No abstract

Nitric oxide (NO) affects many physiological systems by activating cGMP signaling cascades through soluble guanylate cyclase (sGC). In the accepted model, NO binds to the sGC heme, activating the enzyme. Here, we report that in the presence of physiological concentrations of ATP and GTP, NO dissociation from the sGC heme is Ϸ160 times slower than the rate of enzyme deactivation in vitro. Deactivated sGC still has NO bound to the heme, and full activation requires additional NO. We propose an activation model where, in the presence of both ATP and GTP, tonic NO forms a stable heme complex with low sGC activity; acute production of NO transiently and fully activates this NO-bound sGC.heme ͉ nucleotide regulation N itric oxide (NO) mediates blood vessel relaxation, complex aspects of myocardial function, perfusion and function of all major organs, synaptic plasticity in the brain, platelet aggregation, skin function, and numerous other physiological processes, by targeting and activating soluble guanylate cyclase (sGC) (reviewed in refs. 1-5). Dysregulation of NO signaling, then, contributes to many types of disease state, from erectile dysfunction and heart disease, to neurodegeneration, stroke, hypertension, and gastrointestinal disease, to name a few (reviewed in refs. 6-8).In vivo and ex vivo tissue studies of NO have revealed two fundamentally distinct and paradoxical signaling modes: tonic and acute. Tonic NO describes the continual low-level production of NO that elicits a long-lasting low-level cGMP signal (9). Under resting conditions such as normotension, inhibition of nitric oxide synthase (NOS) or sGC results in vasoconstriction; therefore, tonic NO-elicited cGMP production maintains homeostatic vascular tone. Relaxation of smooth muscle requires an acute burst of NO synthesis, typically triggered by acetylcholine, and cGMP levels rise rapidly (10). These data, which describe two separate effects of NO, cannot be explained by the established binary model for NO regulation of sGC: that NO activates sGC solely by binding to its heme cofactor.The N-terminal Ϸ180 aa of the ␤1 subunit of sGC form an evolutionarily conserved protoporphyrin-IX heme domain with spectral properties similar to the holoenzyme (11-13). NO binding to the sGC heme is diffusion-limited; however, oxygen does not bind to sGC, and carbon monoxide (CO) binding is at least 10 6 -fold weaker than NO. Thus, the sGC heme environment is a specific NO sensor. The C-terminal domains of each subunit are homologous to the catalytic domains of adenylate cyclase and fold together to form the active site of the enzyme (14). In the existing activation model (Fig. 1, black scheme), NO binds to the heme of sGC (15), forming a six-coordinate intermediate (16)(17)(18). The rate of conversion of this intermediate to the final five-coordinate ferrous-nitrosyl species depends on the concentration of NO; thus, nonheme NO accelerates rupture of the proximal histidine-iron bond. Breaking of this bond is thought to result in a conformational change in the cataly...

The bacterial toxin protein A from Staphylococcus aureus (SpA) interacts with B cell antigen receptors encoded by variable region heavy chain (VH) clan III genes via a V region framework surface that has been highly conserved during the evolution of the adaptive immune system. We have investigated the consequences of exposure to this prototypic B cell superantigen, and found that treatment of neonates or adults induces a T cell–independent deletion of a large supraclonal set of susceptible B cells that includes clan III/VH S107 family–expressing lymphocytes. In studies of different SpA forms, the magnitude of the induced deletion directly correlated with the VH-specific binding affinity/avidity. Upon cessation of SpA exposure, the representation of conventional splenic (B-2 subset) lymphocytes normalized; however, we found that the VH family–restricted deficit of peritoneal B-1 cells persisted. SpA treatment also induced a persistent loss of splenic S107-μ transcripts, with a loss of certain natural antibodies and specific tolerance to phosphorylcholine immunogens that normally recruit protective antimicrobial responses dominated by the S107-expressing B-1 clone, T15. These studies illustrate how a B cell superantigen can exploit a primordial Achilles heel in the immune system, for which B-1 cells, an important source of natural antibodies and host immune responses, have special susceptibility.