Activated Rho/Rho kinase and modified calcium sensitivity in cryopreserved human saphenous veins.
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.
Müller-Schweinitzer
Else
E
Department of Cardiac Surgery, University Hospital, CH-4031 Basel, Switzerland. dremueller36@bluewin.ch
Reineke
David C
DC
Glusa
Erika
E
Ebeigbe
Anthony B
AB
Grapow
Martin T R
MT
Carrel
Thierry P
TP
D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D015925:Cryopreservation; D003451:Cryoprotective Agents / Q000378:metabolism / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000378:metabolism / Q000494:pharmacology; D005260:Female; D006801:Humans; D008297:Male; D018613:Microscopy, Confocal; D020110:Pinacidil / Q000494:pharmacology; D012501:Saphenous Vein / Q000201:enzymology / Q000378:metabolism; D015398:Signal Transduction; D019311:Staurosporine / Q000494:pharmacology; D054460:rho-Associated Kinases / Q000378:metabolism*
D003451:Cryoprotective Agents; D020110:Pinacidil; D054460:rho-Associated Kinases; D019311:Staurosporine; D002118:Calcium; D004121:Dimethyl Sulfoxide