Does the Cellular Redox State Play a Role in the Regulation of cADPR Accumulation in Pulmonary Artery Smooth Muscle

We were drawn to investigate the possibility that cADPR may be a mediator of HPV for two reasons. As mentioned previously, cADPR had been proposed as an endogenous regulator of RyRs (29, 55), and we had obtained evidence of a role for smooth muscle SR Ca2+ release via RyRs in maintained HPV (23). An additional attraction, however, was the fact that cADPR is a P-NAD+ metabolite. This was because hypoxia had been shown to increase p-NADH levels in all 02-sensing cells studied to date (5, 11, 79, 92). When taken together, these findings suggested that cADPR synthesis itself may, in some way, be sensitive to changes in the metabolic state of pulmonary artery smooth muscle, and that it may thereby play a role in HPV.

As discussed previously, a fall in Po2 likely inhibits oxidative phosphorylation due to the high affinity of the terminal cytochrome oxidase for 02. Consequent reduction in the rate of p-NADH oxidation by mitochondria and acceleration of anaerobic glycolysis, likely results in increased accumulation of in the cytoplasm and mitochondria. Consistent with this view, chemiluminescence studies (5) and direct measurement (79) have demonstrated that hypoxia reduces the cellular redox state in pulmonary artery smooth muscle, in line with classical theory (15). It was therefore suggested that a general reduction in the cellular redox state may mediate signaling by hypoxia in pulmonary arteries, and a variety of redox couples were considered to play a role, namely GSH, p-NADPH, and P-NADH, respectively (86).

Subsequent investigations have, however, brought into question the idea that inhibition of P-NADPH oxidase by hypoxia, and subsequent P-NADPH accumulation represent the primary 02-sensing pathway. In fact, Marshall et al. (58) demonstrated that the activity ofNADPH oxidase may actually be increased by hypoxia in pulmonary artery smooth muscle. In addition, in knock-out mice lacking the neutrophil NADPH oxidase, HPV remained unaffected (7). Moreover, direct extraction and measurement of P-NADPH and P-NADP+, respectively, in pulmonary artery smooth muscle has revealed that the P-ratio is high under normoxic conditions and remains fairly constant in the presence of hypoxia (79). Given the above, it may not be surprising to discover that extremely high concentrations (10 mM) of P-NADPH are without effect on cADPR synthesis from a fixed concentration of P-NAD+ in pulmonary artery smooth muscle homogenates (unpublished observation).

In contrast, paired measurement of P-NAD+ and p-NADH levels, respectively, in extracts from pulmonary arteries yielded quite different findings. P-NAD+ levels were found to be high (> 1 mM) under normoxic conditions whilst P-NADH levels were very low (<80 jiM). Most significantly, however, moderate hypoxia (-30 Torr) increased p-NADH levels at least 5 fold (79). This is consistent, therefore, with the proposal that hypoxia, by inhibiting P-NADH oxidation and oxidative phosphorylation by mitochondria, and accelerating anaerobic glycolysis, leads to an increase in cytoplasmic P-NADH concentration. The increase in cytoplasmic P-NADH is likely allied to saturation of lactate dehydrogenase and/or limitations with respect to the removal oflactate from the cell, as mentioned previously. Consistent with this view, Leach et al. (52) measured an increase inNAD(P)H autofluorescence by hypoxia before and after block of P-NADH oxidation by the mitochondrial electron transport chain.

Initially, and perhaps naively, we considered the possibility that P-NADH maybe abetter substrate forcADPR synthesis than P-NAD+. This proposal could not have been further from the truth, in that we found P-NADH to be a poor substrate for cADPR synthesis. In fact, when applied to pulmonary artery smooth muscle homogenates, 25mM P-NADH produced a total cADPR yield that amounted to no more than 30% of that derived from 2.5 mM p-NAD+ (Fig. 3A) (90). We therefore considered the possibility that the P-NADH : p-NAD+ ratio may be a significant factor, rather than the absolute level of either component. Thus, we investigated the concentration-dependent effects of p-NADH on cADPR production from a fixed concentration of p-NAD+. Surprisingly, P-NADH induced a concentration-dependent and synergistic increase in cADPR production from P-NAD+ (Fig. 3B) (90). Moreover, the range over which a change in the ratio augmented cADPR

accumulation was equivalent to the ratio change predicted from direct measurement of P-NAD+ and P-NADH in extracts from pulmonary arteries during normoxia and moderate hypoxia (79,90). In short, p-NADH may shift the curve for cADPR production from P-NAD+to the left and raise maximal cADPR accumulation (Fig. 3B, inset).

Figure 3. Regulation of cADPR accumulation by ß-NADH. A: Relative cADPR accumulation from 2.5 mM ß-NAD+ and 25 mM ß-NADH, respectively, in pulmonary artery smooth muscle homogenates. B: cADPR accumulation from 2.5 mM ß-NAD+versus ß-NADH concentration in pulmonary artery smooth muscle homogenates. Inset: predicted effect of increased ß-NADH concentration on cADPR accumulation versus ß-NAD+concentration. C: cADPR metabolism in pulmonary artery smooth muscle homogenates versus ß-NADH concentration.

