyperosmotic solution (empty symbols at ∏ = 1178 mosmol/kg), thus implying that more negative membrane potentials are needed to activate the same current at higher osmolality. V1/2(hypo) = -37.2 ± 1.3 mV and V1/2(hyper) = -82.1± 3.1 mV, z(hypo) = 1.8± 0.1 and (z(hyper) = 1.7±0.1).
Fig 4.
The time dependent currents are mediated by chloride channels.
A) Tail currents elicited by voltages ranging from -80 mV to + 90 mV in 10 mV steps after a main pulse to -80 mV from a holding potential of +40 mV. In the standard pipette solution and hyposmotic bath solution, NMDG-chloride replaced 530 mM and 50 mM KCl, respectively. Clearly the tail currents inverted at potentials comprised between V = +40 mV and V = +50 mV (indicated by the two lines), i.e. at a value compatible with the Nernst potential for chloride in this working conditions (VNernst(Cl-) = +46 mV). B) Instantaneous values of the tail currents (extrapolated at t = 0 s) are plotted as a function of the tail potentials in the range from +32 mV to +52 mV, incremented by 2 mV steps after the main pulse to -80 mV.
Fig 5.
Micromolar ZnCl2 reduces the amplitude of the time-dependent current.
A) The decrease of a typical inward time-dependent current induced by the addition of 100 μM ZnCl2 to the bath solution. A series of step voltages to -120 mV were applied to the cell with an interval of 15 s in the presence and in the absence of 100 μM ZnCl2; the holding potential was +20 mV, tail voltage was -50 mV. Control and recovery: hyposmotic solution. Each current trace represents the average of at least 3 different traces obtained in the same conditions. B) The Boltzmann distribution is shifted towards more negative membrane potentials (ΔV = -10.3 ± 2.3 mV) on the addition of 30 μM ZnCl2 (empty circles) to the bath solution with respect to the control conditions (filled circles, hyposmotic bath solution). It can be observed that the current decrease can be ascribed to a shift to the left of the Boltzmann distribution. Data were obtained averaging at least 4 different current records in the different conditions. V1/2(hypo) = -37.2 ± 1.3 mV and V1/2(Zn2+ = 30 mM) = -47.5± 1.0 mV, z(hypo) = 1.8 ± 0.1 and z(Zn2+ = 30 mM) = 1.7± 0.1.