Nitric oxide synthases (NOSs) are classified functionally, based on whether calmodulin binding is Ca 2؉ -dependent (cNOS) or Ca 2؉ -independent (iNOS). This key dichotomy has not been defined at the molecular level. Here we show that cNOS isoforms contain a unique polypeptide insert in their FMN binding domains which is not shared with iNOS or other related flavoproteins. Previously identified autoinhibitory domains in calmodulin-regulated enzymes raise the possibility that the polypeptide insert is the autoinhibitory domain of cNOSs. Consistent with this possibility, three-dimensional molecular modeling suggested that the insert originates from a site immediately adjacent to the calmodulin binding sequence. Synthetic peptides derived from the 45-amino acid insert of endothelial NOS were found to potently inhibit binding of calmodulin and activation of cNOS isoforms. This inhibition was associated with peptide binding to NOS, rather than free calmodulin, and inhibition could be reversed by increasing calmodulin concentration. In contrast, insert-derived peptides did not interfere with the arginine site of cNOS, as assessed from [ 3 H]N G -nitro-L-arginine binding, nor did they potently effect iNOS activity. Limited proteolysis studies showed that calmodulin's ability to gate electron flow through cNOSs is associated with displacement of the insert polypeptide; this is the first specific calmodulin-induced change in NOS conformation to be identified. Together, our findings strongly suggest that the insert is an autoinhibitory control element, docking with a site on cNOSs which impedes calmodulin binding and enzymatic activation. The autoinhibitory control element molecularly defines cNOSs and offers a unique target for developing novel NOS activators and inhibitors.Nitric oxide is a ubiquitous cell-signaling molecule, with protean roles in physiology and pathophysiology (1-3). Encoded by distinct genes, mammalian NO synthases (NOSs) 1 comprise a family of three calmodulin-dependent biopterohemoflavoproteins that are functionally distinguished by their modes of regulation (4). The two constitutively expressed isoforms of NOS (cNOSs), first identified in neuronal cells (nNOS) and endothelial cells (eNOS), remain dormant until calcium/calmodulin (Ca 2ϩ /CaM) binding is actuated by transient elevations in intracellular Ca 2ϩ . This Ca 2ϩ -dependent mode of regulation provides pulses of NO for moment-to-moment modulation of vascular tone and neurosignaling. In contrast, activity of the immunostimulant-induced isoform of NOS (iNOS) is Ca 2ϩ -independent, providing continuous high output NO generation for host defense. A remarkably high affinity for CaM, even at basally low levels of intracellular calcium, is responsible for the Ca 2ϩ independence of iNOS (5). Whether a given NOS isoform binds CaM in a Ca 2ϩ -dependent or -independent manner has been assumed to be a property solely of the amino acid sequence specified by a 20 -25-amino acid CaM binding site. However, this restrictive view is challenged by findings that ...

ABSTRACT:The free radical nitric oxide (NO), generated through the oxidation of L-arginine to L-citrulline by NO synthases (NOSs), has been shown to inhibit steroidogenic pathways. NOS isoforms are known to be present in rat and human testes. Our study examined the sensitivity of Leydig cells to NO and determined whether NOS activity resides in Leydig cells or in another cell type such as the testicular macrophage. The results showed a low level of L-[ 14 C]arginine conversion in purified rat Leydig cell homogenates. Administration of the NOS inhibitor L-N G -nitro-arginine methyl ester (L-NAME), or the calcium chelator ethylenebis (oxyethylenenitrilo)tetraacetic acid (EGTA), had no effect on L-[ 14 C]citrulline accumulation. Increased intracellular Ca 2ϩ concentrations that were induced by a calcium ionophore, or the addition of luteinizing hormone (LH), failed to affect NO formation in intact cells that were cultured in vitro. Introduction of a high concentration of the NO precursor L-arginine did not decrease testosterone (T) production, and NOS inhibitors did not increase T biosynthesis. However, exposing Leydig cells to low concentrations of the NO donor S-nitrosoglutathione (GSNO) induced a dramatic blockade of T production under basal and LH-stimulated conditions. DNA array assays showed a low level of expression of endothelial NOS (eNOS), while the neuronal and inducible isoforms of NOS (nNOS and iNOS) were below detection levels. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses confirmed these findings and demonstrated the presence of high iNOS messenger RNA (mRNA) levels in activated testicular macrophages that produced large amounts of NO. These data suggest that, while T production in rat Leydig cells is highly sensitive to NO and an endogenous NO-generating system is not present in these cells, NOS activity is more likely to reside in activated testicular macrophages.

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