The PI3K/Akt pathway is involved in endothelial NO production [6,7,22]. Similar to previous studies, the current study found that the PI3K inhibitor wortmannin (10-7 M) abolished mild hypothermia-induced NO-mediated inhibition of phenylephrine-induced maximal contraction (Fig. 2A) [6,7,23]. Furthermore, wortmannin caused the concentration-response curve for phenylephrine-induced contraction to shift to the left at 38°C (Fig. 2A), whereas it greatly increased phenylephrine-induced maximal contraction at 33°C (Fig. 3A). This wortmannin-induced increase in the enhancement of phenylephrine-induced maximal contraction at 33°C seems to be associated with the inhibition of PI3K-induced NO production. However, wortmannin had no effect on L-NAME (10-4 M)-induced enhancement of phenylephrine (10-5 M)-induced maximal contraction at 33°C (Fig. 3B). In agreement with the results from the tension study, wortmannin inhibited the enhancement of eNOS phosphorylation induced by combined hypothermia and phenylephrine treatment or phenylephrine alone (Fig. 6). Taken together, these results suggest that hypothermia-induced PI3K activity contributes to enhanced NO production, which leads to decreased phenylephrine-induced contraction of endothelium-intact aortae. However, pretreatment with L-NAME followed by treatment with wortmannin in HUVECs attenuated the hypothermia-induced enhancement of phenylephrine-induced eNOS phosphorylation compared with L-NAME alone (Fig. 7A). This inconsistency between the tension study and the Western blot analysis may be due to differences in species (human versus rat) and vessels (aorta versus umbilical artery). Rho-kinase in the vascular smooth muscle induces vasoconstriction by inhibiting MLCP, which leads to increased phosphorylation of the 20-kDa regulatory light chain of myosin [24]. Additionally, endothelial Rho-kinase activation decreases endothelial NO release via inhibition of PI3K/Akt [7,11]. Thus, hypothermia-induced Rho-kinase activation produces vasoconstriction via both inhibition of MLCP and attenuation of NO release [7,11,13]. The Rho-kinase inhibitor Y-27632 potently decreased phenylephrine-induced maximal contraction under mild hypothermia compared with 38°C (Fig. 4A). Cotreatment with Y-27932 and wortmannin has been reported to partially reverse Y-27632-induced inhibition of phenylephrine-induced contraction in endothelium-intact rat aortae but not in endothelium-denuded rat aortae or endothelium-intact aortae pretreated with L-NAME [6]. This suggests that Rho-kinase inhibitor-mediated inhibition of phenylephrine-induced contraction is associated with uninhibited PI3K-induced endothelial NO production [6]. Thus, considering previous reports, the increased Y-27632-induced inhibition of phenylephrine-induced maximal contraction observed at 33°C (Fig. 4A) may be due mainly to increase NO production via increased activation of PI3K [6]. As in a previous report, subsequent treatment with wortmannin abolished the enhancing effect of Y-27632 on the inhibition of maximal phenylephrine-induced contraction observed at 33°C (Fig. 4A) [6]. Taken together, these results suggest that, as hypothermia enhanced endothelial Rho-kinase membrane translocation induced by phenylephrine (Fig. 8A), mild hypothermia (33°C)-induced endothelial Rho-kinase membrane translocation contributes to enhanced contraction via PI3K inhibition-mediated decreased NO production [6,7,11,13]. This response may be associated with a compensatory mechanism to counterbalance hypothermia-induced endothelial NO production. In endothelium-intact aortae pretreated with Y-27632 and wortmannin, the subsequent addition of L-NAME enhanced phenylephrine-induced contraction under conditions of mild hypothermia compared with 38°C. This result suggests that other NO production pathways that are not mediated by PI3K may contribute to the decrease in phenylephrine-induced maximal contraction in mild hypothermia. L-NAME enhanced phenylephrine-induced maximal contraction in mild hypothermia compared with 38°C, but after addition of the Rho-kinase inhibitor Y-27632, contraction decreased to similar levels at both 33°C and 38°C (Fig. 4B). This lack of significant difference in phenylephrine-induced maximal contraction between 38 and 33°C with the addition of Y-27632 following pretreatment with L-NAME (Fig. 4B) may be due to increased inhibition of mild hypothermia-induced, Rho-kinase-mediated enhancement of vascular smooth muscle cell contraction caused by the relative activation of MLCP in the vascular smooth muscle [13,24]. Consistent with the tension study, Y-27632 attenuated the hypothermia-induced enhancement of endothelial Rho-kinase membrane translocation induced by phenylephrine (Fig. 8A). However, pretreatment with L-NAME caused greater inhibition of hypothermia- and phenylephrine-induced enhancement of endothelial Rho-kinase membrane translocation compared with pretreatment with Y-27632 (Fig. 8A). Reciprocally, Y-27632-mediated inhibition of phenylephrine-induced eNOS phosphorylation was enhanced in mild hypothermia conditions compared with 37 °C (Fig. 7B). These results suggest that the putative underlying mechanism for L-NAME's inhibition of endothelial Rho-kinase membrane translocation increased by phenylephrine and hypothermia (Fig 8A) and Y-27632's inhibition of phenylephrine- and hypothermia-induced eNOS phosphorylation (Fig. 7B) is as follows [25]. Given that mild hypothermia (33 °C) enhanced phenylephrine-induced eNOS phosphorylation and ROCK-2 membrane transloncation (Fig 6 and 8A) and that the ability of mild hypothermia to attenuate phenylephrine-induced contraction involves PI3K-mediated endothelial NO release (Fig. 3A and B) and Rho-kinase activation (Fig. 4A), enhanced NO release in hypothermia may activate endothelial Rho-kinase to counterbalance excessive NO-mediated attenuation of phenylephrine-induced contraction (Fig. 9) [7,13]. Thus, pretreatment with L-NAME at 33°C may contribute to decreased Rho-kinase membrane translocation through the inhibition of NO production induced by mild hypothermia. On the other hand, activation of the NO-cGMP pathway inhibits the RhoA/ROCK pathway [25]. The relationship between the Rho-kinase pathway and the NO-cGMP pathway in the mildly hypothermic endothelium remains to be characterized in detail.. PG produced tuberous well developed root system (Figure 1e).

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