Regulation of soluble guanylyl cyclase: looking beyond NO.

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 and a heme-containing -sGC subunit. sGC 1/ 1-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 sGC 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 NOindependent 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), CCT (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 -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 -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 Address for reprint requests and other correspondence: W. J. Perkins, Mayo Clinic College of Medicine, 200 1st St. SW, Rochester, MN 55905 (e-mail: [email protected]). Am J Physiol Lung Cell Mol Physiol 291: L334–L336, 2006; doi:10.1152/ajplung.00158.2006.