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Results: 5

1.
Fig. 4.

Fig. 4. From: ?1-Adrenoceptor stimulation potentiates L-type Ca2+ current through Ca2+/calmodulin-dependent PK II (CaMKII) activation in rat ventricular myocytes.

Localization of activated CaMKII in response to α1ARS. Immunofluorescence images of ventricular myocytes labeled with active CaMKII antibody (red; A and B) and the plasma membrane marker WGA-FITC (green; C and D) before and after α1ARS. (E and F) Overlay images demonstrate that active CaMKII localized near the T-tubules and the sarcolemma after α1ARS (F). However, immunoreactivity of active CaMKII was found only at the plasmalemma before α1ARS (E). (Scale bars in F, 10 μm.)

Jin O-Uchi, et al. Proc Natl Acad Sci U S A. 2005 June 28;102(26):9400-9405.
2.
Fig. 2.

Fig. 2. From: ?1-Adrenoceptor stimulation potentiates L-type Ca2+ current through Ca2+/calmodulin-dependent PK II (CaMKII) activation in rat ventricular myocytes.

Effect of CaMKII inhibition and PKC inhibition on ICa,L in the presence of phenylephrine. (A) Time-dependent changes of ICa,L after the application of 10 μM phenylephrine in the presence of 0.5 μM KN-93 (▵, n = 12) or 0.5 μM KN-92 (▴, n = 7). After establishing a new steady state for ICa,L by 10-min application of KN-93 or KN-92, the effect of 10 μM phenylephrine on ICa,L was observed in the continuous presence of KN-93 or KN-92. The amplitude of the current at each period was normalized to the current before the application of phenylephrine. (Bars indicate SD.) *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with the normalized current in the presence of KN-92 at each time. Phe, phenylephrine. (B) Time-dependent changes of ICa,L after the application of 10 μM phenylephrine in the presence of 10 μM chelerythrine (○, n = 9) and in the absence of chelerythrine (▵, n = 12). The amplitude of current at each period was normalized to the current before the application of phenylephrine. (Bars indicate SD.) *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with the current after the application of 10 μM phenylephrine in the absence of chelerythrine at each time.

Jin O-Uchi, et al. Proc Natl Acad Sci U S A. 2005 June 28;102(26):9400-9405.
3.
Fig. 3.

Fig. 3. From: ?1-Adrenoceptor stimulation potentiates L-type Ca2+ current through Ca2+/calmodulin-dependent PK II (CaMKII) activation in rat ventricular myocytes.

Activation of CaMKII in response to α1ARS. (A)(Top and Middle) Western immunoblot analyses showing the activation levels of CaMKII in response to various concentration of phenylephrine (n = 8). Although the level of total CaMKII protein was not changed, the level of active CaMKII significantly increased at concentrations of phenylephrine ≥1 μM. (Bottom) Bar graphs show the intensity of the active CaMKII band, normalized to the control, indicating the change of CaMKII activation level. (BD)(Top and Middle)Western immunoblot analyses showing the activation level of CaMKII during α1ARS in the presence of prazosin, KN-93, and chelerythrine, respectively. (Bottom) Increase of the level of active CaMKII during α1ARS was completely blocked by prazosin (n = 6), KN-93 (n = 9), and chelerythrine (n = 7), showing the percentage of increase in CaMKII activation. (Bars indicate SD.) *, P < 0.05, compared with the control. N.S., no significant difference between the two experimental results; CTL, control; Phe, phenylephrine; Pra, prazosin; Che, chelerythrine.

Jin O-Uchi, et al. Proc Natl Acad Sci U S A. 2005 June 28;102(26):9400-9405.
4.
Fig. 5.

Fig. 5. From: ?1-Adrenoceptor stimulation potentiates L-type Ca2+ current through Ca2+/calmodulin-dependent PK II (CaMKII) activation in rat ventricular myocytes.

Activated CaMKII is preferentially localized in T-tubules after α1ARS. (AD) Immunoelectron microscopy images of myocytes labeled with 15 nm of gold-active CaMKII before and after α1ARS. After α1ARS, a higher intensity of gold labeling was observed directly under T-tubule membranes (A and B), and a lower intensity of gold labeling was observed just beneath the non-T-tubular surface sarcolemmal membrane (C). (D) No gold labeling was observed directly under T-tubules membrane before α1ARS. CM, cell membrane; MT, mitochondrion; Z, Z line. (Scale bar, 500 nm.) (E) Normalized gold particles density in T-tubules (n = 108 areas counted before α1ARS and n = 125 areas counted after α1ARS) and sarcolemma (n = 30 areas counted before α1ARS and n = 53 areas counted after α1ARS) relative to the surface areas on the myofilaments (n = 110 areas counted before α1ARS and n = 183 areas counted after α1ARS). The calculation method for normalized gold particles density is described in Materials and Methods. (Bars in E indicate SD.) ***, P < 0.001.

Jin O-Uchi, et al. Proc Natl Acad Sci U S A. 2005 June 28;102(26):9400-9405.
5.
Fig. 1.

Fig. 1. From: ?1-Adrenoceptor stimulation potentiates L-type Ca2+ current through Ca2+/calmodulin-dependent PK II (CaMKII) activation in rat ventricular myocytes.

Effect of 10 μM phenylephrine on ICa,L using perforated patch. (A) Representative time course of ICa,L amplitude changes during application of 10 μM phenylephrine. ICa,L was elicited by a 200-msec depolarizing pulse from a holding potential of -40 to 0 mV (cell capacitance, 76.1 pF) every 10 sec. (Bar indicates the period of the application of phenylephrine.) (Inset) Superimposed original current traces at the points indicated along the current traces. Phe, phenylephrine. (B) Mean current–voltage relationships (n = 10) of ICa,L before (○) and 15 min after application of 10 μM phenylephrine (▪). (Bars indicate SD.) *, P < 0.05; **, P < 0.01, compared with the current before phenylephrine at each voltage. (C) Time-dependent changes of ICa,L after the application of 10 μM phenylephrine (▵, n = 12) and in the absence of phenylephrine (▵, n = 10). The amplitude of current at each time was normalized to the current before the application of phenylephrine. (Bars indicate SD.) *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with the current before phenylephrine. (D) Time-dependent changes in ICa,L after the application of 10 μM phenylephrine in cells preincubated with 20 μM BAPTA–acetoxymethyl ester (♦, n = 10). The amplitude of current at each time was normalized by the current before the application of phenylephrine. (Bars indicate SD.)

Jin O-Uchi, et al. Proc Natl Acad Sci U S A. 2005 June 28;102(26):9400-9405.

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