Figure 3. Regulation of cADPR accumulation by ß-NADH. A: Relative cADPR accumulation from 2.5 mM ß-NAD+ and 25 mM ß-NADH, respectively, in pulmonary artery smooth muscle homogenates. B: cADPR accumulation from 2.5 mM ß-NAD+versus ß-NADH concentration in pulmonary artery smooth muscle homogenates. Inset: predicted effect of increased ß-NADH concentration on cADPR accumulation versus ß-NAD+concentration. C: cADPR metabolism in pulmonary artery smooth muscle homogenates versus ß-NADH concentration.

Considering our findings with respect to the facilitation ofcADPR synthesis by P-NADH in a little more detail, however, we can gather further significant information. Firstly, we demonstrated that P-NADH inhibited cADPR metabolism in pulmonary artery smooth muscle homogenates in a concentration-dependent manner, equivalent in range to that by which p-NADH augmented cADPR synthesis. However, the reduction in cADPR metabolism was not sufficient to account for the total increase in cADPR synthesis by P-NADH (Fig. 3C). This is clear from the fact that 2mM P-NADH increased cADPR synthesis from2.5mM P-NAD+by ~ 12 nmoles mg protein' hr'1, but only reduced cADPR metabolism by ~ 3 nmoles mg protein"1 hr'1 (90). Most significantly, however, our findings show that the maximal increase in cADPR accumulation by p-NADH from a fixed concentration of P-NAD+ was approximately 2 fold. This is despite the fact that the level of substrate available was sufficient to promote almost maximal cADPR synthesis from P-NAD+ in the absence of P-NADH. In contrast, the maximal increase in smooth muscle cADPR content by hypoxia was 10 fold, in equal quantities of pulmonary artery smooth muscle. Clearly, the latter measurements were taken under conditions where the increase in cellular p-NADH would lead to a consequent reduction in substrate (P-NAD+) availability for cADPR synthesis, whilst the in vitro measurements did not allow for a fall in P-NAD+ levels as p-NADH was increased. In short, it seems unlikely that P-NADH acts as the primary mediator of cADPR accumulation by hypoxia.

13.1 A Po2Window for cADPR Accumulation by Hypoxia

Further consideration of our developing model for regulation of cADPR synthesis by hypoxia in pulmonary artery smooth muscle, offered yet more insights into the process ofHPV. As mentioned above, arise in P-NADH levels will result in a consequent fall in P-NAD+ availability, the substrate for cADPR synthesis. This would be exacerbated if re-oxidation of P-NADH to P-NAD+ is tightly coupled to the maintenance of anaerobic glycolysis. Our model would predict, therefore, that severe hypoxia may trigger a fall in cADPR levels due to limited substrate availability. In short, as p-NADH rises, cADPR levels would begin to fall at a point determined by the Po2, P-NAD+ availability, and the kinetics of the enzymes for cADPR synthesis and metabolism, respectively. We would therefore expect to observe a Po2 window within which hypoxia promotes cADPR accumulation in pulmonary artery smooth muscle.

To examine this hypothesis, we measured the levels of cADPR in pulmonary artery smooth muscle from third order branches of the pulmonary arterial tree under normoxic, hypoxic and near anoxic conditions. As mentioned previously, we observed a 10-fold increase in cADPR levels by hypoxia (16-21 Torr) when compared to levels measured under normoxia (155-160 Torr) (Fig. 4A). Consistent with the proposal that there may be a P02 window for cADPR accumulation by hypoxia, however, we measured a fall in cADPR levels under near anoxic conditions (5-8 Torr), to a level not significantly different from that observed in the presence of normoxia (Fig. 4A) (24). Furthermore, the Po2 window for cADPR accumulation by hypoxia, was associated with a concomitant rise in ß-NADH levels that was inversely related to the Po2 of the bath solution between normoxia (150-160 Torr) and near anoxia (5-8 Torr) (Fig. 4B) (Evans, unpublished data). We can conclude, therefore, that cADPR accumulation by hypoxia may be limited by ß-NAD+ availability. Furthermore, we have shown that the fall in cADPR levels by anoxia was associated with a failure of HPV in intact arteries (see Chapter 6). This further strengthens our proposal that cADPR-dependent SR Ca2+ release in pulmonary artery smooth muscle is a primary mediator of maintained HPV.

Figure 4. Po2 window for cADPR accumulation. Relative cADPR (A) and 0-NADH (B) levels in pulmonary artery smooth muscle during normoxia (20% 02; 155-160 Torr), hypoxia (2% 02; 16-21 Torr) and anoxia (0% 02; 5-8 Torr), respectively.

In summary, increased p-NADH may effect a rise in cADPR synthesis and concomitant reduction in cADPR metabolism within a P02 window determined by substrate (P-NAD+) availability. However, our findings also suggest that another mechanism may be required to mediate the extent of cADPR accumulation by hypoxia observed in pulmonary artery smooth muscle, and that this mechanism and not P-NADH may perhaps represent the primary effector in 02-sensing cells.

Tips and Tricks For Boosting Your Metabolism

Tips and Tricks For Boosting Your Metabolism

So maybe instead of being a pencil-neck dweeb, youre a bit of a fatty. Well, thats no problem either. Because this bonus will show you exactly how to burn that fat off AS you put on muscle. By boosting your metabolism and working out the way you normally do, you will get rid of all that chub and gain the hard, rippled muscles youve been dreaming of.

Get My Free Ebook


Post a comment