neurotransmission, platelet function, skeletal muscle and vascular and nonvascular smooth muscle, and numerous other cellular functions are significantly regulated by the principal enzyme initially transducing nitric oxide (NO) signals, soluble guanylyl cyclase (sGC) (8). The better-characterized active forms of this enzyme exist as a heterodimer, comprising an α- and a heme-containing β-sGC subunit. sGC α1/β1-heterodimer, the principal active form in vascular smooth muscle, converts GTP to cGMP at a low rate in the NO free state. sGC activity is markedly increased by NO binding to the heme iron center on the sGCβ subunit, and this is the salient manner by which sGC activity is regulated. cGMP produced by sGC mediates physiological effects through a highly integrated and multifactorial system with NO and cGMP production/breakdown and cGMP-regulated proteins and their associated targets as the primary determinants of the ultimate physiological effect.
Although NO is the best-characterized mechanism by which sGC activity is regulated, other mechanisms that may fine tune sGC activity in physiological systems are known to exist. These can be broadly classified as NO bioavailability, sGC expression, and sGC activity per unit of enzyme present (specific activity for the purposes of this discussion). This brief discussion focuses on factors regulating NO-induced and NO-independent sGC specific activity. It should be pointed out that, with the exception of the purified enzyme, a change in specific activity is typically inferred when sGC activity is changed and an immunoblot band intensity with equal protein loading is either not changed or is not linearly proportional to the change in activity observed.
Although this NO-responsive enzyme is found largely in the soluble fraction of cells, sGC is nonetheless found associated with a number of different proteins, including postsynaptic density protein 95 (14), heat shock protein (HSP)90 (15), AGAP1 (11), HSP70 (1), CCTη (6), and Src (10). These protein interactions regulate sGC function either by localizing the enzyme adjacent to nitric oxide synthase, stabilizing the enzyme, or by modulation of specific activity by as yet unclear mechanisms. The extent to which these interactions are recruitable by specific conditions is a subject of continuing investigation.
sGC specific activity is also modulated by a number of other processes independent of NO, including phosphorylation (10), thiol redox (19), tissue redox state (12), and intracellular ATP (13). The finding that sGC activity acutely and significantly decreases, despite constant NO levels, strongly suggests that its activity is under regulation by factors other than NO under normal conditions (2).
Of critical importance to sGC activity is the presence and oxidation state of the heme group on the β-subunit (3, 16). Without heme in the Fe(II) state, there is no NO-induced sGC activation. The catalytic domain functions without heme as demonstrated by the presence of a basal GTP-to-cGMP conversion rate, but this is much lower than that attained by the NO-activated heme-containing enzyme. Earlier work performed in Louis Ignarro's laboratory, to which the senior author of the Mingone et al. study, one of the current articles in focus (Ref. 12a, see p. L337 in this issue), significantly contributed, indicated that the immediate biological precursor to heme, iron free protoporphyrin IX (PpIX), activated purified sGC at concentrations that were biologically relevant (9). Other heme biosynthetic precursors did not have this effect, and heme itself actually competitively inhibited both basal and PpIX-induced increases in sGC catalytic activity (18), until NO is bound to the heme Fe(II) center. The sGC-inhibiting effect of heme and conversion of the enzyme from an NO-unresponsive to NO-responsive enzyme are central to regulation of the NO/sGC/cGMP signaling system. Competitive heme inhibition of PpIX-induced increases in purified sGC activity indicated that the two porphyrins bound to the same site on sGC and also indicated that sGC-bound porphyrin is exchangeable. The possibility for a PpIX role in regulating sGC activity in intact tissue was suggested in this earlier work but had never been examined until the present study.
The first committed precursor to heme biosynthesis is δ-aminolevulinic acid (ALA) (17). Whereas ALA biosynthesis is subject to feedback inhibition by heme, exogenous ALA bypasses this feedback control and results in increased PpIX and heme biosynthesis. Under normal circumstances, the final step in heme biosynthesis, ferrochelatase-mediated insertion of Fe(II) into PpIX, efficiently converts most of the PpIX into heme. In the presence of a large excess of ALA that might overwhelm the bioavailable Fe(II), however, PpIX accumulation may occur. This is particularly true for certain tumor tissues and, coupled with PpIX spectroscopy and photochemistry, has been used in human subjects as a photosensitizer for photodynamic targeting of tumor cells with laser ablation (5). Tissue containing increased levels of PpIX have characteristic epifluorescence, and this can be used to qualitatively assess the presence of increased PpIX levels in a tissue following exogenous administration of ALA. Taking advantage of these findings and methods established for photodynamic treatment of tumors, Mingone et al. (12a) first determined whether exogenous ALA administration results in increased PpIX accumulation in pulmonary arteries (PA). They then tested the hypothesis that PpIX accumulation results in findings consistent with sGC activation in intact vascular smooth muscle.
