1. The Na-Ca exchange current was investigated in single ventricular cells from guinea-pig hearts by combining the techniques of whole-cell voltage clamp and intracellular perfusion. 2. The membrane conductance was minimized by blocking Ca and K channels as well as the Na-K pump. Under these conditions, when Ca2+ was loaded internally by a pipette solution containing 430 nM-Ca2+, changing the Li+-rich external solution to a Na+-rich one induced a significant inward current. Applying external Na+ in the absence of internal Ca2+ did not appreciably change the current. 3. In contrast, perfusing 1 mM-external Ca2+ in the presence of internal Na+ which was loaded by a 20 mM-Na+ pipette solution, induced a marked outward current. Ca2+ superfusion in the absence of internal Na+ caused only a small current change. 4. The current-voltage relation of external-Ca2+- and external-Na+-induced current showed almost exponential voltage dependence as given by the equation i = a exp (rEF/RT), where a is a scaling factor that determines the magnitude of the current and r is a partition parameter used in the rate theory and represents the position of the energy barrier in the electrical field, which indicates the steepness of the voltage dependence of the current. E, F, R and T have their usual meanings. The value of a was 1-2 microA/microF and r about 0.35 for the Ca2+-induced outward current. At very positive or negative potentials, the current magnitude became smaller than expected from an exponential relation. 5. The current was blocked by heavy metal cations, such as La3+, Cd2+, Mn2+ and Ni2+ and partially blocked by amiloride and D600. 6. The temperature coefficient (Q10) value of the Ca2+-induced outward current was 3.6 +/- 0.4 (n = 4) at 0 mV and 4.0 +/- 0.9 at 50 mV in the range between 21 and 36 degrees C. 7. The outward current magnitude showed a sigmoidal dependence upon the external Ca2+ concentration with a half-maximum concentration, K1/2 of 1.38 mM and a Hill coefficient of 0.9 +/- 0.2 (n = 5). 8. Sr2+ could replace Ca2+ with K1/2 of 7 mM. Mg2+ and Ba2+, however, did not replace Ca2+. 9. The inward current component also showed a sigmoidal external Na+ dependence with K1/2 of 87.5 +/- 10.7 mM and a Hill coefficient of 2.9 +/- 0.4 (n = 6). 10. The reversal potential of the current was obtained near the values expected for 3 Na+:1 Ca2+ exchange.(ABSTRACT TRUNCATED AT 400 WORDS)

other currents as a competitive inhibitor with external Ca2+. This effect is in contrast to DCB which preferentially inhibits the inward rather than the outward Na+/Ca2+ exchange current.

Electrogenic Na-Ca exchange has been known to act in the cardiac sarcolemma as a major mechanism for extruding Ca ions. Ionic flux measurements in cardiac vesicles have recently suggested that the exchange ratio is probably 3 Na:1 Ca, although a membrane current generated by such a process has not been isolated. Using the intracellular perfusion technique combined with the whole-cell voltage clamp, we were able to load Na+ inside and Ca2+ outside the single ventricular cells of the guinea pig and have succeeded in recording an outward Na-Ca exchange current while blocking most other membrane currents. The current is voltage-dependent, blocked by La3+ and does not develop in the absence of intracellular free Ca2+. This report presents the first direct measurement of the cardiac Na-Ca exchange current, and should facilitate the study of Ca2+ fluxes during cardiac activity, together with various electrical changes attributable to the Na-Ca exchange and the testing of proposed models.

The multiple skeletal components of the skull originate asynchronously and their developmental schedule varies across amniotes. Here we present the embryonic ossification sequence of 134 species, covering all major groups of mammals and their close relatives. This comprehensive data set allows reconstruction of the heterochronic and modular evolution of the skull and the condition of the last common ancestor of mammals. We show that the mode of ossification (dermal or endochondral) unites bones into integrated evolutionary modules of heterochronic changes and imposes evolutionary constraints on cranial heterochrony. However, some skull-roof bones, such as the supraoccipital, exhibit evolutionary degrees of freedom in these constraints. Ossification timing of the neurocranium was considerably accelerated during the origin of mammals. Furthermore, association between developmental timing of the supraoccipital and brain size was identified among amniotes. We argue that cranial heterochrony in mammals has occurred in concert with encephalization but within a conserved modular organization.

The Na/K pump usually pumps more Na+ out of the cell than K+ in, and so generates an outward component of membrane current which, in the heart, can be an important modulator of the frequency and shape of the cardiac impulse. Because it is electrogenic, Na/K pump activity ought to be sensitive to membrane potential, and it should decline with hyperpolarization. However, such voltage dependence of outward pump current has yet to be demonstrated, one reason being the technical difficulty of accurately measuring pump current over a sufficiently wide voltage range. The whole-cell patch-clamp technique allows effective control of both intracellular and extracellular solutions as well as membrane voltage. Applying this technique to myocardial cells isolated from guinea pig ventricle, we have measured Na/K pump current between -140 mV and +60 mV, after minimizing passive currents flowing through Ca2+, K+ and Na+ channels. We report here that strongly activated pump current shows marked voltage dependence; it declines steadily from a maximal level near 0 mV, becoming very small at -140 mV. Pump current-voltage relationships will provide essential information for testing models of the Na/K pump mechanism and for predicting pump-mediated changes in the electrical activity of excitable cells.