Comprehensive Study of Vascular Mitochondria

Both the production of AOS and the opening of the MTP are directly related to mitochondrial respiration and mitochondrial A^. Therefore, measuring AOS production (using chemiluminescence, AmplexRed and DCF assays), respiration (with classic polarographic methods) and A^ (using À ^-sensitive dyes and dynamic multiphoton confocal imaging, which allows for measurement of A^ in situ in living cells) allows for a functional study of mitochondria. Along with perfused organs, tissue baths and isolated vascular SMC electrophysiology, these methods can be used for a comprehensive study of the role of mitochondria in vascular function.

We used these methods and addressed the hypothesis that mitochondrial diversity exists within the vasculature and specifically between the pulmonary and systemic vascular beds (37). We speculated that such diversity might explain why the pulmonary and systemic circulations respond in opposing ways to hypoxia. In the oxygen sensing model described by Moudgil et al. (see Chapter 9), PASMC mitochondria (the sensors) respond to changes in 02 tension by the production of AOS (the mediator) that tonically regulate the function of PASMC voltage-gated K+ channels (Kv, the effector) (3). Since Kv channels are present in both the pulmonary and renal circulations (19, 21, 36) we speculated that the opposing responses to hypoxia might be due to differential production of AOS

between the PA and systemic vascular SMC in response to hypoxia. Furthermore potential differences in the tonic production of AOS at baseline (normoxia) might even help explain why the baseline pressure of the pulmonary circulation is lower compared to the systemic circulation.

This hypothesis was based on the following previously described observations. First, the mechanism of 02 sensing lies within the vascular SMC and although modulated by endothelial or circulating factors, it occurs independent of them. Madden et al. have shown that pulmonary SMC contract and have increased intracellular Ca2+ levels in response to hypoxia, while cerebral vascular SMC relax and have decreased Ca2+ levels (35). Second, the only class of drugs that can mimic hypoxia in a variety of 02-sensitive tissues (PA, ductus arteriosus, carotid body) is the group of inhibitors of the proximal ETC, i.e., rotenone and antimycin-A which block the AOS-producing complexes I and III, respectively (3). Both hypoxia and these inhibitors cause PASMC contraction, decreased AOS levels, and inhibition of K+ currents (1, 4).

Rotenone mimics hypoxia constricting the pulmonary while dilating the renal circulation. Rotenone (5 ^M) and physiological hypoxia (Po2, -40 mmHg) both constrict pulmonary arteries (PA) whereas they dilate the renal arteries (RA) (Fig. 3 A). As shown in Figure 3A, the lungs and kidneys of the rat are perfused in series with the same perfusate and while the flow is kept constant with pumps. This difference is independent of nitric oxide and prostaglandins since the experiments were performed in the presence of inhibitors of the endothelial nitric oxide synthase, and cyclooxygenase. In contrast, both vascular beds constrict to angiotensin n and 4-aminopyridine (4-AP, 5 mM), a Kv channel blocker.

Further evidence that the opposing response to hypoxia and rotenone is intrinsic to the SMC and independent of the endothelium, comes from tissue bath experiments. In endothelium-denuded vessels, rotenone and hypoxia causes PA constriction and RA dilation, while 4-AP constricts both artery types (37).

6.1. Rotenone Mimics Hypoxic Inhibition of K+ Current in PASMC

Whole-cell patch clamping in freshly isolated SMC from resistance PA and RAs shows that rotenone inhibits outward K+ current (/K) in PA while it activates /K in RASMC (Fig. 3B). The effects of both hypoxia end rotenone are rapid, occurring within 5 minutes. These data are in agreement with earlier work showing that metabolic inhibitors decrease PASMC but not systemic (mesenteric) vascular SMC current (58).

The facts that most of the 02-sensitive channels are present in both the PA and RA, and that 4-AP constricts both vessels (19, 21, 36), suggest that the opposing responses to hypoxia are likely not due to a differential expression of Kv channels. Rather, the opposing responses to hypoxia might be due to differences in the 02 sensor (i.e., the mitochondria) that perhaps results in the differential regulation of the effector of 02 sensing (i.e., AOS).

