Abstract:
:In the present study, we investigated the actions of calmodulin (CaM) and CaM-dependent protein kinase II (CaMK-II) on the L-type Ca2+ currents (ICa(L)) of cultured vascular smooth muscle (VSM) cells (A7r5 cell line), using the whole-cell voltage clamp method. The peak IBa (Ca2+ channel using 5 mM Ba2+ 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 IBa, 1 microM calmidazolium (CMZ), an inhibitor of CaM, was added to the pipette solution (pCa of 6.5 or 300 nM [Ca]i). The amplitude of maximally activated IBa 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 IBa was not abolished when 5 microM 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 IBa. On the other hand, the CMZ stimulation of IBa 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 Ca2+/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 IBa: 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 mM 8Br-cAMP, a membrane permeable cAMP analogue, stimulated IBa by +42%: before, -3.7 +/- 0. 7 pA/pF; after, -5.2 +/- 1.0 (n = 6). In conclusion, Ca2+ channels of VSM cells might not be directly regulated by the CaM/CaMK-II pathway. Therefore, the CMZ stimulation of IBa might occur due to the increase in intracellular concentration of cAMP produced by inhibition of CaM-dependent PDE.
journal_name
J Vasc Resjournal_title
Journal of vascular researchauthors
Sunagawa M,Yokoshiki H,Seki T,Sperelakis Ndoi
10.1159/000025599subject
Has Abstractpub_date
1998-09-01 00:00:00pages
303-9issue
5eissn
1018-1172issn
1423-0135pii
jvr35303journal_volume
35pub_type
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