The authors used cultured bovine PA preparation in these studies. Although this preparation introduces diffusion barriers to the uptake of ALA and tissue opacity and light scattering into the fluorescence detection method used to assess PpIX, epifluorescence in intact tissue has been correlated with PpIX levels in these tissues. In addition, use of cultured PA permits assessment of functionally significant effects associated with increased PpIX, such as isometric force response to contractile agonist. Another advantage to this approach over that of cultured vascular smooth muscle cells is that the smooth muscle cells in the organ culture preparation are unambiguously the contractile phenotype over the time frame used by the authors.
The epifluorescence method used in this study demonstrates a time-dependent and ALA concentration-dependent increase in signal in PA. The increase in epifluorescence observed at 24 h of treatment with 100 μM ALA was comparable to that obtained with 3 μM PpIX. The authors appropriately avoid assigning this increase in epifluorescence exclusively to PpIX, since the emission filter used in these studies would also detect other heme precursors, such as uroporphyrinogen III and coproporphyrinogen III. The observations are, however, consistent with an exogenous ALA induced increase in heme precursor levels in PA. The ALA-induced increase in epifluorescence reflects an increase in heme precursors and not in heme. Addition of exogenous Fe(II) with ALA resulted in an increase in heme, but not in epifluorescence, which suggests that ALA alone increases heme precursors due to a limitation in the amount of Fe(II) available to ferrochelatase. To test the possibility that reduced Fe(II) availability could result in an increase in heme precursor accumulation at lower ALA concentrations, the authors added the iron-chelating agent deferoxamine to the culture medium and found that this was the case.
Mingone et al. (12a) then demonstrated that ALA treatment resulted in a reduction in isometric force in response to the PA contractile agonist serotonin, resulting in submaximal and maximal contractions. It is possible that high concentrations of myoplasmic ALA itself might have contributed to this finding. This is unlikely, however, since exogenous Fe(II) eliminated both the ALA-induced increase in epifluorescence and reduction in serotonin-induced contraction. This was consistent with the possibility that ALA-induced increase in heme precursor levels was related to the reduction in serotonin-induced contraction.
The soluble fraction of PA tissue homogenates were used to determine the amount of cGMP produced per unit time in PA that had been treated with ALA, Fe(II), and ALA + Fe(II). These studies were performed in the presence of a phosphodiesterase (PDE), which decreases the likelihood that observed findings could be accounted for by a possible interaction between ALA or heme precursors and the PDE system. ALA, but not Fe(II), increased cGMP production by ∼50%. Fe(II) prevented the ALA-induced increase in cGMP production. The interesting possibility that ALA treatment increased steady-state cGMP levels in intact PA was not determined. Together, these results suggest that ALA treatment results in a heme precursor-related relaxant effect that was associated with an increase in cGMP production. The possibility that this might be accounted for by an ALA-induced increase in sGC protein expression was addressed by Western blot analysis. sGC protein expression was decreased by ALA, which suggests that the ALA-induced increase in cGMP production reflects an even greater increase in sGC specific activity.
In the soluble fraction of a PA tissue homogenate, the only enzyme known to produce cGMP is sGC and the only heme precursor known to increase sGC activity is PpIX. Several predictions would be made if the ALA-mediated effects on contraction occur via PpIX sGC activation. The first is that an sGC inhibitor that oxidizes the heme Fe(II) (20) should have no effect on ALA-induced reductions on agonist-induced contraction, since PpIX sGC contains no Fe(II) central metal ion. A second and related, but independently testable, prediction is that cGMP-dependent protein kinase target phosphorylation should be similarly unaffected by the sGC inhibitor. Both predictions were borne out and were in distinct contrast to findings obtained when an NO donor rather than ALA was used to reduce agonist induced contraction.
The findings by Mingone et al. (12a) support the earlier suggestion made by Louis Ignarro and the senior author that protoporphyrin IX “could play a biological role in altering guanylate cyclase activity” (9). Whereas the heme precursor detection methods used by Mingone et al. are not specific for PpIX and sample only the tissue surface, the findings are consistent with their hypothesis. Direct spectroscopic demonstration of PpIX/sGC formation in small tissue samples is not currently feasible. The results by Mingone et al. suggest, however, that exchange of PpIX for heme occurs in the more complex tissue environment. Under what circumstances might PpIX/sGC might comprise a significant fraction of the sGC in a tissue? ALA is being administered to patients to increase tumor photosensitivity to laser ablation. A significant reduction in systemic and pulmonary artery pressures has been reported in these patients (7), which may relate to formation of PpIX/sGC. It is unclear whether the rapid onset of these hemodynamic findings are consistent with formation of PpIX/sGC and further work with animal models will be required to clarify this. Patients with erythropoietic protoporphyria have mutations in their ferrochelatase gene, which results in accumulation of PpIX and might also be expected to have significant levels of PpIX/sGC in some tissues (4).
The article by Mingone et al. (12a) is a welcome addition to the literature on factors regulating sGC specific activity and is a field to which the senior author has made sustained and significant contributions. It is becoming increasingly clear that sGC specific activity is regulated by factors other than NO, and we can look forward to greater insights into contributions from sGC-associated proteins, from a variety of posttranslational sGC modifications, and from sGC heme content and state.
This work was supported by National Heart, Lung, and Blood Institute Grant HL-69968.
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