alexa Activated Rho Rho kinase and modified calcium sensitivity in cryopreserved human saphenous veins.

Author(s): MllerSchweinitzer E, Reineke DC, Glusa E, Ebeigbe AB, Grapow MT,

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Abstract BACKGROUND: We have shown previously that cryopreservation of human internal mammary arteries activates protein kinase C and enhances intracellular Ca(2+) [Ca(2+)](i). We now present evidence that in human saphenous veins (HSV) cryoinjury is associated with activation of the Rho/Rho kinase signaling pathways and enhanced [Ca(2+)](i). METHODS: HSV were investigated in vitro either unfrozen within 12h after removal or after storage at -196 degrees C in a cryomedium containing 1.8M dimethyl sulfoxide and 0.1M sucrose as cryoprotectant additives. RESULTS: Cryostorage diminished responses to receptor-mediated contractile agonists such as noradrenaline, 5-HT and endothelin-1 by up to 30\% whereas responses to KCl were attenuated by about 50\%. Concentration-response curves for CaCl(2) on unfrozen and cryopreserved HSV revealed similar inhibitory activities of both blocking 1,4-dihydropyridine derivatives nifedipine and the (-)-(R) enantiomer of SDZ 202-791 whereas the Ca(2+) channel activating (+)-(S) enantiomer of SDZ 202-791 was 10 times less effective at enhancing contractions to CaCl(2) when tested after cryostorage. These functional effects were reflected by changes in [Ca(2+)](i) as demonstrated by fluorescence of Fluo-3AM loaded veins. The diminished activity of (+)-(S) SDZ 202-791 in cryopreserved HSV was reversed partially when the potassium channel opener pinacidil (1 microM) was present during the freezing/thawing process. Blockade of Rho kinase by HA-1077 proved to be significantly more effective at attenuating contractile responses to both endothelin-1 and KCl after cryostorage. CONCLUSIONS: Data suggested that cryopreservation modified [Ca(2+)](i) of venous smooth muscle cells (1) through depolarization-induced changes in Ca(2+) influx and (2) through activation of Rho kinase signaling pathways. This article was published in Cryobiology and referenced in

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