Figure 3. Divergent effects of rotenone and hypoxia on renal (RAP) versus pulmonary (PAP) arterial pressure (A) and on K+ currents (IK) in PASMC and MASMC (B). A: A representative trace and mean data from the isolated perfused lung-kidney model are shown. Rotenone (5 nM) and hypoxia (Po2, 40 mmHg) increase PAP and decrease RAP in the presence of L-NAME and meclofenamate. 4-AP (5 mM) and angiotensin II (All, 0.1 ng/50 nl bolus) increase both RAP and PAP. B: Hypoxia and rotenone increase whole-cell IK in PASMC but decrease IK in RASMC. In contrast, 4-AP inhibits IK in both PASMC and RASMC (From Ref. 37).

Figure 3. Divergent effects of rotenone and hypoxia on renal (RAP) versus pulmonary (PAP) arterial pressure (A) and on K+ currents (IK) in PASMC and MASMC (B). A: A representative trace and mean data from the isolated perfused lung-kidney model are shown. Rotenone (5 nM) and hypoxia (Po2, 40 mmHg) increase PAP and decrease RAP in the presence of L-NAME and meclofenamate. 4-AP (5 mM) and angiotensin II (All, 0.1 ng/50 nl bolus) increase both RAP and PAP. B: Hypoxia and rotenone increase whole-cell IK in PASMC but decrease IK in RASMC. In contrast, 4-AP inhibits IK in both PASMC and RASMC (From Ref. 37).

6.2. Differential Regulation of Mitochondrial AOS and H202 Production between Pulmonary and Renal Arteries

We measured the baseline levels of AOS production (during normoxia) as well as the effects of hypoxia and ETC inhibitors in isolated, denuded, resistance PA and RA rings using three independent methods:

a). Lucigenin-enhanced chemiluminescence, which preferentially detects superoxide anion levels (34), shows that baseline AOS levels are significantly higher in the PA compared to the RA, even after incubation with 1 JJ.M diphenyliodonium (DPI), an inhibitor of NAD(P)H oxidase (16) (Fig. 4). Rotenone's effects on the production of AOS parallel those of hypoxia. They both decrease AOS production in isolated PA rings, confirming previous studies on whole lung preparations (1). In contrast, hypoxia and rotenone increased AOS production in the RA (Fig. 4), confirming studies in other systemic arteries, such as coronary arteries (29).

b). The AmplexRed assay and the DCF (2',7'-dichlorofluorescin diacetate) fluorescence assay, which are specific for H202 show that, concordant with the chemiluminescence data, the production of is higher in the PA versus the RA at baseline. Once again, proximal ETC inhibitors (rotenone and antimycin A, 50 (J.M) mimic hypoxic effect and decrease the production of H202in the PA. However, neither hypoxia nor the proximal ETC blockers alter the production of H202 in the RA (Fig. 4). Cyanide (1 ^M), a distal ETC complex IV blocker, does not affect H202 production in the PA and RA. Interestingly, we observed a significant auto-fluorescence of the aqueous sodium cyanide solution itself at a higher dose (10 jxM) which prevented us from using this dose (37).

Figure 4. Differences of mitochondria-derived activated oxygen species (AOS) production in pulmonary (PA) and renal (RA) arteries. A: Lucigenin-enhanced chemiluminescence at normoxic baseline is higher in PA than in RA rings without endothelium (J /MLOOl) in the absence or presence of the NAD(P)H oxidase inhibitor DPI (* P<0.05). Rotenone and hypoxia (Po2, 40 mmHg) decrease AOS in PA (t^O.Ol) and increase AOS in RA (** P<0.05). B: PA produces more H202 than RA (} P<0.01). Hypoxia and antimycin A decrease H202 production in PA (f P<0.05) but not in RA (From Ref. 37).

Figure 4. Differences of mitochondria-derived activated oxygen species (AOS) production in pulmonary (PA) and renal (RA) arteries. A: Lucigenin-enhanced chemiluminescence at normoxic baseline is higher in PA than in RA rings without endothelium (J /MLOOl) in the absence or presence of the NAD(P)H oxidase inhibitor DPI (* P<0.05). Rotenone and hypoxia (Po2, 40 mmHg) decrease AOS in PA (t^O.Ol) and increase AOS in RA (** P<0.05). B: PA produces more H202 than RA (} P<0.01). Hypoxia and antimycin A decrease H202 production in PA (f P<0.05) but not in RA (From Ref. 37).

