| Inhibiting the Na/K pump has been believed to be the classical mechanism that glycosides exert their positive inotropic effects to treat the patient with congestive heart failure. However, a number of studies have suggested that the effective therapeutic concentrations of glycosides in plasma of patients with the congestive heart failure are much lower than those to inhibit the Na/K pump in vitro. Even these low concentrations of glycosides could not inhibit, but stimulate the activity of the Na/K pump in heart. Thus, the theory of the Na/K pump inhibition seems to be insufficient to explain the therapeutic mechanism of glycosides for the congestive heart failure at effective concentration. The present study was undertaken to determine the Na/K pump current (Ip) and calcium current (ICa) by the whole-cell patch-clamp technique and to measure the intracellular calcium concentration ([Ca2+]i) by confocal microscopy in isolated guinea pig ventricular myocytes in order to explore the effects of glycosides in the therapeutic concentration on the Na/K pump and their underlying mechanisms for increasing the cardiac contractibility. 1. Low concentrations of glycosides stimulate the Na/K pump current (Ip)(1) When myocytes were perfused with 5×10-6 mol·L-1 dihydroouabain (DHO), due to depression of Ip, an inward current was observed. However, an outward current was recorded when the myocytes were perfused with 10-10 mol·L-1 DHO. (2) When Na-aspartic acid in the pipette solution was replaced byaspartic acid to remove intracellular Na+ from myocytes, DHO 10-10 mol·L-1 and 5×10-6 mol·L-1 induce neither outward nor inward current described above, respectively. (3) When myocytes were superfused with K+-free solution to remove the external K+, DHO 10-10 mol·L-1 did not induce the outward current described above.(4) Vanadate, an inhibitor of the Na,K-ATPase activity, could eliminate the outward current induced by 10-10 mol·L-1 DHO and the inward current induced by 5×10-6 mol·L-1 DHO.It is well-known that 5×10-6 mol·L-1 DHO-induced inward current is an inhibitory Ip, however, some characteristics of the outward current induced by 10-10 mol·L-1 DHO were similar to those of inward current induced by 5×10-6 mol·L-1 DHO, suggesting that this inward current is possessed of outstanding characteristic of Ip. So, it may be thought that 10-10 mol·L-1 DHO-induced outward currents are a stimulatory Ip. (5) The β-adrenergic agonist isoproterenol (ISO), which act selectively on the α1-isoform of the Na/K pump, did not influence the stimulatory Ip induced by 10-10 mol·L-1 DHO (Ip (ISO)/ Ip (Con) = 1.0 ± 0.4).(6) The α-adrenergic agonist norepinephrine (NE), which act selectively on the α2-isoform of the Na/K pump, increased the stimulatory Ip induced by 10-10 mol·L-1 DHO in presence of β-blocker propranolol (PROP) (Ip (NE)/ Ip (Con) = 1.7±0.5).(7) The ratio of the stimulatory Ip induced by 10-10 mol·L-1 DHO in presence of 8 mmol·L-1 and 4 mmol·L-1 [K+]o was 1.1 ± 0.3, showing that the stimulatory Ip are unaffected by the change of external K+, indicating that the stimulatory Ip induced by low [DHO] is associated with the α2-isoform but not the α1-isoform of the Na/K pump.(8) Low concentration of ouabain (OUA, 10-10 mol·L-1) also produced the stimulatory Ip in guinea pig ventricular myocytes.All results above show that low concentrations of glycosides (DHO and OUA, 10-10 mol·L-1) stimulate rather than inhibit Ip, and theirstimulatory effects on Ip are specifically mediated through α2-isoform of the Na/K pump.2. Low concentrations of DHO elevate [Ca2+]i in ventricular myocytesThe effects of low concentrations of DHO on [Ca2+]i was investigated in guinea pig ventricular myocytes. [Ca2+]i was detected by confocal microscopy and represented by fluorescent intensity (FI). The results are as follows: (1) DHO 10-13 ~ 10-9 mol·L-1 elevated [Ca2+]i, especially, 10-11 mol·L-1 DHO could obviously increase [Ca2+]i by 73% ± 12% (P<0.01). (2) After m...
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