FUNCTIONAL-ACTIVITY AND TRANSMEMBRANE SIGNALING MECHANISMS AFTER CRYOPRESERVATION OF HUMAN INTERNAL MAMMARY ARTERIES

Purpose: Cryopreserved human blood vessels are important tools in bypa ss surgery. However, several in vitro studies have demonstrated dimini shed postthaw functional activity. Therefore the aim of this study was to investigate the consequences of various freezing/thawing protocols and the role of protein kinase C in the postthaw functional activity of cryopreserved human arteries. Methods: In vitro responses of frozen /thawed human internal mammary arteries (IMA) were used to investigate the functional activity after thawing at 15 degrees, 30 degrees, and 100 degrees C/min and after different prefreezing equilibration times (10, 60, 120, 240 minutes) with the cryomedium (Krebs-Henseleit soluti on containing 1.8 mol/L dimethyl sulfoxide and 0.1 mol/L sucrose) at r oom temperature followed by cryostorage at -196 degrees C. Results: Pr efreezing equilibration for 10 to 120 minutes diminished maximal a-adr enoceptor-mediated responses to noradrenaline to approximately 60%, an d equilibration for 240 minutes attenuated noradrenaline effects to le ss than 25% of that produced by unfrozen controls. Contractile respons es were slightly better when thawing was performed at 15 degrees C/min compared with 100 degrees C/min. The postthaw sensitivity to direct a ctivation of protein kinase C by phorbol 12,13-dibutyrate was enhanced . Compared with unfrozen tissues (pD(2) = 7.36 +/- 0.07, n = 32) maxim al sensitization to phorbol 12,13-dibutyrate was observed in IMA that had been frozen after 60 minutes of equilibration with the cryomedium (pD(2) = 8.31 +/- 0.09, n = 30). Responses to phorbol 12,13-dibutyrate of cryopreserved IMA were highly susceptible to blockade of calcium i nflux by nifedipine, whereas those of unfrozen IMA were resistant to n ifedipine. Against noradrenaline nifedipine was equipotent in cryopres erved (pD'(2) = 7.75 +/- 0.15, n = 8) and unfrozen IMA (pD'(2) = 7.70 +/- 0.10, n = 6). Endothelium-dependent relaxant responses to acetylch oline were significantly attenuated after cryopreservation (E-max = 26 % +/- 5%, n = 4) compared with unfrozen IMA. (E-max = 71% +/- 4%, n = 4, p < 0.001); endothelium-independent relaxant responses to sodium ni troprusside were unchanged. Conclusions: Cryopreservation of human IMA under the conditions applied in this study (1) attenuated endothelial cell function and (2) induced an activation of protein kinase C, ther eby increasing calcium influx through dihydropyridine-sensitive calciu m channels. These experimental data suggest that postoperative adminis tration of calcium channel blockers alone or combined with long-acting nitrates should effectively prevent the development of spasms in arte rial grafts.