The major findings of the present study include (i) NTG-induced increase of PKCα activity is critically involved in the induction of NTG tolerance in rat aortas and (ii) both in vivo and in vitro treatment with berberine reverses NTG tolerance through inhibiting the PKCα activity in VSMCs.
Nitrates are the NO donors widely used to treat patients with angina pectoris, congestive heart failure, and myocardial infarction. However, chronic nitrate administration results in nitrate tolerance in patients while the mechanisms underlying this deleterious effect are not fully understood [27]. Although it has been always challenging to present a uniform hypothesis for the development of NTG tolerance, the most causative studies on NTG tolerance focused on the role of endothelial cells and endothelial function [28], while less was directed to the role of VSMCs despite the fact that NTG and all other NO donors are the endothelium-independent vasodilators. The NTG-induced overproduction of superoxide anions was used to explain eNOS uncoupling and endothelial dysfunction in nitrate tolerance [8, 7], while other studies argued that superoxide anion is probably not the primary mediator of nitrate tolerance because neither tiron nor dimethylsulfoxide, the intracellular ROS scavengers, affects nitrate tolerance [29, 30]. Furthermore, NTG tolerance still developed in NADPH oxidase subunit knockout mice receiving chronic administration of NTG [31]. In addition, some reports indicate that eNOS may not be critically involved in nitrate tolerance as eNOS deficient mice also developed NTG tolerance [32]. Taken together, these studies suggest that other more important mechanisms are yet to be revealed during the development of NTG tolerance in blood vessels. In the present study, acetylcholine-induced endothelium-dependent relaxations were not affected in aortas from rats with 3-day NTG-patch treatment, suggesting that NTG tolerance was more likely to occur in VSMCs rather than in endothelial cells. Therefore, in this study we chose VSMCs to investigate the mechanisms underlying NTG tolerance.
PKC signaling is known to play important roles in regulating vascular tone through phosphorylating the actin filament-associated proteins, calponin and caldesmon in VSMCs [13]. Previous studies suggested that PKC signal might be implicated in NTG tolerance [11, 12]. However, these studies did not reveal the isoform(s) of PKC involved in NTG tolerance. The present study demonstrates that the activity of PKCα was induced by NTG treatment as evidenced by the elevated levels of both phosphorylation and membrane bound PKCα in NTG-treated VSMCs, which can be reversed by PKC inhibitor GF109203X or by selective PKCα inhibitor Go6976. More importantly, we found that genetic knockdown or the pharmacological inhibition of PKCα inhibited or even reversed NTG tolerance in rat aortas both in vivo and in vitro, thus supporting a critical role of PKCα activation during the development of NTG tolerance in rat arteries and suggesting that PKCα is likely a molecular target for intervention to inhibit NTG tolerance.
Berberine, an isoquinoline alkaloid possesses a wide spectrum of pharmacological properties, including anti-inflammation, anti-oxidative stress and anti-apoptosis [14, 33]. In addition, berberine also exerts cardiovascular protective action to inhibit atherosclerosis [18], cardiac hypotrophy [19], and aortic stiffness [21]. The present demonstrates for the first time that both in vitro and in vivo treatment with berberine is effective to reverse the development of NTG tolerance in rat aortas through normalizing the increased membrane bound PKCα and PKCα phosphorylation in NTG-treated VSMCs or in the aortas from NTG patch-treated rats. These new results clearly indicate that PKCα inhibition is most likely to mediate the beneficial effect of berberine to prevent the development of NTG tolerance. Consistent with our results, other studies also showed a suppressive effect of berberine on PKC or PKCα signaling in non-vascular tissues such as lumbar spinal cords and breast cancer cells [34, 17], although one study described an stimulatory effect of berberine on PKC in heptacytes [35], indicating the effect of berberine on the PKC activity may be tissue- or disease-dependent.
Due to the multiplicity of NTG tolerance-inducing mechanisms, a growing list of compounds appear to be effective in preventing the development of NTG tolerance and they include vitamin C [36], vitamin E [37], thiols [38]), hydralazine [39], L-arginine [40], adrenergic receptor blockers[41], phosphodiesterase inhibitor[42], angiotensin-converting enzyme inhibitor [43], and diuretics [44]. However, most of these drugs were only tested in basic experiments or were administered to healthy volunteers and they are yet to be tested clinically in patients who develop NTG tolerance. In addition, NTG and other drugs such as hydralazine, adrenergic blockers, angiotensin-converting enzyme inhibitors or diuretics share the similar effect to dilate blood vessels and lower blood pressure through different mechanisms of actions. These drugs are commonly use as monotherapy or in combination to decrease cardiac afterload and/or preload and thus to improve cardiac performance, and they have rarely been used to ameliorate NTG tolerance. Our new findings strongly suggest that berberine is a promising drug to inhibit NTG tolerance acting through mechanisms that are distinct from other agents reported in literature and that berberine could be potentially developed into an adjunct agent to prevent NTG tolerance for patients receiving nitrates therapy. Nevertheless, the therapeutic value of berberine needs to be confirmed in future clinical studies.
Although we provide new evidence demonstrating that berberine inhibits NGT tolerance through suppressing PKC activity in VSMCs and rat aortas, we are also aware of several limitations in the present study. First, both in vitro and in vivo results in rodents cannot be confirmed by the effect of berberine in patients. Second, although we confirmed that berberine reversed NTG tolerance both in vivo and in vitro through PKCα inhibition, we still cannot discount other PKCα inhibition-independent mechanism, albeit in a lesser degree mediating the effect of berberine against NTG tolerance. Third, we only used male SD rats in the present study and do not know whether there is any gender difference in the pharmacological effect of berberine in inhibiting NTG tolerance.
In summary, the present study provides new mechanistic insights into the important role of PKCα activation in the development of NTG tolerance. Berberine is potent and effective to prevent the development of NTG tolerance through inhibiting the PKCα activity in VSMCs. The present study not only deepens the understanding of VSMC-associated mechanisms underlying NTG tolerance by revealing the critical role of PKCα, but also highlights the therapeutic potential of berberine as an adjuvant agent to enhance clinical efficacy of NO donors in the treatment of coronary heart disease.