To further show that the differences of the AOS production are intrinsic to the mitochondria, we isolated mitochondria from lungs and kidneys. The production of AOS and H202 from isolated mitochondria was measured with lucigenin-enhanced chemiluminescence and AmplexRed. Once again, the lung mitochondria produced more AOS and H202 than the kidney mitochondria and, while hypoxia and proximal ETC inhibitors decrease AOS and H202 in the lung, they do not in the kidney (Fig. 5A). In addition, in agreement with the vessel data, the complex IV ETC blocker cyanide (CN") does not alter AOS production, confirming previous reports (1) (Fig. 5A). These data are also consistent with complexes I and III, but not IV, being the major sites of AOS production within the ETC (18).

Figure 5. Lung mitochondria produce more AOS and are less active than kidney mitochondria. A: Lucigenin-enhanced chemiluminescence (a) indicating AOS production in the lung and kidney mitochondria in the absence or presence of cyanide (CN) or antimycin (* P<0.05). The catalase-sensitive production of H202 (b) measured by AmplexRed in the lung and kidney mitochondria (*P<0.05). B: The baseline respiration is greater in kidney mitochondria than in lung mitochondria. Rotenone and antimycin A inhibit respiration in both organs (f P<0.01, * P<0.05). Glutamate (10 mM) and succinate (2.5 mM) were used as substrates (From Ref. 37).

Figure 5. Lung mitochondria produce more AOS and are less active than kidney mitochondria. A: Lucigenin-enhanced chemiluminescence (a) indicating AOS production in the lung and kidney mitochondria in the absence or presence of cyanide (CN) or antimycin (* P<0.05). The catalase-sensitive production of H202 (b) measured by AmplexRed in the lung and kidney mitochondria (*P<0.05). B: The baseline respiration is greater in kidney mitochondria than in lung mitochondria. Rotenone and antimycin A inhibit respiration in both organs (f P<0.01, * P<0.05). Glutamate (10 mM) and succinate (2.5 mM) were used as substrates (From Ref. 37).

6.3. The RA and Kidney Mitochondria Have Higher Respiratory Rates and More Hyperpolarized Membrane Potentials Than Those in the PASMC and Lungs

Why would the two groups of mitochondria have different levels of AOS production? Since the AOS production is linked with mitochondrial respiration, we studied 02 consumption in both lung and kidney mitochondria. We show that lung mitochondria have significantly lower rates of respiration, compared to kidney mitochondria (Fig. 5B). Rotenone and antimycin A inhibit respiration in both lung and kidney mitochondria. Care was taken to study the same amount of mitochondria for both tissues, based on the protein content. Because of the tissue characteristics of lungs versus kidneys, the mitochondria isolation process could theoretically have resulted in preferential damage of one preparation versus the other, complicating any comparison. We therefore used two different assays to ensure that the quality of the mitochondria is similar in both preparations. Both preparations have almost identical respiratory control ratios and lysed/intact NADH-supported respiration ratios (17, 41). We isolated organ mitochondria because it is impossible to isolate adequate mitochondria from the small resistance rat PA and RA. Interestingly the data are in agreement with the measurements in isolated vessels, perhaps suggesting that the redox and metabolic environments of each organ parallel those of their vasculature.

We did however study vascular mitochondria in situ in freshly isolated PASMC and RASMC. As discussed above, the AOS production correlates with A*Fm and respiration, in that lower respiration is associated with depolarized A^ and higher AOS levels. Using two different A ^-sensitive dyes (JC-1 and TMRM), we showed that mitochondria from RASMC have significantly higher than mitochondria from PASMC under identical loading and imaging conditions. Extensive filamentous networks of mitochondria throughout the cytoplasm are shown in the representative pictures in Figure 6A. When the SMC were superfused with a hypoxic solution, the PASMC mitochondria hyperpolarized whereas the RASMC mitochondria depolarized (Fig. 6B).

Figure 6. Mitochondrial membrane potential (A^J is more negative in RASMC than in PASMC. A: JC-l-loaded PASMC and RASMC. B: Mean data (as the ratio of the relative fluorescent units in the green and red channels) showing baseline A*Fm in PASMC and RASMC (a). Hypoxia hyperpolarizes PASMC mitochondria and depolarizes RASMC mitochondria (b). *P<0.05, **P<0.0001 vs. PASMC (From Ref. 37).

Figure 6. Mitochondrial membrane potential (A^J is more negative in RASMC than in PASMC. A: JC-l-loaded PASMC and RASMC. B: Mean data (as the ratio of the relative fluorescent units in the green and red channels) showing baseline A*Fm in PASMC and RASMC (a). Hypoxia hyperpolarizes PASMC mitochondria and depolarizes RASMC mitochondria (b). *P<0.05, **P<0.0001 vs. PASMC (From Ref. 37).

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