Beta-adrenergic regulation of voltage-dependent calcium currents in cultured skeletal myocytes of the frog Rana temporaria.

Effects of beta-agonists isoproterenol (Isp) and adrenaline (Adr) and beta-adrenoblocker obsidan (Obs) on the voltage-dependent calcium currents in cultured embryonic skeletal myocytes were studied at various stages of development ranging from day 2 to 10, using the whole-cell patch-clamp technique at 19-21 degrees C. Adr (or Isp) in concentrations 0.1-10 mumol/l increases the amplitude of both the slow dihydropyridine(DHP)-sensitive calcium current (ICa) and the fast-activated DHP-insensitive ICa. From day 2 to 6 after myoblast plating, Adr and Isp did not change the amplitude of ICa at all or slightly increased it. Obvious strong positive effects (an approximately twofold amplitude increase) on the calcium channels have been observed in 7-10-day-old myocytes only. beta-adrenoblocker obsidan known to abolish the positive beta-agonist effect, had a positive effect on membrane calcium currents. It may have been a result of the immaturity of the beta-adrenergic regulatory system of the myocytes. It is concluded that the beta-adrenergic regulatory complex can stimulate the activity of the fast and the slow voltage-dependent calcium channels of the frog skeletal myocytes, and that there is a distinct developmental stage at which a functioning beta-adrenergic regulatory complex appears in the membrane of skeletal myocytes.

Pharmacological analysis of voltage-dependent potassium currents in cultured skeletal myocytes of the frog Rana temporaria.

Previously, the existence of nine types of outward potassium current (IK) was shown. The whole family of IK may be divided into two groups: fast transient currents (f) with time to peak less than 70 ms (at test potential near 0 mV), and slow (s) components (Lukyanenko et al. 1993). The latter were completely blocked by 4-aminopyridine (4-AP) and the former were more sensitive to TEA than slow IK. In the present study we analyzed the effects of calcium blockers on different types of IK using the whole-cell patch-clamp technique. One to seven-day-old myocytes without slow calcium current and without contact with nerve cells were examined. Extracellullar application of 40-80 mumol/l dihydropyridine (DHP) antagonist nifedipine did not change maximal conductance of K-channels, but induced a parallel shift by 5-10 mV of chord conductance curve along the voltage axis in the direction of more negative potentials. Quinidine in concentrations 30-200 mumol/l caused a reversible block of the fast and the slow IK (C0.5 = 75 mumol/l), and enhanced the current decay (2-3-fold at 150 mumol/l). Verapamil (VP) in concentrations 100-700 mumol/l reduced IK with dose-dependent effect (C0.5 = 200 mumol/l) and changed its kinetic properties. VP 100 mumol/l caused a complete irreversible block of the slow IK. VP reduced the time inactivation constant of fast IK with a dose-dependent effect (8-10-fold at 300 mumol/l), and this effect was stronger during depolarizing pulses. The latter points to the possibility that the fast K-channels preferentially bind VP in open state. An analysis of the effects suggests that K-channels of the frog myocytes could be divided into 2 groups: 1) K-channels which irreversibly blocked by VP and 4-AP (slow), and 2) those reversibly inhibited by VP and 4-AP (fast potassium channels).

Self-trapped interstitial-type defects in iron.

Small interstitial-type defects in iron with complex structures and very low mobilities are revealed by molecular dynamics simulations. The stability of these defect clusters formed by nonparallel {110} dumbbells is confirmed by density functional theory calculations, and it is shown to increase with increasing temperature due to large vibrational formation entropies. This new family of defects provides an explanation for the low mobility of clusters needed to account for experimental observations of microstructure evolution under irradiation at variance with the fast migration obtained from previous atomistic simulations for conventional self-interstitial clusters.

Development of muscle-specific features in cultured frog embryonic skeletal myocytes.

To study the development of muscle-specific features during myogenesis, we analysed the ultrastructure and voltage-dependent currents of frog embryonic skeletal myocytes maintained in culture for 10 days. The cells were maintained under culture conditions that prevented cell division, fusion and cell contacts with neuroblasts. The cell surface was estimated morphometrically and from cell capacity and the values obtained were used to calculate ion current densities. It was shown that the expression of all main types of voltage dependent ionic currents occurs during the first 3-5 days. Na+ maximum specific conductance at days 1-2 was low but by day 7 it showed a 20-fold increase. The magnitude of Na+ current densities increased 16-fold from day 1 (3.6 microA/cm) to the day 7 (58.1 microA/cm). The maximum specific K+ conductance increased almost 3-fold during the first 5 days. In contrast to the other types of currents, I(K) undergoes qualitative changes. Sodium action potentials, whose amplitude and time course depend on gNa/gK ratio, appeared from day 4 in culture, when myofibrils and the T-system also developed. The amplitude of DHP-sensitive slow I(Ca) increased in parallel with the development of the T-membrane. I(Ca,S) density per unit of T-membrane area reached an equilibrium of ca., 17 microA/cm2 on the day 4 and then remained stable until the end of the period of observation. These studies demonstrate that muscle-specific characteristics including morphology and excitatory properties begin to develop on the third day and resemble those of adult muscle cells by the sixth day in culture.