Ultraviolet (UV) radiation is the main physiological stimulus for human skin pigmentation. Within the epidermalmelanin unit, melanocytes synthesize and transfer melanin to the surrounding keratinocytes. Keratinocytes produce paracrine factors that affect melanocyte proliferation, dendricity, and melanin synthesis. In this report, we show that normal human keratinocytes secrete nitric oxide (NO) in response to UVA and UVB radiation, and we demonstrate that the constitutive isoform of keratinocyte NO synthase is involved in this process. Next, we investigate the melanogenic effect of NO produced by keratinocytes in response to UV radiation using melanocyte and keratinocyte cocultures. Conditioned media from UV-exposed keratinocytes stimulate tyrosinase activity of melanocytes. This effect is reversed by NO scavengers, suggesting an important role for NO in UVinduced melanogenesis. Moreover, melanocytes respond to NO-donors by decreased growth, enhanced dendricity, and melanogenesis. The rise in melanogenesis induced by NOgenerating compounds is associated with an increased amount of both tyrosinase and tyrosinase-related protein 1.These observations suggest that NO plays an important role in the paracrine mediation of UV-induced melanogenesis. ( J. Clin. Invest. 1997. 99:635-642.)

G-POEM was efficacious and safe for treating refractory gastroparesis, especially in patients with low pyloric distensibility.

The peptide hormone angiotensin II (AngII) binds to the AT 1 (angiotensin type 1) receptor within the transmembrane domains in an extended conformation, and its C-terminal residue interacts with transmembrane domain VII at Phe-293/Asn-294. The molecular environment of this binding pocket remains to be elucidated. The preferential binding of benzophenone photolabels to methionine residues in the target structure has enabled us to design an experimental approach called the methionine proximity assay, which is based on systematic mutagenesis and photolabeling to determine the molecular environment of this binding pocket. The octapeptide hormone angiotensin II (AngII) 1 (Fig. 1A) is the active component of the renin-angiotensin system. Virtually all known physiological effects of AngII are produced through the activation of the hAT 1 receptor, which belongs to the class A rhodopsin-like family of the heptahelical G proteincoupled receptor (GPCR) superfamily (1, 2). Elucidating the stereochemistry of the ligand-receptor interaction is vital for understanding the mechanism of ligand binding, GPCR activation, and, eventually, rational drug design.In the past, much effort was devoted to identifying the domains or individual residues of a given receptor that may interact with its ligand. Most experiments to address ligandreceptor interactions were performed with series of receptor mutants to identify specific residues critical to ligand binding (3-5). It is, however, speculative to deduce precise structures of ligand-receptor interactions through mutagenesis studies alone. More direct approaches have therefore been used to study ligand-receptor interactions. Among these is photoaffinity labeling, which allows covalent incorporation of the ligand within its binding site, presumably at the contact area of the photolabel in the receptor. This ligand-receptor contact can be identified by specific enzymatic or chemical digestion of the labeled receptor (6) or by mass spectrometry (7). The binding pockets within the transmembrane domains of several bioamine receptors have been identified using this kind of approach. The adenosine A 1 receptor (8) and the ␤ 2 adrenergic receptor (9, 10) are typical examples. Peptidergic receptors such as hAT 1 and hAT 2 (11, 12), neurokinin receptors (13), and several other receptors from the secretin GPCR family B (14) have been also studied using this approach. We previously identified ligandcontact points within the second extracellular loop (ECL) and the seventh transmembrane domain (TMD) of the hAT 1 receptor (12,15,16). Although photoaffinity labeling has been widely used to study peptidergic GPCR binding pockets, generally only a single contact point between a given ligand and its cognate receptor has been identified. The resulting information does not, however, induce sufficient restrictions to generate credible GPCR structures in the ligand-bound state using homology modeling.Labeling studies using benzophenone residues have identified many ligand-receptor contact points with a surpris...

