In:
Journal of Vascular Research, S. Karger AG, Vol. 35, No. 5 ( 1998), p. 303-309
Abstract:
In the present study, we investigated the actions of calmodulin (CaM) and CaM-dependent protein kinase II (CaMK-II) on the L-type Ca 〈 sup 〉 2+ 〈 /sup 〉 currents (I 〈 sub 〉 Ca(L) 〈 /sub 〉 ) of cultured vascular smooth muscle (VSM) cells (A7r5 cell line), using the whole-cell voltage clamp method. The peak I 〈 sub 〉 Ba 〈 /sub 〉 (Ca 〈 sup 〉 2+ 〈 /sup 〉 channel using 5 m 〈 i 〉 M 〈 /i 〉 Ba 〈 sup 〉 2+ 〈 /sup 〉 as charge carrier) was evoked every 15 s by a test potential to +10 mV from a holding potential of –60 mV. To test the effect of CaM on I 〈 sub 〉 Ba 〈 /sub 〉 , 1 µ 〈 i 〉 M 〈 /i 〉 calmidazolium (CMZ), an inhibitor of CaM, was added to the pipette solution (pCa of 6.5 or 300 n 〈 i 〉 M 〈 /i 〉 [Ca] 〈 sub 〉 i 〈 /sub 〉 ). The amplitude of maximally activated I 〈 sub 〉 Ba 〈 /sub 〉 was –4.3 ± 0.5 pA/pF (n = 13) for control and –8.1 ± 0.9 pA/pF (n = 14) in the presence of CMZ. This difference was statistically significant (p = 0.016). The CMZ stimulation of I 〈 sub 〉 Ba 〈 /sub 〉 was not abolished when 5 µ 〈 i 〉 M 〈 /i 〉 KN-62, a specific inhibitor of CaMK-II, was included in the pipette (–9.5 ± 1.1 pA/pF; n = 10). Introduction of CaMK-II itself intracellularly had no effect on the basal I 〈 sub 〉 Ba 〈 /sub 〉 . On the other hand, the CMZ stimulation of I 〈 sub 〉 Ba 〈 /sub 〉 was prevented by both H-7, a nonspecific protein kinase inhibitor, and H-89, a specific inhibitor of protein kinase A (PK-A). Since CMZ is a strong inhibitor of Ca 〈 sup 〉 2+ 〈 /sup 〉 /CaM-dependent phosphodiesterase (type I PDE), we studied the effect of 8-methoxymethyl-3-isobutyl-1-methylxanthine (MIBMX), another specific inhibitor of the PDE. MIBMX, like CMZ, stimulated I 〈 sub 〉 Ba 〈 /sub 〉 : control, –4.6 ± 0.4 pA/pF (n = 10); MIBMX, –9.6 ± 1.2 (n = 8), and CMZ, –7.9 ± 0.9 (n = 15). 0.1 m 〈 i 〉 M 〈 /i 〉 8Br-cAMP, a membrane permeable cAMP analogue, stimulated I 〈 sub 〉 Ba 〈 /sub 〉 by +42%: before, –3.7 ± 0.7 pA/pF; after, –5.2 ± 1.0 (n = 6). In conclusion, Ca 〈 sup 〉 2+ 〈 /sup 〉 channels of VSM cells might not be directly regulated by the CaM/CaMK-II pathway. Therefore, the CMZ stimulation of I 〈 sub 〉 Ba 〈 /sub 〉 might occur due to the increase in intracellular concentration of cAMP produced by inhibition of CaM-dependent PDE.
Type of Medium:
Online Resource
ISSN:
1018-1172
,
1423-0135
Language:
English
Publisher:
S. Karger AG
Publication Date:
1998
detail.hit.zdb_id:
1482726-8
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