Endotoxic shock, one of the most prominent causes of mortality in intensive care units, is characterized by pulmonary hypertension, systemic hypotension, heart failure, widespread endothelial activation/injury, and clotting culminating in disseminated intravascular coagulation and multi-organ system failure. In the last few years, studies in rodents have shown that administration of low concentrations of carbon monoxide (CO) exerts potent therapeutic effects in a variety of diseases/disorders. In this study, we have administered CO (one our pretreatment at 250 ppm) in a clinically relevant, well-characterized model of LPS-induced acute lung injury in pigs. Pretreatment only with inhaled CO significantly ameliorated several of the acute pathological changes induced by endotoxic shock. In terms of lung physiology, CO pretreatment corrected the LPS-induced changes in resistance and compliance and improved the derangement in pulmonary gas exchange. In terms of coagulation and inflammation, CO reduced the development of disseminated intravascular coagulation and completely suppressed serum levels of the proinflammatory IL-1beta in response to LPS, while augmenting the anti-inflammatory cytokine IL-10. Moreover, the effects of CO blunted the deterioration of kidney and liver function, suggesting a beneficial effect in terms of end organ damage associated with endotoxic shock. Lastly, CO pretreatment prevents LPS-induced ICAM expression on lung endothelium and inhibits leukocyte marginalization on lung parenchyma.

Activation of G protein-coupled receptors by agonists involves significant movement of transmembrane domains (TMD) following agonist binding. The underlying structural mechanism by which receptor activation takes place is largely unknown but can be inferred by detecting variability within the environment of the ligand-binding pocket, which is a water-accessible crevice surrounded by the seven TMD helices. Using the substituted-cysteine accessibility method, we identified the residues within the third TMD of the wild-type angiotensin II (AT 1 ) receptor that contribute to the formation of the binding site pocket. Each residue within the Ile 103 -Tyr 127 region was mutated one at a time to a cysteine. Treating the A104C, N111C, and L112C mutant receptors with the charged sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA) strongly inhibited ligand binding, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT 1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD3 reporter cysteines engineered in a constitutively active AT 1 receptor. Indeed, two additional mutants (S109C and V116C) were found to be sensitive to MTSEA treatment. Our results suggest that constitutive activation of the AT 1 receptor causes a minor counterclockwise rotation of TMD3, thereby exposing residues, which are not present in the inactive state, to the binding pocket. This pattern of accessibility of residues in the TMD3 of the AT 1 receptor parallels that of homologous residues in rhodopsin. This study identified key elements of TMD3 that contribute to the activation of class A G protein-coupled receptors through structural rearrangements.

GPCRs (G-protein-coupled receptors) are preferentially N-glycosylated on ECL2 (extracellular loop 2). We previously showed that N-glycosylation of ECL2 was crucial for cell-surface expression of the hAT1 receptor (human angiotensin II receptor subtype 1). Here, we ask whether positioning of the N-glycosylation sites within the various ECLs of the receptor is a vital determinant in the functional expression of hAT(1) receptor at the cell surface. Artificial N-glycosylation sequons (Asn-Xaa-Ser/Thr) were engineered into ECL1, ECL2 and ECL3. N-glycosylation of ECL1 caused a very significant decrease in affinity and cell surface expression of the resulting receptor. Shifting the position of the ECL2 glycosylation site by two residues led to the synthesis of a misfolded receptor which, nevertheless, was trafficked to the cell surface. The misfolded nature of this receptor is supported by an increased interaction with the chaperone HSP70 (heat-shock protein 70). Introduction of N-glycosylation motifs into ECL3 yielded mutant receptors with normal affinity, but low levels of cell surface expression caused by proteasomal degradation. This behaviour differed from that observed for the aglycosylated receptor, which accumulated in the endoplasmic reticulum. These results show how positioning of the N-glycosylation sites altered many properties of the AT1 receptor, such as targeting, folding, affinity, cell surface expression and quality control.

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